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Structural alterations of the rat iris associated with function and growth Lim, Wan Cheng 1973

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STRUCTURAL ALTERATIONS OF THE RAT IRIS ASSOCIATED WITH FUNCTION AND GROWTH by WAN CHENG LIM B.A., Wellesley College, 1968 M.Sc., University of British Columbia, 1970  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  in the Department of ANATOMY  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA September 1973  In presenting  this thesis i n p a r t i a l fulfilment of the requirements for  an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t freely available for reference  and study.  I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may by his representatives.  be granted by the Head of my Department or  It i s understood that copying or publication  of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission.  Department of  , '  /HN/TTOM^  The University of B r i t i s h Columbia Vancouver 8, Canada  Date *cT.  *t  H7^>  ABSTRACT 1.  The  s t r u c t u r a l a l t e r a t i o n s of the a d u l t r a t i r i s  w i t h i t s f u n c t i o n of d i l a t i n g and The  associated  c o n s t r i c t i n g the p u p i l were examined.  r a t eyes were r e g u l a r l y d i l a t e d or c o n s t r i c t e d w i t h a few drops of a  m i x t u r e of p h e n y l e p h r i n e h y d r o c h l o r i d e iodide, respectively. light,  The  t r a n s m i s s i o n and  and  cyclopentolate,  or e c h o t h i o p h a t e  eyes were p r e p a r e d f o r e x a m i n a t i o n w i t h  the  s c a n n i n g e l e c t r o n m i c r o s c o p e s by w e l l - k n o w n methods.  In p u p i l l a r y d i l a t i o n ,  scanning e l e c t r o n microscopic  studies  reveal  t h a t the p o s t e r i o r e p i t h e l i a l c e l l s a r e a r r a n g e d i n c i r c u m f e r e n t i a l r i d g e s w h i c h b i f u r c a t e , t a p e r down and s u r f a c e of the i r i s , bulge prominently  j o i n with adjacent ridges.  the a n t e r i o r  blood v e s s e l s , g e n e r a l l y c i r c u m f e r e n t i a l l y o r i e n t e d ,  outwards.  In transmission  e l e c t r o n m i c r o g r a p h s the e p i t h e l i a l c e l l s a r e  and a r e d i s c r e t e l y s e p a r a t e d from each o t h e r . of the n u c l e a r  On  envelope.  The  n u c l e i show  Bundles of i n t r a c e l l u l a r f i l a m e n t s  'hammock' around the n u c l e u s .  The  indentations  form a  d i l a t o r muscle l a y e r i s t h i c k .  h i l l o c k s and  d i l a t o r p r o c e s s e s a r e found a l l a l o n g  the stroma.  The  high  Dilator  the boundary zone w i t h  n u c l e i of the d i l a t o r muscle c e l l s a l s o show n u c l e a r  indentations. W i t h the l i g h t m i c r o s c o p e , i t i s c l e a r l y shown t h a t the components and perpendicular  stromal  the c o l l a g e n network appear to be a r r a n g e d i n columns t o the p o s t e r i o r s u r f a c e of the  iris.  A low m a g n i f i c a t i o n s c a n n i n g e l e c t r o n m i c r o g r a p h of the r a t  iris  i n p u p i l l a r y c o n s t r i c t i o n shows t h a t most of the p o s t e r i o r s u r f a c e of iris  i s smooth.  mere p i n h o l e ,  the  I n extreme p u p i l l a r y c o n s t r i c t i o n , where the p u p i l i s a  the p o s t e r i o r e p i t h e l i a l c e l l s around the p u p i l a r e a r r a n g e d  i n r a d i a l r i d g e s which p e t e r out p e r i p h e r a l l y .  Bulbous s t r u c t u r e s  are  often seen in amongst the radial epithelial ridges.  On the anterior sur-  face of the i r i s the blood vessels do not bulge out as prominently, as they zig-zag from the periphery to the pupillary margin.  The morphology of the  iridic crypts and pores are well illustrated. In transmission electron micrographs, the posterior epithelial cells in pupillary constriction are low and they form a continuous layer. The nuclei, with smooth nuclear outlines, and the intracellular filaments are disposed parallel to the length of the layer is low.  cells.  The dilator muscle c e l l  Dilator hillocks and dilator processes are absent.  The  nuclei of the dilator muscle cells have a smooth outline and l i e along the length of the cells. The stromal elements and the connective tissue framework are oriented parallel to the posterior surface of the i r i s , as seen light microscopically. 2.  The overall developmental changes in the structure of the  various components of the fetal and post-natal rat iris were observed on toluidine blue stained plastic sections.  In the immature i r i s , the brick-  shaped posterior epithelial cells form a continuous layer.  By two weeks  after birth, the epithelial cells have acquired characteristics of the adult i r i s .  The anterior epithelium develops to give rise to the sphincter  and dilator muscles.  The stromal elements stream into the i r i s parallel to  the posterior surface of the i r i s in very close association with the stromal surface of the developing dilator muscle cells.  With development, the  stromal elements move away from the dilator. A scanning electron microscopic  study of the posterior surface of  the developing rat i r i s shows the changes in the surface configurations of the posterior epithelial cells.  I n i t i a l l y the posterior surface of the  iris is smooth.  By a gradual process, the epithelial cells begin to bulge  out posteriorly.  By two weeks after birth, the epithelial cells are be-  ginning to be arranged in rows. The topography of the pupillary membrane and its relationship with the hyaloid system is shown. Most of the blood vessels of the pupillary membrane appear to come from the i r i s stroma with perhaps some contribution from the hyaloid system.  The thin-walled  blood vessels are suspended within  a scaffolding of connective tissue fibers.  The rat pupillary membrane is  s t i l l present during the f i r s t few days after birth. Changes in the permeability  of the i r i s blood vessels to an intra-  vascularly injected solution of HRP were investigated.  The i r i s capilla-  ries of the fetal and early post-natal rats, up to two weeks after birth, are readily permeable to HRP. the adult i r i s .  They then become impermeable to HRP, as in  TABLE OF CONTENTS Page ABSTRACT  i i  TABLE OF CONTENTS  v  LIST OF TABLES  x  LIST OF FIGURES ACKNOWLEDGMENT  xi „  xiii  DEDICATION  xiv  EXPLANATORY NOTE  xv  INTRODUCTION  1  A. Historical  1  B.  Functions of the Iris  .  C.  Iris Colors  4  D.  Pupillary Patterns  7  E.  Comparative Anatomy of the Vertebrate Iris  13  F.  Areas of Research on the Iris  22  1. General Consideration of the Iris  23  2. Posterior Pigment Epithelium  27  .  2  3.  Anterior Epithelium and Dilator Muscle  30  4.  Sphincter Muscle  33  5.  Stroma  35  6.  Iris Blood Vessels  39  7.  Innervation of the Iris  44  . „  8. Miscellaneous Studies on the Iris  54  G. Thesis Proposal  59  MATERIALS AND METHODS  65  A. A Light Microscopic Study of the Rat Iris in Pupillary Dilation and Constriction  <>»..  65  v L.  Page 1.  For Demonstrating the Overall Histology of the Iris  65  2. For Demonstrating the Collagen Network in the Iris Stroma  66  B. A Transmission Electron Microscopic Study of the Rat Iris in Pupillary Dilation and Constriction  66  C. A Scanning Electron Microscopic Study of the Rat Iris in Pupillary Dilation and Constriction  68  1. Camphene Method  68  2. D.  Critical Point Drying Method  „ . .  69  A Light Microscopic Study of the Development of the Rat Iris Using Toluidine Blue Stained Epon Sections and Horse-radish Peroxidase (HRP) Studies of the Iris in Fetal, Post-natal and Adult Rats  70  2.  Post-natal and Adult Rats  71  3. Processing of the Tissues  72  Examination of the Tissues  . . . _  74  A Scanning Electron Microscopic Study of the Posterior Surface of the Developing Rat Iris  74 o . . . . .  RESULTS A.  70  1. Fetal Rats  4. E.  „  .  76  A Light Microscopic Study of the Rat Iris in Pupillary Dilation and Constriction 1. The Iris in Pupillary Dilation (Figures 2-7)  76 ...  76  2. The Iris in Pupillary Constriction (Figures 8-11) .  89  vii  Page B.  A Light Microscopic Study of the Collagen Network in the Stroma of the Rat Iris in Pupillary Dilation and Constriction (Figures 12-13)  C.  D.  ...  96  A Transmission Electron Microscopic Study of the Rat Iris In Pupillary Dilation and Constriction  100  1.  General (Figure 14)  100  2.  The Iris in Pupillary Dilation (Figures 15-23)  3.  The Iris in Pupillary Constriction (Figures 24-30)  . .  126  A Scanning Electron Microscopic Study of the Rat Iris in Pupillary Dilation and Constriction 1. General 2.  . . . . . . .  . .  3.  The Posterior Surface of the Iris in Pupillary 139  The Posterior Surface of the Iris in Pupillary Constriction (Figures 39-56)  4.  150  The Anterior Surface of the Iris in Pupillary Dilation (Figures 57-65)  5.  139 139  Dilation (Figures 31-38)  172  The Anterior Surface of the Iris in Pupillary Constriction (Figures 66-76)  E.  102  0  . . . .  185  A Light Microscopic Study of the Development of the Rat Iris Using Toluidine Blue Stained Plastic Sections  . .  . . . . „ „ . . . .  200  1.  19 Days Fetal (Figures 77-79)  2.  20-21 Days Fetal (Figures 80-82)  210  3.  1-4 Days Post-natal (Figures 83-86)  214  4.  5-10 Days Post-natal (Figures 87-90)  5.  From 11 Days Post-natal On (Figures 91-95)  „  200  225 ....  233  viii  Page.  F.  G.  A Scanning Electrcm Microscopic Study of the Posterior Surface of the Developing Rat Iris  240  1.  17 Days Fetal-Term (Figures 96-105)  240  2.  1-4 Days Post-natal (Figures 106-115)  253  3.  5-10 Days Post-natal (Figures 116-127)  266  4.  From 11 Days Post-natal On (Figures 128-137)  ...  Horse-radish Peroxidase (HRP) Studies of the Iris in Fetal, Post-natal and Adult Rats (Figures 138-150)  . .  DISCUSSION AND SUMMARY OF THE RESULTS A.  282  295 313  A Light and Transmission Electron Microscopic Study of the Rat Iris in Pupillary Dilation and Constriction . .  313  1. General  313  „ „ . .  2. The Histological and Ultrastructural Features of the Iris in Pupillary Dilation and Constriction . . B.  A Scanning Electron Microscopic Study of the Rat Iris in Pupillary Dilation and Constriction 1.  Comments on the Methodology  2.  The Posterior Surface of the Iris in Pupillary Dilation and Constriction  3.  . . . . .  324 „ .  324  .  327  The Anterior Surface of the Iris in Pupillary Dilation and Constriction  C.  332  A Light Microscopic Study of the Development of the Rat Iris Using Toluidine Blue Stained Plastic Sections  D.  316  . .  335  A Scanning Electron Microscopic Study of the Posterior Surface of the Developing Rat Iris  „  342  1. General 2. The Posterior Surface of the Developing Rat Iris  342 .  343  ix. a  Page 3. The Peri-natal Vascular System of the Rat Iris E.  . .  346  Horse-radish Peroxidase (HRP) Studies of the Iris in Fetal, Post-natal and Adult Rats  CONCLUDING RE^1ARKS BIBLIOGRAPHY  .  „  350 .  358  c . o  367  LIST OF TABLES  Table 1  HRP Tracer Studies  x i  LIST OF FIGURES Figure  Page  1  Pupillary Patterns  . . . . „  2-7  The Iris in Pupillary Dilation (LM)  8-11  The Iris in Pupillary Constriction  12 84-S8 (LM)  . . . „ . .  12  The Collagen Network in Pupillary Dilation (LM)  13  The Collagen Network in Pupillary Constriction  14-23  The Iris in Pupillary Dilation (TEM)  106-125  24-30  The Iris in Pupillary Constriction (TEM)  129-138  31-38  The Posterior Surface of the Iris in Pupillary Dilation (SEM)  39-40  154-155  The Posterior Surface of the Iris in Extreme  (SEM)  The Posterior Surface of the Iris in Extreme (SEM)  162-171  The Anterior Surface of the Iris in Pupillary Dilation (SEM)  66-76  156-157  158-163  Pupillary Constriction 57-65  (SEM)  The Posterior Surface of the Iris in Pupillary Constriction  48-56  99  The Posterior Surface of the Iris in Pupillary  Pupillary Constriction 43-47  (LM)  97-98  142-149  Constriction (SEM) 41-42  . .  92-95  175-184  The Anterior Surface of the Iris in Pupillary Constriction (SEM)  188-199  77-79  19 Days Fetal (LM)  .  206-209  80-82  20-21 Days Fetal (LM)  212-213  83-86  1-4 Days Post-natal (LM)  221-224  87-90  5-10 Days Post-natal (LM)  229-232  91-95  From 11 Days Post-natal On (LM)  236-239  Figure  Page  96-105  17 Days Fetal - Term (SEM)  243-252  106-115  1-4 Days Post-natal (SEM)  116-127  5-10 Days Post-natal (SEM)  128-137  From 11 Days Post-natal On (SEM)  138-139  20-21 Days Fetal (HRP)  .  299-300  140-141  1 Day Post-natal (HRP)  . . . . . .  301-302  142-143  4 Days Post-natal (HRP)  144-145  5 Days Post-natal (HRP)  .  305-306  146-147  7 Days Post-natal (HRP)  .  307-308  148-149  12 Days Post-natal (HRP)  150  Adult (HRP)  . . .  256-265 270-281  „ . . .  285-294  303-304  309-310 . . .  311-312  ACKNOWLEDGMENT  Dr. W.A. Webber, my supervisor, gave his much-welcomed advice and suggestions freely throughout the course of this study preparation of the thesis.  and in the  Under his subtle supervision, doing research  was made enjoyable. Dr. C.E. Slonecker was always present with encouragement and with his perspectives on research. Pam G i l l expertly perfused the rats for the transmission electron microscopic studies. A few have been catalysts and sources of encouragement for the completion of the thesis. To a l l the above, I wish to express my appreciation.  (This research was supported by a Medical Research Council Studentship)  for  Explanatory Note: The terms "anterior" and "posterior" are considered with respect to the eye alone and not with respect to the whole rat, as the rat eye does not face directly anteriorly. "Centrally" is used synonymously with "pupillary" to indicate direction towards the center of the pupil. "Peripherally" indicates direction towards the root of the i r i s .  I. A.  INTRODUCTION  Historical Stephen Polyak (1957) has given us an interesting account of the  history of the study of the eye. 370 B.C.)  Even as early as the 4th century B.C.  (460  Democritos of Abdera and Hippocrates described the eye as consis-  ting of a number of concentric tunics; an outer, white, fibrous tunic (the sclera) with an anterior transparent cornea; a delicate, spongy, middle tunic with an anterior colored portion (the i r i s ) , a crown (the ciliary body and i r i s root) and a central opening (the pupil); and an inner (the retina).  tunic  However, i t was not until the time of Herophilos (344-280  B. C.) that the first scientific description of the eye appeared. vations were based on actual animal dissections.  His obser-  Early diagrams of the  structure of the eye are found in not only Greek but also Arab anatomy texts The Arabic diagrams were probably derived from Greek sources as the Arabs, being Muslims, were not allowed to dissect animals.  Over the years,  changes were made to these early eye diagrams as anatomists gained more knowledge of the relative proportions and relationships of the various parts of the eye. Rufos and Galen, two Greek anatomists from Classical times, in studying the eye, described the i r i s as a grape-like tunic. meaning a "rainbow" in Greek was 1961).  The word " i r i s "  f i r s t used by Rufos (Duke-Elder and Wybar,  The i r i s is appropriately so named because of the amazing varia-  tions in color that do occur in this structure. fishes, amphibians, reptiles, birds, and man,  In a large number of  the i r i s exhibits diverse  variegated color schemes. The pupil, the aperture formed by the i r i s , means "maiden" or " l i t t l e g i r l " in Greek.  The reason for this nomenclature is speculative.  It has been suggested that "the pupil was probably so named from the diminutive image seen reflected from the cornea standing out on the black background of the pupil" (Duke-Elder and Wybar, 1961, p. 167).  B.  Functions of the Iris The i r i s is a delicate and movable diaphragm similar to those found  in other optical systems. of the eye.  It separates the anterior and posterior chambers  The size and shape of the pupil, the aperture formed by the  i r i s , is controlled by movements of the i r i s resulting in variations in its area.  The main function of the i r i s is to regulate the amount of light  entering the eye and impinging on the retina u n t i l the retina has satisfactorily adjusted itself to the light.  This is needed as there are variations  in the sensitivity of different retinas to external illumination.  In  general, the image on the retina will be sharper with a decreased aperture or pupil size.  A small pupil prevents light coming from the periphery of  the cornea from passing through the lens.  It channels the light through the  central, optically good part of the lens, thus resulting in a sharp image on the retina. ing  However, with a smaller aperture the amount of light enter-  the eye may not be sufficiently intense to stimulate the retina.  such an instance, the pupil must be capable of being widely dilated.  In The  sharpness and clarity of the retinal image is reduced but at least an image is perceived. The i r i s serves a secondary function in some amphibious vertebrates where by the very nature of their habits and habitats a wide range of accommodation is required (Walls, 1942; Rochon-Duvigneaud, 1943; Prince, 1956).  Having a dual habitat, aquatic and terrestial, a means must be  found to ensure that as perfect a focus is attainable so as to give the  animals reasonable vision both when the head is in the air and when i t is immersed in water.  The range of accommodation has to be greatly increased.  In the turtles (Walls, 1942, pp. 436-437; Rochon-Duvigneaud, 1943, pp. 126, 387-388; Prince, 1956, pp. 237, 374), we see a superb example of how this is accomplished.  The lens is extremely soft and pliable.  It projects  through the pupil so that the i r i s sphincter surrounds the anterior peripheral part of the lens.  In accommodation, the powerful i r i s sphincter  contracts and grips the lens.  The front of the lens is squeezed and dis-  torted to give a very short radius of curvature to the lens.  The i r i s  sphincter also aids in accommodation by deformation of the lens in some aquatic snakes (Prince, 1956, pp. 235, 375), for example the Natrix tessellatus (Walls, 1942, p. 438); in some diving birds, for example, the shags and cormorants (Walls, 1942, p. 440; Rochon-Duvigneaud, 1943, pp. 472473; Prince, 1956, p. 238); and even in an aquatic mammal, the sea otter (Walls, 1942, p. 444; Rochon-Duvigneaud, 1943, pp. 669-670; Prince, 1956, p. 240). The cormorant is an aquatic bird which dives deeply into the water and remains submerged for a considerable length of time. muscular i r i s of a l l the vertebrates so far studied. particularly well-developed.  It has the most  The i r i s muscles are  The circular bundles of the sphincter muscle  fibers are found extending from the pupillary margin to the root of the i r i s (Rochon-Duvigneaud, 1943, pp. 472-473).  At the i r i s root, the sphincter  muscle fibers are found intermingled with some radial fibers of the dilator muscle.  Wherever and whenever warranted, the i r i s not only acts to regulate  the amount of light entering the eye but i t also assists in the accommodation process.  This latter function, though present in only a few verte-  brates, is nevertheless important.  It may oftentimes supercede the other  means of accommodation present in the eye.  C.  Iris Colors Although iris coloration niay appear meaningless in terms of its  physiological importance to man or animal, i t nevertheless has a significant role to play in the expression and ornamentation of the eye.  The varying  color of the vertebrate iris is the result of pigmentation within the i r i s tissue and optical phenomena. Unlike other vertebrates, where the iris may exhibit a range of brilliant hues, the color of the i r i s in man is either light blue or grey to dark brown (Coque, 1927; Walls, 1942; Rochon-Duvigneaud, 1943; Duke-Elder and Wybar, 1961; Hogan, Alvarado and Weddell, 1971). The color of the i r i s is genetically transmitted where blue eyes are recessive to brown or dark eyes. the  The color of the human i r i s depends on  amount and location of the i r i d i c pigment, melanin, as well as on the  density of the stromal tissue.  There are two kinds of pigment in the  human i r i s , that found in the posterior pigment epithelial cells and that contained in the branched melanocytes of the i r i s stroma.  The pigment  of the posterior epithelial cells is very opaque and shows a certain resistance to chromic acid decoloration (Rochon-Duvigneaud, The pigment granules w i l l turn yellow but not white.  1943, p. 36).  Pigment is almost always  present in the posterior pigment epithelial c e l l s except in extremely rare cases of true albinism. epithelial cells. amounts of pigment.  In blue eyes, pigment is present mainly in the  A few of the stromal cells may also contain varying The deep blue color of the i r i s of Caucasions at birth  is the result of the absorption of the long wavelengths of light by the s t i l l fairly translucent iris stroma. are  Thus only the shorter blue wavelengths  transmitted to the observer's eyes after being reflected from the  posterior pigment epithelial layer.  I f the i r i s stroma is thick but s t i l l  devoid of melanocytes, the iris appears grey.  In dark irises, pigment  is present in varying amounts in the stromal melanocytes.  The  coloration of the i r i s may not be uniform due to differences in the degree of pigmentation in various parts of the i r i s .  Freckles or dark brown spots  on the i r i s denote areas of hyperpigmentation, that is, where there are clusters of melanocytes. mottled appearance.  A large number of such spots w i l l give the i r i s a  Heterochromia is also a result of the uneven distribu-  tion of stromal melanocytes.  In such cases, one part of the i r i s is of a  different color from that of the other parts.  There may even be striking  differences between the two irises of the same individual.  It appears that  there are documented cases of freaks of nature where the arrangement of the pigmented spots and markings on the i r i s resemble letters or numbers.  As  is clearly apparent, the possible variations in the coloration of the human i r i s are enormous.  Attempts have been made to classify the colors of the  individual irises and to use such a classification as a means of identifying criminals (Duke-Elder and Wybar, 1961). When compared to the relatively sober colors of the human i r i s , those of the other vertebrates appear almost ostentatious.  Rochon-Duvigneaud  (1943) in his vertebrate ocular anatomy text "Les yeux et la vision des vertebres" discusses the whole gamut of i r i s colors - shades of orange, yellow, green, blue, red, white, gold, etc. - encountered in the different groups of vertebrates.  Some have a metallic silvery or gold sheen to the  i r i s while others show only a silver or gold lining on the pupillary edge. What is present in these irises to impart to them such brilliant and variegated color schemes?  In man and the Cyclostomes (Rochon-Duvigneaud, 1943,  p. 185), only melanin pigment is present in the stromal melanocytes.  How-  ever, in the other vertebrate groups, a varying quantity of crystals and pigments, other than melanin, are found within some of the other stromal cells.  The type and quantity of these other stromal elements is what makes  the difference.  In the Elasmobranchs  (Rochon-Duvigneaud, 1943, p. 214) the  6.  color and metallic lustre of the i r i s is not as distinctive as in the higher Teleosts (Rochon-Duvigneaud, 1943, pp. 245-275).  There is no definitive  guanophore layer although some guanine crystals are present.  The golden or  bronzed color of the anterior surface of the amphibian eye (Rochon-Duvigneaud, 1943, p. 340) owes its coloration to both carotinoid pigments and guanine crystals.  The carotinoid pigments are seen as red or yellow (in the  oxidised state) granules in certain stromal cells. belong to other cell types. entering the eye.  The guanine crystals  These crystals diffract and reflect the light  It was once thought that there were xantholeucophores in  the i r i s but the research of Schmidt and Millot have refuted this.  The  melanocytes beneath the b r i l l i a n t l y colored layer also help to modify the color of the i r i s . or yellow irises.  Crocodiles (Rochon-Duvigneaud, 1943, p. 368) have buff They only have a thin and discontinuous layer of cells  containing guanine on the anterior i r i s surface.  In many reptiles (Rochon-  Duvigneaud, 1943, pp. 393-394) guanine-containing cells cover the anterior surface of the i r i s and impart to i t a brilliant and metallic hue. Differences occur according to the species.  Birds (Rochon-Duvigneaud,  1943, p. 472) generally have deep brown or black irises because of the melanocytes in the stroma. eyes.  However, some birds do have brightly colored  Then, the i r i s shows a layer of vesiculated cells which contain red  or yellow pigments.  Birds are interesting, in that of a l l the vertebrates,  with a few exceptions, sexual differences are expressed in the colors of the irises.  The female pheasant and condor have brown eyes while their respec-  tive males have yellow and red eyes.  In the booby (Walls, 1942) the male  has a yellow i r i s but the female has,  in addition, a ring of black spots  along the pupillary margin.  Other than the birds, the irises of the male  adder and box turtle are red in color while those of the females are light brown and yellow brown, respectively.  Among the birds, too, the intensity  of the i r i s colors changes with heightened sexual activity.  Various other  pheonmena also seem to affect the color of the i r i s in some vertebrates (Walls, 1942, p. 550; Rochon-Duvigneaud, 1943, p. 245), for example, emotion, age, the seasons and noxious stimuli.  D.  Pupillary Patterns The anatomy of the eye of man or animal is adapted so as to best  meet the needs of the individual in performing i t s life-sustaining activities.  This is also subject to the demands of the environment, whether i t  be water, a i r or the ground.  The information in the following discussion  has been gleaned from numerous sources (Walls, 1942; Rochon-Duvigneaud, 1943; Prince, 1956; Prince, Diesen, E g l i t i s and Ruskell, 1960). The whole animal world can very broadly be divided into four main activity groups; those which are diurinal, those which are nocturnal, those which are arrhythmic and those which are crepuscular. much overlapping of the categories. to interact are varied.  Naturally there is  The conditions wherein the animals have  Anatomically differences are seen in the eye.  fications occur so as to allow the animal to function efficiently.  Modi-  The  shape, size and mobility of the pupil partly does signify to which activity group an animal belongs.  However, this is only partly so.  The pupillary  form must always be considered in relationship to the structure of the retina. Both i r i s and the retina work together in harmony to ensure that as clear a retinal image as possible is obtained. Certain requirements must be met in order for an animal to-operate effectively within a particular activity group.  Animals which are diurnal,  for example, man and a large number of birds, move about chiefly during the daytime.  There is an increase in visual acuity owing to the greater number  of cones relative to the number of rods in the retina.  Animals which feed  on insects or seeds, for example, lizards, primates and birds, must of necessity be diurnal.  They must be able to discriminate between that which  is suitable and that which is unsuitable as food.  Predators and prey ani-  mals also need the acute vision afforded by a cone rich retina. animals usually have a circular pupil.  Diurnal  In dim light, the pupil dilates  widely to allow as much light as possible to enter the eye to compensate for the paucity of the rods in the retina. Nocturnal animals, like owls, bats, crocodiles and nocturnal foxes, are more active at night. they may  During the day they either hide from the light or  bask in the sun with eyes closed.  They are totally passive during  the day and have no need of the use of their eyes.  Nocturnal animals norm-  ally have a much more sensitive retina where the number of rods predominate over the number of cones.  Being so sensitive to light, there must be an  efficient means of pupillary control to regulate the amount of light striking the retina especially during the hours of daylight.  Glare must be  eliminated, or at least diminished as much as possible.  The s l i t pupil  is associated with nocturnal habits.  The s l i t pupil, as compared with the  circular pupil, is capable of a greater degree of closure owing to the arrangement of the sphincter muscle fibers within the substance of the i r i s . At times a s l i t pupil can be closed completely, or a pin-hole or two remain.  may  Complete closure of the pupil is not possible with circular pupils.  As the pupil constricts, the tissue around the pupillary edge, namely, the sphincter and the overlying stroma, becomes bunched together.  This bunching  together of the tissue is the limiting factor in preventing a maximum degree of closure.  S l i t pupils do not encounter this mechanical problem.  Slit  pupils which can close completely in bright daylight, can also on the other hand, dilate widely in dim light conditions.  Thus i t is the most efficient  type of pupil and is best suited to nocturnal animals.  Although the generalisation is made that circular pupils are associated with diurnality, and s l i t pupils with nocturnality, this is not always so.  In nature, there can be no hard and fast rules.  always occur.  Exceptions  Among the mammals, the Monotremes are nocturnal and yet they  possess round pupils. Arrhythmic animals, for example, the larger terrestial animals like the ungulates and the large carnivores like the wolves, lions and cougars, have a twenty-four hour habit.  They can be active both during the day or  during the night, although there is usually a preference as to the maximum period of activity.  Here the retina is not highly specialised for acuity  as in s t r i c t l y diurnal animals, or for sensitivity as in nocturnal animals. However, the retina is sensitive enough for its purposes. pupil which can be precisely controlled is very important.  A highly mobile It may be c i r -  cular or i t may be some form of a s l i t pupil. Crepuscular animals, for example, badgers and foxes, show l i t t l e or no adaptation of the eye to cope with extremes of illumination of night and day.  They carry out most of their activities only during the hours of dawn  and dusk. In discussing the habits of the animal world, we have only dealt with how the visual apparatus has adapted to serve the individual effectively in its quest for food and also in performing other of its life's activities.  But a l l animals do not depend entirely, or at least to a great  extent, on vision alone, even though some would be incapacitated i f vision were removed.  The other senses, notably hearing and smell, work in harmony  with vision. The form of the pupil, circular or s l i t , with its countless variations, is the result of the arrangement of the sphincter and dilator muscles within the i r i s tissue (Figure 1 - composite of Walls, 1942, p. 218; and  Prince, 1956, p. 186).  In an iris with a round pupil, the sphincter is  arranged concentrically around the pupillary margin. contracts,  When the sphincter  the pupil size is reduced without a change in pupil shape. As  afore mentioned, because of the bunching together of the tissue around the pupillary edge, an i r i s with a round pupil is incapable of complete closure. The dilator muscles are arranged symetrically in a radial fashion. these fibers contract, the pupil dilates uniformly.  When  The degree of dilation  attainable w i l l , of course, depend on the amount of dilator muscle fibers present.  The round pupil is seen in man as well as in the rat. The simple s l i t pupil is characteristic of many nocturnal animals.  The s l i t may be disposed vertically, horizontally or even at an angle.  Some  of the sphincter muscle fibers do surround the pupillary aperture. The majority form two bundles which criss-cross above and below and are anchored at the periphery of the i r i s .  When these muscle fibers contract, they exert  a "scissor-action" on the pupil.  The pupil is constricted into a s l i t .  There is no bunching of the tissue as in circular pupils, and thus there is no mechanical hindrance for complete pupillary closure.  The dilator fibers  are symetrically radially arranged so that in dilation the pupil assumes a circular form. conditions.  The s l i t pupil is capable of extreme dilation in nocturnal  In strictly nocturnal animals, like the lemur, tarsier and  chinchilla, this ability of the pupil to dilate maximally is coupled with a large cornea.  In dim light, the iris is pushed out of sight of the observer  and a large round pupil comprises the whole visible eye. Other pupil forms are present.  In ungulates, the pupil constricts  to a horizontal rectangle but dilates to a circle because of the radial positioning of the dilator muscle fibers.  The sphincter fibers are also  radial but are anchored laterally in i r i s  connective tissue which is free  of dilator muscle.  In contracting,  they draw the pupil into a rectangular  form.  In some fish, neither the dilator nor the sphincter encircles the  pupil completely so that unusual pupil shapes occur in dilation or constrict ion. There is a multiplicity of pupillary patterns.  It is not feasible  nor entirely useful to deal with them here in too much detail, except to mention that pupils, being round or a s l i t , may be diamond-, dumbell-, or tear-drop-shaped.  Also, the pupillary shape may change with the extent of  dilation or constriction. happens.  The king penguin is one instance where this  In the constricted state the pupil is a perfect square whereas in  the dilated state the pupil is a large circle.  In dilating from its square  form, the i r i s assumes a series of polygonal shapes until the. circle is attained. functions.  Prince (1956) deals extensively with pupillary patterns and their In Walls (1942), Rochon-Duvigneaud (1943) as well as in Prince,  Diesem, Eglitis and Ruskell pupillary shapes.  (1960), there are relevent sections dealing with  Taxonomically, the form of the pupil is not used as an  element in classification simply because i t is not a very constant characteristic in a particular group of animals.  Pupil patterns may be very  similar in different families and yet differ in very closely related families.  The over-riding rule is that the pupil w i l l take on a form which  w i l l be most advantageous to the individual in its environment, disregarding its taxonomic or evolutionary position. The pupil changes in size in response to changes in the intensity of the external illumination. accommodation for near vision.  In man, the pupil also constricts during Pupils also respond to emotional states -  fear, anger, or excitement - as well as to respiratory conditions.  The  horse's pupils dilate when i t is being exercised and the oxygen content of the blood is reduced. anger or excitement.  The pupils of a cat dilate in fear but constrict in A l l these pupillary excursions occur automatically.  However, i t appears that certain amphibians, reptiles and birds can vol tarily control the size of their pupils (Walls, 1942).  The intracacies  the mechanisms of how a l l this takes place are not entirely known.  Figure 1 Pupillary Patterns (Walls, 1942, p. 218 and Prince, 1956, p. 186)  13.  E.  Comparative Anatomy of the Vertebrate Iris It is not only for intellectual but also for practical reasons that  the human eye has been extensively and intensively studied since the earliest days of scientific endeavors.  An adequate understanding of the normal  morphology and physiology of the human eye is very useful when encountering the eye in diseased states.  Besides, the human eye can serve as an example  of a typical vertebrate eye.  The main area of interest here is the his-  tology of the iris (Coque, 1927; Walls, 1942; Rochon-Duvigneaud, 1943; Duke-Elder and Wybar, 1961; Hogan, Alvarado and Weddell, 1971).  The micro-  scopic anatomy of the human i r i s w i l l serve as the basis on which we w i l l compare the other vertebrate irises since i t is devoid of any outstandingly unusual features. The i r i s is the forward and centrally directed non-sensory portion of the retina and of the uvea.  The i r i s is attached by its root to the  c i l i a r y body, which intervenes between the sensory retina and the mobile iris.  In the i r i s , the uvea and the neuroectoderm are very intimately  associated.  The posterior surface of the i r i s is covered by a double layer  of epithelium, the posterior pigment epithelium which is the forward extension of the sensory retina, and the anterior epithelium, which is the extension of the retinal pigment epithelium.  The posterior pigment epi-  thelium is the innermost posterior layer nearest to the lens.  It is so  heavily pigmented that cellular details are not visible except in depigmented specimens.  In very light-colored eyes, the pigmentation of the posterior  epithelium makes up almost the totality of the pigmentation present in the iris.  At the pupillary edge the posterior pigment epithelium may bulge and  be apparent anteriorly.  This results in a thin, deeply pigmented rim or  ruff at the pupillary margin which becomes more prominent when the pupil is constricted.  It may almost be non-visible when the pupil is widely dilated.  At the periphery of the i r i s , the posterior pigment epithelium loses its pigments as i t becomes continuous with the non-pigmented c i l i a r y epithelium. A thin basement membrane, or posterior limiting membrane as i t is also called, covers a l l of the posterior surface of the pigment epithelium. Closely adherent to the anterior surface of the posterior pigment epithelium is the anterior epithelium.  It is the direct continuation of the  retinal and c i l i a r y pigment epithelium.  However, unlike these, the anterior  epithelium contains few, i f any, pigment granules. thelium.  It is a modified epi-  It has retained its epithelial character in its apical portion.  Cell organelles and pigment granules, whenever present, are found within this portion of the anterior epithelium.  The basal portion has differen-  tiated into smooth muscle tissue, the dilator, and is f i l l e d with myofilaments.  Some myofilaments may also be found in the apical epithelial portion  of the epithelium.  The anterior epithelium is thus, by definition, a myo-  epithelial layer.  The dilator processes are oriented radially in a number  of layers, depending on the degree of dilation or constriction of the pupil.  By its contractile action, the dilator muscle causes the pupil to  open widely, as in the response to dim light conditions, pain or excitement. A thin f i b r i l l a r  layer, the basement membrane of the anterior epithelium,  separates the dilator processes from the i r i s stroma.  This basement mem-  brane layer is also variously called the membrane of Bruch or the membrane of Henle (Duke-Elder and Wybar, 1961).  However, in some anatomy texts  (Coque, 1927), the term the membrane of Henle embraces the dilator processes as well. The antagonist of the dilator muscle is the sphincter muscle, a band of smooth muscle cells in the stroma oriented concentrically around the pupillary margin.  As has been mentioned previously, the disposition of the  sphincter muscle fibers and of the dilator muscle fibers within the i r i s  tissue determines the shape of the pupil in dilation or constriction.  Un-  like the dilator muscle fibers, which are the contractile portions of myoepithelial cells, the sphincter muscle cells are fully developed smooth muscle fibers. the pupil.  Medially the sphincter does not extend to the very edge of  Laterally i t merges with the fibers of the dilator.  In amongst  the sphincter muscle mass are collagen bundles, nerves and blood vessels. A basement membrane is also found in the sphincter. Embryologically, both the sphincter and dilator in man are derived from neuroectoderm, as is usual in most vertebrates.  The sphincter muscle  cells have become detached from the lining epithelium of the i r i s and have developed into typical smooth muscle cells located in the stroma.  However,  the dilator has only gone half-way in the process of differentiation.  For  the longest time, there was much controversy as to whether the sphincter was not actually a mesodermal rather than a neuroectodermal derivative, when i t is considered in terms of its innervation and its position in the stroma in the adult i r i s (Duke->Elder and Wybar, 1961).  However, recent  transmission electron microscopic studies of the developing human eye (Ruprecht and Wulle, 1973) have shown that myofilaments are very early detected in the basal parts of the anterior layer of neuroepithelial cells at the rim of the optic cup. ter,  These cells later formed a bundle, the sphinc-  within the i r i s stroma. The mesodermal portion of the i r i s , the stroma, consists of a loose  connective tissue, variably pigmented, highly vascular and well innervated by both sympathetic and parasympathetic nerves.  The supporting  connective  tissue framework consists of a delicate, loosely arranged network of collage fibers.  It is debatable whether elastic fibers are present (Duke-Elder and  Wybar, 1961) or are absent (Hogan, Alvarado and Weddell, 1971).  Fibro-  blasts, melanocytes, clump cells and mast cells comprise the cellular  components of the stroma.  The varying shades and patterns of coloration  seen in the human i r i s is attributed to the number and distribution of the melanocytes, f i l l e d with melanin pigments, within the stroma. of different calibers are present.  Blood vessels  Fine plexuses of mainly unmyelinated  nerves are found associated with the blood vessels and the musculature of the iris. The above brief discussion of the histology of the human i r i s w i l l serve adequately as a means of comparison in the following survey of the i r i s in the other vertebrate groups.  We w i l l be able to note the similari-  ties and differences between these other irises and that of the "typical vertebrate i r i s " .  Walls (1942) and Prince (1956) deal with certain compara-  tive aspects of the vertebrate i r i s .  Prince, Diesem, Eglitis and Ruskell  (1960) are primarily concerned with domestic animals.  However, Rochon-  Duvigneaud" s "Les yeux et la vision des vertebres" (1943), represents a gold mine of comparative vertebrate ocular histology, including the histology of the i r i s .  The following discussion is based on information obtained  from the above sources. At the other end of the evolutionary scale from man are the Cyclostomes, namely the hag fishes and the lampreys. immobile pupils. lature.  This is understandably  These have relatively  so since there is no i r i s muscu-  The scheme of pigmentation harks back to that of the retina.  The  anterior epithelium is, like the retinal pigment epithelium, deeply pigmented and cellular details are not discernible without prior depigmentation. The posterior epithelium, in reverse to that in man, cept in the peripupillary zone. apparent.  is not pigmented ex-  A marginal sinus may be more or less  The thin stroma which contains a blood vessel layer and l i t t l e  or no pigment, is not as adherent to the posterior epithelia as in the other vertebrates.  There are also some radial collagenous fibers in the  stroma.  An annular ligament, consisting of a mass of large, irregularly  polyhedral cells, is seen at the irido-corneal angle. Although i t is not really an integral part of the i r i s , i t is nevertheless very closely associated with i t morphologically and may at times affect the movements of the iris.  The i r i s has a slight metallic lustre owing to the presence of  guanine crystals in some of the stromal cells. pale, yellow powder.  Guanine, a purine, is a  However, when i t is deposited in cells either as  simple guanine or as its calcium salt, i t imparts to the tissue a silvery . or golden metallic lustre. The i r i s of the Chondrosteans (sturgeons), Holosteans (fresh water dogfish) and Dipnoans (Protopterus) is very much like that of the Cyclostomes.  There are no i r i s muscles.  The anterior epithelium is squamous  and pigmented whereas the posterior epithelium shows no pigmentation. stroma on the whole is thin.  The  There is an argentea, a guanine-laden layer  of cells, towards the anterior surface of the i r i s . argentea varies in the different sectors of the eye.  In sturgeons, the The annular ligament,  containing glycogen deposits, is present in the Holosteans. Elasmobranchs in general, have irises very similar to those of the Cyclostomes.  There are a few sphincter and dilator muscle fibers but these  may be non-functional as the pupils are observed to be quite immobile. Their epithelial origin was ascribed to as early as 1897 by Vialleton.  The  anterior epithelium is wholly pigmented although the posterior epithelium is only pigmented at the pupillary region.  The stroma consists of a con-  nective tissue network, small non-pigmented cells, large melanocytes,  nerves  and blood vessels. Cells laden with guanine crystals may be seen scattered in the i r i s stroma, or as an argentea.  Annular ligaments are absent.  Some  Elasmobranchs, for example, the skates and rays, have pupillary opercula to protect the eyes from the effects of direct sunlight.  They are amuscular  prolongations of the superior pupillary border shaped to look like the palm of a hand with the digits spread apart. The Teleosts, or bony fishes, show no or restrained movements of their irregularly circular or oval pupils. of the i r i s histology.  This may be due to two features  Firstly, although there are dilator fibers (so  named because of their radial direction), as well as a sphincter, which may indeed be quite large, these appear to be non-contractile.  Secondly, there  is almost always a well-developed annular ligament at the i r i s angle.  It  consists of an accumulation of swollen, vesiculated cells, which may contain glycogen granules, pigment cells, blood vessels and an arcade of fibrils.  It is a relatively firm structure holding the i r i s at a fixed  angle to the cornea. the i r i s .  It would mechanically hinder any great excursion of  There is a thick argentea just below the mesothelium lining the  anterior surface of the i r i s . a l l fish eyes.  The argentea is what gives the glint seen in  The stroma is pigmented and highly vascularised.  The an-  terior epithelium and the peripupillary zone of the posterior epithelium are heavily pigmented. In dilation, the amphibian pupils are circular but in constriction, they assume numerous forms, vertical and horizontal s l i t s , triangles, etc. The color schemes of the i r i s follow quite closely the pigmentation patterns of the skin, namely, black and gold.  The i r i s is most often large and thin.  Both the anterior and posterior epithelium contain pigment granules, unlike the situation seen in the fishes.  The dilator, derived from the anterior  epithelium, appears as a thin line with delicate f i b r i l s which are neatly, radially oriented.  The sphincter is a small compact mass which rests in  close relationship to the anterior epithelium.  These cells, besides being  f i l l e d with myofilaments, are also f i l l e d with pigment granules.  The stroma  is very thin so that blood vessels often bulge from the anterior surface of  the  iris.  In places the stroma may be absent so that the i r i s is reduced to  two epithelial layers.  There is no argentea in the stroma but melanocytes  and other cells containing yellow pigments or guanine crystals are present. One distinguishing characteristic of the amphibian i r i s is the pupillary nodules, which are variously placed along.the pupillary margin.  These  nodules are, in essence, a mass of epithelial cells resulting from the proliferation of the posterior epithelium of the pupillary border.  It w i l l  later be seen that ruminants have a similar structure known as the corpora nigra.  It is speculated that the function of the pupillary nodules is to  l i f t the i r i s off the lens so as not to impede the flow of aqueous humor. A pigmented and vascular pectinate ligament is present in the i r i s angle. In the reptilian i r i s both the anterior and posterior epithelial layers are pigmented.  There is some controversy over the question of the  musculature of the i r i s .  It is generally agreed that a sphincter muscle, of  neuroectodermal origin, is present in a l l reptilian irises. presence of a dilator is s t i l l in debate.  However, the  The sphincter consists of  striated muscle fibers, unlike that in many vertebrates which consists of smooth muscle fibers.  This perhaps suggest  that there might be some  voluntary control of the sphincter in reptiles.  The sphincter muscle  fibers, oriented mostly circularly but a few are oriented obliquely, are seen throughout the whole extent of the i r i s but are concentrated at the pupillary margin, except in the Sphenodon where i t is concentrated at the periphery of the i r i s .  In the tortoise eye, the i r i s sphincter muscle is  especially powerful and aids in accommodation by squeezing on the lens. The presence of a dilator in tortoises, crocodiles and lizards is questionable. In the Sphenodon, i t has been suggested that there are some muscle fibers derived from the sphincter which possess the function of a dilator. It could conceivably be that the dilator is present in a rudimentary form,  that is, i t is comprised of cells containing some myofilaments which are not  readily visible with the light microscope.  studies would help to elucidate this question. pigmented.  Electron microscopic The stroma is vascular and  Blood vessels, nerves, lipophores (in crocodiles), melanocytes  and cells containing red or yellow pigments or guanine crystals, are also present in the stroma.  There is a loose connective tissue network, the  pectinate ligament, at the irido-corneal angle. Snakes show certain characteristics which are different from those in the other reptilian irises, especially in relation to the musculature. The dilator consists of a thin layer of striated muscle fibers pressed flat against the anterior surface of the anterior epithelium.  There is also a  network of fine striated muscle fibers seen throughout the whole i r i s .  They  increase in number in two regions of the i r i s to form two compact bundles. One bundle is found at the pupillary border and is morphologically correctly placed to act as a sphincter. of the i r i s .  The other muscle bundle is found at the root  These muscle fibers run obliquely as well as circularly.  This is used in accommodation to press on the lens to push i t towards the cornea.  Unlike the condition in the other vertebrates, both the sphincter  and dilator are surmised to be of a mesodermal origin. There is a multiplicity of colors in the bird iris due to the pigments and crystals "in the i r i s stroma. a l l y thinner than the colored irises. the  The dark or black irises are generIn most birds, the i r i s is thin at  pupillary margin and at the root, where i t is attached to the cornea by  a pectinate ligament. The middle of the i r i s is thick. and posterior epithelia are pigmented.  Both the anterior  The anterior epithelium is myoepi-  thelial in nature, consisting of an apical pigmented portion and a basal muscular portion.  The striated muscle fibers are radially arranged and  thus act as a dilator.  In most cases, the sphincter muscle is well-  developed, as is exemplified by the cormorant.  The circularly and obliquely  disposed striated muscle fibers act as a strong sphincter for light reflexes, as well as for accommodation in certain instances.  Blood vessels, nerves,  collagen fibers and melanocyte processes are found in amongst the muscle fibers.  In some birds, there are no muscle fibers and the i r i s is then re-  duced to two layers of epithelia and a thin stroma.  The stroma is often-  times so thin that blood vessels protrude from the anterior surface of the iris. Of the mammals, the Monotremes and Marsupials have simple irises consisting of the anterior and posterior epithelia (either one or both of which may be pigmented), a large sphincter of smooth muscle fibers, no d i lator, and a simple pigmented and vascularised stroma. The i r i s of the Placentals is very similar to that in man with the exception of a few differences which are noted here.  The most distinctive feature is the modi-  fication that occurs along the pupillary edge. possess an umbraculum or flocculi.  The camel and giraffe  These are folds or pleats of the  superior and inferior borders of the i r i s which f i t into each other when the pupils are constricted.  The dolphin and porpoises have an operculum. It  is generally held that the umbraculum is amuscular whereas the opercula of the dolphins and porpoises are essentially enlargements of the sphincter. Pigmented epithelial cysts, formed from the proliferations of the posterior epithelium are known as corpora nigra.  They belong almost exclusively to  ruminants with horizontally oval pupils. the horse, cattle, sheep and goats.  They are seen in the irises of  A l l these modifications of the pupil-  lary margins may serve as eye shades to shield the retina from glare when the pupils are open. When the pupils are constricted, they may ensure a more complete closure of the pupillary aperture in situations where the sphincter is not powerful enough or, when, because of the nature of the  pupillary shape, complete pupillary closure is not inherently possible.  F.  Areas of Research on the Iris Various aspects of the structure of the i r i s have been of interest  to researchers over the years and continue to be of interest at the present time.  The basic histology of the i r i s as being made up of two layers of  epithelium, some contractile elements, namely the dilator and the sphincter muscles, and a mesodermally-derived stroma consisting of blood vessels, nerves and stromal cells, has been known for a very long time.  But interest  was and is focussed on various particular aspects of the i r i s structure, for example, the epithelium, the pigments, the muscles, and the blood vessels.  This is based on the premise that a greater and more detailed know-  ledge of the i r i s structure i t s e l f would help elucidate the role or roles the i r i s plays in the overall functioning of the eye. been carried out using the light microscope.  Such studies have  In addition, recent advances  in the field of electron microscopy have resulted in an instrument with a greatly increased magnification factor and resolving power compared with the light microscope.  Studies using the electron microscope have given us an  insight into the fine ultrastructural details of the cytology of the i r i s , not only in man but also in the other vertebrates. vation of the i r i s is a viable field of research.  The study of the innerHisto-chemical methods  at the light and electron microscopical levels have attempted to work out the intricate details of i r i s innervation. reted with regard to pharmacological  These observations are interp-  studies of the i r i s .  Other investiga-  tors are interested in the dynamics of i r i s movements and have brought a mathematical approach towards the understanding  of a biological problem.  This, in brief, is what is happening in the field of i r i s research, at the present time.  Each aspect of the above mentioned w i l l be treated in greater  detail in the following discussion.  1. General Considerations of the Iris The human i r i s is an easily observable pigmented structure. In certain diseased conditions of the eye, for example, uveitis, gross changes occur in the i r i s .  Norn (1971a, 1971b) in studying the human i r i s observed  that certain pigment defects are associated with uveitis (Norn, 1371b) as well as in normal eyes (Norn, 1971a).  In normal eyes, defects of the pig-  ment layer are only found in people who are over 45 years of age.  These  defects, seen as small depigmented holes, are found near the pupillary ruff infero-nasally, or they may actually be at the ruff i t s e l f .  If such de-  fects are seen in the irises of young people they are considered to be pathological and indicative of a diseased state.  In older people these de-  fects would have to be large to be pathological.  It is postulated that  normal physiological movements of the i r i s over the years expose the pigment epithelium to much wear and tear and result in the destruction of the epithelial cells.  Regeneration does not or cannot keep up so that depigmented  areas are grossly seen.  Uveitis accelerates the depigmentation process in  young people and aggravates that in old people. Using a gonioscope, an observer is able to look at different parts of the i r i s , especially towards the root and at the chamber angle.  Processes  of i r i s tissue (Lichter, 1969) are seen attaching to the angle wall.  The  number of such i r i s processes may vary from one individual to the next, being more prominent in dark irises than in pale irises, and also within an individual eye. There may be areas with an abundant number of i r i s processes, and yet other areas in the same eye are devoid of i r i s processes.  The i r i s  processes can insert at any level on the angle wall although in any one eye they a l l tend to insert at about the same level.  Presumably,  the i r i s  processes are mesodermal remnants from cleavage of the chamber angle during development.  These processes, when present in large numbers, may be of  diagnostic importance when considering glaucomatous conditions of the eye. As with a l l human organs, and the i r i s is no exception, individual variations are common. However, in our discussions of the human i r i s , the average most generally occurring structure of the i r i s is taken as the norm. Hervouet (1962) divides the human i r i s as belonging to one of three categories depending on the amount of stromal tissue that is present.  The normal  eye would have an average amount of stroma, melanocytes and numerous easily visualised crypts. Then there are the other two extremes.  On the one hand,  there are irises where the stroma is very dense, heavily pigmented and where there are very few crypts. On the other hand, the i r i s may possess very l i t t l e stroma, as to be almost non-existent, and crypts which are both large and numerous. Before considering the fine ultrastructural details of the various components of the i r i s , i t would be of interest to f i r s t consider the i r i s as a whole in its milieu.  The i r i s is completely bathed by aqueous humor.  In the adult eye the aqueous humor is produced by the c i l i a r y processes and released into the posterior chamber of the eye.  The aqueous humor than  circulates forward centrally through the pupil into the anterior chamber.' It leaves the eye via the trabecular meshwork and the canal of Schlemm. The aqueous humor is continuously produced at a constant rate. too, ure.  Its outflow,  is well regulated so as not to unduly increase the intra-ocular pressOf course, in disease, this balance is disturbed.  It is reasonable  to conjecture that the i r i s i t s e l f is in some way intimately involved with this constant flow of aqueous humor, especially since there are communications between the i r i s stroma and the surrounding aqueous humor by means of the iridic crypts and i r i d i c pores (Vrabec, 1952; Gregersen, 1958a, 1958b,  25.  1959a, 1959b, 1961; Coulombre, 1961; Klika and Kloucek, 1962; Purtscher, 1962; Newsome and Loewenfeld, 1971).  Such crypts and pores are not only found in  human beings but are also commonly seen in the irises of many vertebrates (Rohen and Voth, 1960; Rohen, 1961).  At one time i t was thought that there  was a continuous endothelial layer covering the anterior surface of the i r i s so that the i r i s tissue was, in essence, structurally cut off from the surrounding aqueous humor. However, Vrabee, (1952) showed that in fetal human eyes and in human eyes at birth, the anterior surface of the i r i s is indeed covered by a continuous endothelium. occur and crypts and pores are formed. c e l l layer.  With development,  discontinuities  Fibroblasts replace the endothelial  Through these crypts and pores, aqueous humor can easily c i r -  culate into the stroma, and conversely, materials from the i r i s may be added to the aqueous humor.  In other words, the i r i s may be involved in  aqueous humor dynamics (Gregersen, 1958a). Most of the studies on the relationship between the i r i s stroma and the  aqueous humor were done by Gregersen (1958a, 1958b, 1961).  S l i t lamp  observations of the i r i s shows that the i r i s very often looks like a sponge saturated with fluid.  This aspect of the appearance of the i r i s may be lost  in histological preparations.  Gregersen perfused the anterior chambers of  human and rabbit eyes with the polysaccharide Dextran (Gregersen, 1958a), killed cocci (Gregersen, 1958b), or a suspension of red blood cells (Gregersen, 1958b). the  Dextran molecules are about the same order of magnitude as  proteins found in the aqueous humor. The Dextran is very rapidly  imbibed by the iris tissue of both human and rabbit irises.  The Dextran  passes through the anterior border layer via the i r i d i c crypts and pores and is widely distributed in the stroma, including the spaces around the blood vessels.  The pigment epithelium does not participate in this imbi-  bition of the iridic tissue by Dextran, but instead acts as a barrier to the  further spread of the Dextran. being \ - Ifi in size. the  Cocci are larger than Dextran molecules,  In human eyes, the cocci are found in the region of  crypts, and often, throughout the stroma as well.  Where the stroma is  dense, no cocci are present. Rabbit irises, unlike human irises, lack crypt Cocci are only found on the anterior i r i d i a l surface but not within the stroma.  This demonstrates clearly that i r i d i c crypts are necessary for the  penetration of large molecules or particles into the i r i s stroma, whereas, i r i d i c pores are sufficiently large enough to let protein molecules through. Red blood cells, 7-8u in size, rarely enter the i r i s tissue. the  Even then,  red blood cells are located toward the anterior border layer of the  iris.  Thus, the i r i s crypts and i r i s pores must act as openings into a  system of inter-communicating, probably widely branched tissue channels in the  i r i s stroma.  These tissue channels are f i l l e d with aqueous humor.  During pupillary movements, there is, conceivably, a free passage of fluid between the tissue spaces of the i r i s stroma and the anterior chamber. Whether the aqueous humor is modified in some way while in its passage through the i r i s tissue is not known. the  Such fluid movements in and out of  i r i s stroma would necessarily considerably reduce the tension changes  that would otherwise occur in the i r i s stroma during pupillary dilation and constriction. It seems that this system of tissue channels is organised in some sort of fashion.  Francois, Neetens and Collette (1960) perfused human eyes  for 10. minutes with Thorotrast and then examined the i r i s by microradiography.  They found a seemingly canalicular network which is completely  different from the vascular network of the i r i s . is localised in the stromal layer.  This canalicular network  It consists of two relatively distinct  parts; in the peripupillary region, the canaliculi are seen as parallel radial marks, whereas in the peripheral region they are seen as circular  marks.  In the middle of the i r i s , the canaliculi are radial with some  interconnecting circular canaliculi.  This canalicular network revealed by  microradiography most probably represents the system of tissue channels in the  iris stroma capable of imbibing aqueous humor from the anterior chamber.  2.  Posterior Pigment Epithelium  The fine structure of the pigment epithelium covering the posterior surface of the i r i s has been studied in various vertebrates, for example, man (Tousimis and Fine, 1959a; Mizuno, 1961; Tomita, Matsuo and Kato, 1961; Tousimis and Fine, 1961; Tousimis, 1963; Kaczurowski, 1965; Hogan, Alvarado and Weddell, 1971), monkey (Tousimis and Fine, 1959a, 1961; Tousimis, 1963), rabbit (Tousimis, 1963; Richardson, 1964; Hvidberg-Hansen, 1971a), ox and gecko (Tucker, 1971), newt (Dumont and Yamada, 1972), Elasmobranch (Kuchnow and Martin, 1970), and skates (Kuchnow and Martin, 1972).  However, the  electron microscopic picture of the pigment epithelial cells from these diverse vertebrates, is amazingly similar.  Embryologically, the posterior  pigment epithelium of the i r i s is the forward and central extension of the retina and c i l i a r y epithelium.  Unlike the anterior epithelium which is  transformed into myoepithelial and muscle elements, the posterior epithelium has maintained its epithelial nature even in adult irises.  In pigmented  eyes, this layer is heavily pigmented, which is not so in albinos.  The  overall shape of the posterior epithelial cells is variously described as cuboidal (Tousimis and Fine, 1961; Dumont and Yamada, 1972), columnar (Kuchnow and Martin, 1972), cylindrical (Tousimis and Fine, 1961), rectangular,  truncated and pyramidal (Hogan, Alvarado and Weddell, 197;).  Naturally  the  shape of the posterior epithelial cells would vary somewhat according to  the  location on the iris i t s e l f .  In the human iris there are structural  folds and furrows and the shape of the epithelial cells varies to f i t their  positions (Hogan, Alvarado and Weddell, 1971). Also during the excursion of the i r i s in miosis and mydriasis changes in the shape of the epithelial cells would occur to accommodate the changes in the total surface area of the  i r i s (Alphen, 1963; Kaczurowski, 1965).  Thus, the described shape of  the epithelial cell would very much depend on the degree of pupillary dilation or constriction of the i r i s when the tissue was removed and placed in the  fixative.  Kaczurowski (1965) has tried to reconstruct a three dimen-  sional picture of the i r i s pigment epithelial c e l l based on numerous observations.  He visualises an epithelial cell with a varyingly polygonal base  whose walls are neither parallel nor flat as a result of the numerous and continuous movements of the i r i s . Posteriorly, the i r i s epithelium is bounded by a typical basement membrane which is continuous with that covering the c i l i a r y body. o  The  basement membrane may vary in thickness from 120-140A (Hvidberg-Hansen, 1971a) to 250$ (Kuchnow and Martin, 1970). Rarely, a series of fine, electron dense particles may be seen at the interface between the basement membrane and the aqueous humor, the significance of which is not known (Richardson, 1964). The basal plasma membrane of the epithelial cells shows many infoldings which may extend quite deeply into the cell cytoplasm. foldings are irregular in shape, size and orientation.  These in  The basement mem-  brane, however, does not follow the course of the infoldings but only covers the external surface of the epithelium.  Most often there is a clear  looking space between the filamentous basement membrane and the epithelial plasma membrane.  The basement membrane does not appear to be tightly or  closely adherent to the plasma membrane.  The lateral walls of the epithe-  l i a l cells show deep, complicated and irregular outlines of the interdigita o tions between adjacent cells. The intercellular spaces may be 200A (Hogan,  Alvarado and Weddell, 1971), or as much as Lu in width (Tousimis and Fine, 1961).  This discrepancy may be due to the mode of fixation as well as, per-  haps, the degree of miosis or mydriasis of the tissue under observation. There may be some maculae adherentes or occludentes here and there along the lateral wall.  The apical cell wall, that is, the surface in apposition with  the anterior epithelium, may be relatively smooth and in close contact with the anterior epithelium (Richardson, 1964), or undulating (Hogan, Alvarado and Weddell, 1971). At intervals between the anterior and posterior epithelial layers, there would be relatively large spaces with microvilli projecting into them from both the epithelial layers (Tomita, Matsuo and Kato, 1961; Richardson, 1964; Hogan, Alvarado and Weddell, 1971).  In skates  (Kuchnow and Martin, 1972) there is a heterogenous intercellular matrix between the anterior and posterior pigment epithelial layers. The most conspicuous components of the pigment epithelium in pigmented eyes are the large number of pigment granules.  Tousimis (1963) com-  pared the pigment granules in the pigment epithelium and stromal melanocytes in man, rabbit, cat, dog and monkey. He found that the pigment granules in the epithelium of a l l these five species are very similar, being round or oval in shape and larger than those in the stromal melanocytes.  Shearer  (1969), with the scanning electron microscope, has given us a three dimensional view of isolated pigment particles in man, rabbit, sheep, ox, pig, rat, pigeon, crab, fish and lizard.  There is some diversity in the shape  and size of the pigment particles but there is also an underlying basic similarity between them a l l . The nuclei are round and smooth and may at times be indented, or they may be elongated (Dumont and Yamada, 1972),  There is generally a paucity  of intracellular cytoplasmic organelles. The mitochondria are few in number. Smooth and rough endoplasmic reticulum is present in limited amounts.  An  occasional Golgi apparatus may be discerned.  Glycogen is also present in  normal human i r i s pigment epithelium (Berkow and Fine, 1970).  Most often,  i t is extracted during the usual preparative procedures for electron microscopy.  The glycogen particles are widely distributed, although they might  have a tendency to accumulate near to dense melanin granules. In man  i t is unfortunate that the i r i s pigment epithelium is unable  to contribute towards regeneration of the lens, but in the newt this is observed following lentectomy (Dumont and Yamada, 1972).  Iris epithelial  cells from the mid-dorsal margin of the i r i s are able to f i r s t dedifferentiate and then redifferentiate to form lens cells.  Changes in cell morphology  and physiology occur during this process. (Yamada,and Roesel, 1969).  3.  Anterior Epithelium and Dilator Muscle  The anterior epithelial layer of the i r i s is myoepithelial in nature in various mammals that are commonly studied in the laboratory, for example, man  (Tousimis and Fine, 1961; Hogan, Alvarado and Weddell, 1971), monkey  (Tousimis and Fine, 1961), cat (Geltzer, 1969), rat (Nilsson, 1964; Roth and Richardson, 1969; Kelly and Arnold, 1972) and rabbit (Richardson,  1964).  However, in Elasmobranchs the question is under dispute (Kuchnow and Martin, 1970).  In the adult Elasmobranch i r i s the anterior epithelium and the  dilator form two distinctly separate layers.  Other investigators do con-  sider this layer as being myoepithelial. The anterior epithelial layer consists of an apical epithelial and a basal contractile portion.  It is, in common usage, usual to refer to the  epithelial portion as the anterior epithelium and the contractile portion as the dilator muscle, although they are both really part of one cell layer. This will be the terminology that w i l l be used in this discussion. The anterior epithelial cells are generally not as high as the cells  in the posterior epithelium.  The nucleus is flattened or elongated in a  radial direction and situated towards the apical or posterior poles of the cell.  Sometimes the nucleus may be irregularly indented  1970).  (Nishida and Sears,  Numerous pigment granules similar to those in the posterior epithe-  lium are present.  The usual cell organelles are seen.  Rough endoplasmic  reticulum in stacks and smooth endoplasmic reticulum in the form of tubules or vesicles are present.  In man  (Hogan, Alvarado and Weddell, 1971), the  Golgi apparatus is not well developed, whereas in the hen (Nishida and Sears, 1970), the Golgi apparatus can be quite sizeable.  Free ribosome clusters  are observed over most of the cytoplasm, with a greater concentration in the perinuclear region.  Mitochondria are large (Tousimis and Fine, 1961), rod-  shaped (Nishida and Sears, 1970)  but are generally not plentiful.  There is  a moderate amount of interdigitation of the lateral c e l l membranes. At the apices of the anterior epithelial cells there are desmosomes and tight junctions which hold the anterior epithelium to the posterior epithelium.  As  mentioned before, there are large intercellular gaps between the two epithelial layers into which microvilli and occasional c i l i a from both epithel i a l layers project.  Myofilaments may also be found in the anterior epi-  thelium. The dilator muscle consists of a series of muscular processes from the anterior portions of the anterior epithelium projecting into the stroma. These dilator muscle processes are radially arranged in numerous stacks. The dilator muscle has a highly complicated  outline so that i t is extremely  d i f f i c u l t to trace out a l l the dilator muscle processes that stem from any one individual c e l l .  The dilator muscle processes are separated o  other by an intercellular gap of about 200A (Richardson, Alvarado and Weddell, 1971).  from each  1964; Hogan,  However, the dilator muscle processes also  adhere to each other by junctional specialisations. It appears that only  zonulae occludentes and zonulae adherentes are present. rarely found (Richardson, 1964; Nishida and Sears, 1970).  Desmosomes are The dilator  muscle processes are f i l l e d with contractile myofilaments oriented radially in the i r i s .  The myofilaments are of two types - the thin myofilaments are  o about 30A in diameter (Hogan, Alvarado and Weddell, 1971) while the thick o filaments are about 180A in diameter (Kelly and Arnold, 1972).  Dense bodies  are found amongst the myofilaments as well as along the plasma membranes. At times, the myofilaments appear to insert into these dense bodies. The sarcoplasm is quite electron-dense.  In the human dilator muscle processes  there are a few pinocytotic vesicles (Hogan, Alvarado and Weddell, 1971) but Geltzer (1969) in his observations of the cat dilator found many pinocytotic vesicles at the borders of the cells. a l l y within a matrix of myofilaments  Long mitochondria are oriented radi(Tousimis and Fine, 1961).  Elasmobranch, the presence of pinocytotic  In the .  vesicles is used as a means of  identifying the dilator muscle processes which are widely dispersed in the i r i s stroma (Kuchnow and Martin, 1970).  A typical basement membrane covers  a l l of the surfaces of the dilator processes but stops short at the epithelial portion of the anterior epithelium. As with the basement membrane covering the posterior surface of the i r i s , there is a clear space of about o 500A (Nishida and Sears, 1970) which separates the basement membrane from the plasma membrane of the dilator muscle processes. Embryologically, the dilator muscles are derived from neuroectodermal cells which have remained in their original position but have acquired a myoepithelial structure (Duke-Elder and Wybar, 1961; Lowenstein and Loewenfeld, 1969; Mann, 1964; Hogan, Alvarado and Weddell, 1971; Imaizumi and Kuwabara, 1971; Lai, 1972b).  33.  4.  Sphincter Muscle  The sphincter muscle, as its name suggests, acts as the constrictor of an orifice, namely, the pupil.  It consists of an annular band of typical  smooth muscle fibers, concentric with the pupillary margin, in man (Tousimis and Fine, 1961; Lowenstein and Loewenfeld, 1969; Hogan, Alvarado and Weddell, 1971), monkey (Tousimis and Fine, 1961), rat (Hokfelt and Nilsson, 1965; Kelly and Arnold, 1972), guinea pig (Nishida and Sears, 1969b), newt (Tonosaki and Kelly, 1971), Elasmobranch (Kuchnow and Martin, 1970), and skate (Kuchnow and Martin, 1972).  However, in birds and reptiles, for ex-  ample, the alligator (Reger, 1966), the sphincter muscle consists of an admixture of both smooth and striated muscle cells.  It w i l l be considered  separately from the more usually occurring smooth muscle sphincter of most vertebrates. The sphincter is located more or less in the i r i s stroma at the pupillary margin.  These muscle cells show a remarkable degree of contrac-  t i l i t y as compared to the other smooth muscle cells.  The sphincter muscle  cells are elongated and spindle-shaped and are oriented concentrically around the pupil.  The muscle cells are intimately and firmly attached to  each other and to the surrounding stroma, so that i f a segment of the i r i s is removed surgically due to disease, the rest of the sphincter can function unimpaired (Lowenstein and Loewenfeld, 1969; Hogan, Alvarado and Weddell, 1971).  A basement membrane separates the individual muscle cells from the  surrounding connective tissue stroma.  This basement membrane follows the  contours of the cells except in regions where two -cells are in very close apposition, as at a nexus or tight junction.  Hogan, Alvarado and Weddell  (1971), in their observations of the human i r i d i a l sphincter with the electron microscope, note that sphincter muscle cells associate together, by means of tight junctions, in groups of five to eight cells.  Each muscle  cell may have more than one region of close apposition with another c e l l . It is postulated that possibly such morphological groupings represent functional units, where the cells can contract in synchrony in response to a stimulus.  The elongated nucleus is located centrally within the spindle-  shaped c e l l .  These muscle.cells also contain a Golgi apparatus, an endo-  plasmic reticulum, which may be quite extensive in some cases (Kuchnow and Martin, 1972), some free polyribosomes and elongated mitochondria which are oriented along the long axes of the cells.  There are also some melanin pig-  ment granules, or, as in the West Coast newt, a cluster of special R granules.  Large irregular vesicles are distributed throughout the cytoplasm.  Besides, there are many pinocytotic vesicles along the c e l l membrane. Some vesicles open into the extracellular space. along the c e l l membrane.  Sometimes, there are densities  Being a contractile tissue, the most abundant  components within the c e l l cytoplasm are the myofilaments.  It was once  thought that only one type of myofilaments is present (Reger, 1966; Hogan, Alvarado and Weddell, 1971).  In the Elasmobranch sphincter, Kuchnow and o  Martin (1970) found myofilaments with transverse diameters of 40-100A, depending on the species.  These would comprise the thin filaments.  However  Kelly and Arnold (1972) found that i f the rat i r i s tissue is well fixed by perfusion, both thick and thin filaments are consistently observed. The o o thin filaments are 70-80A while the thick filaments are 220-230A in diameter The sphincter of the Alligator mississippiensis (Reger, 1966) contains not only smooth but also striated muscle c e l l s .  In addition, there  are some myoblast-like cells which probably represent a stage in the differentiation of striated muscle.  Smooth and striated muscle cells are often in  contact, as are myoblast-like cells with striated c e l l s .  The smooth muscle  c e l l components of the sphincter possess the characteristics of typical smooth muscle cells, as described above.  The striated muscle cells show  features of a slow-acting muscle, that is, a poorly organised reticulum and T system.  sarcoplasmic  Thick and thin filaments and Z lines are present.  1  Myoblast-1ike cells are in many respects similar to the striated muscle cells. Any discussion of the sphincter must necessarily include some comments on its embryological. derivation.  In the adult i r i s , the sphincter is  structurally a smooth muscle but embryologically derm (Mann, 1964)  i t develops from neuroecto-  rather than from mesoderm. This was the generally accepted  view although some were not convinced, their arguments being based on the position of the sphincter in the adult (in the stroma), and the innervation of the sphincter muscle cells (parasympathetic, which is similar to the innervation of the mesodermally-derived c i l i a r y muscles). tions on the development of the sphincter muscle in man  Recent investiga-  (Ruprecht and Wulle,  1973), rabbit (Tamura and Smelser, 1973), rat (Lai, 1972a; Imaizumi and Kuwabara, 1971) and newt (Tonosaki and Kelly, 1969,  1970,  1971) with the  electron microscope have shown definitively that the mature smooth muscle cells of the sphincter do arise from the neuroepithelial cells of the anterior layer of the optic cup near to its rim.  Except for minor differ-  ences, in terms of the sequence and timing of the events that take place during the differentiation of the epithelial layer into muscle tissue, this process essentially involves the formation of myofilaments within the cells. The shape and orientation of the cells also alter to best f i t its function in adult l i f e .  The sphincter muscle as a whole may also shift to occupy a  stromal position. The degree of movement depends on the species.  5.  Stroma  Apart from the posterior and anterior epithelial layers and the two muscles, the rest of the bulk of the i r i s comprises the stroma.  The width  of the stroma from the anterior i r i d i a l surface to the dilator varies from individual to individual (Hervouet, 1962) as well as with the degree of pupillary dilation or constriction (Alphen, 1963).  There are also species  differences in terms of the density of the stroma (Rohen, 1961).  The number  and distribution of the cellular and intercellular components of the i r i s stroma is characteristic for each species, as is amply shown by Rohen (1961). The cells of the i r i s stroma are the non-pigmented fibroblasts and mast cells (Hogan, Alvarado and Weddell, 1971) and the pigmented melanocytes and clump cells (Iwamoto, 1961, 1962; Tousimis, 1963; Kaczurowski, 1965; Hogan, Alvarado and Weddell, 1971). stromal cells (Vrabec, 1971). ing through the stroma. the  In addition, there are some ciliated  Blood vessels and nerves are also seen cours-  The blood vascular supply and the innervation of  i r i s w i l l be considered separately.  A l l the cells are surrounded by a  mucopolysaccharide ground substance and fluid milieu.  The intercellular  spaces are also f i l l e d with a connective tissue framework of filaments and collagen f i b r i l s  (Ringvold, 1970b).  The structure of the connective tissue  and the arrangement of the cells within this connective tissue framework varies with the species (Rohen, 1961). At the anterior surface of many primate irises, the stroma is modified to form the anterior border layer.  This is a dense accumulation of  cells and connective tissue (Rohen, 1961; Hogan, Alvarado and Weddell, 1971; Purtscher, 1972).  The thickness of this layer varies for different segments  of the same eye (Hogan, Alvarado and Weddell, 1971),. This variation is also observed between eyes from different species of mammals (Rohen, 1961).  The  anterior border layer, according to current thinking, does not perform any important functions but does influence eye color to a certain extent.  The  principal types of cells found in the anterior border layer are fibroblasts and melanocytes. These are oriented parallel to the anterior surface of the  i r i s and may form a relatively continuous layer.  An endothelium covers the  anterior i r i d i a l surface incompletely in the adult (Vrabec, 1952).  Local-  ised defects of the anterior border layer and the anterior endothelium give rise to the i r i d i a l crypts (Vrabec, 1952; Gregersen, 1959a; Rohen and Voth, 1960).  Gregersen (1959a) has made a detailed study on the structural varia-  tions seen in the crypts of the human i r i s .  Spanning the openings of some,  but not a l l , of the crypts are the so-called "bridge trabeculae". non-cellular f i b r i l l a r  These are  (presumably collagenous) tissue components.  They may  be remnants of the fetal pupillary membrane. Fibroblasts are seen throughout the stroma but they very often congregate around blood vessels and nerves. elles. are  The melanocytes (Iwamoto, 1962; Tousimis, 1963; Kaczurowski,  1965)  also distributed throughout the stroma and especially towards the anter-  ior surface in man.  The melanocytes are small and elongated and possess a  few branching processes. the  They possess the usual cell organ-  It appears that the melanocytes situated towards  anterior i r i d i a l surface and at the crypts may possess heavily pigmented  "globes" at the ends of the cytoplasmic processes (Kaczurowski, 1965).  This  feature is lacking in melanocytes found in the other more posterior parts of the  stroma.  The melanocytes are nucleated and possess mitochondria, ribo-  somes and both smooth and rough endoplasmic reticulum.  Melanin granules in  different stages of differentiation are observed within the melanocytes. Unlike the pigment granules of the epithelial cell layers, there are distinct differences between the pigment granules of the stromal cells of different mammalian species (Tousimis, 1963). are  Generally, the stromal pigment granules  smaller than those in the epithelium and are more rod-like in shape.  They may be specifically or randomly oriented within the cell cytoplasm. Clump cells (Iwamoto, 1962; Tousimis, 1963; Kaczurowski, 1965; Hogan, Alvarado and Weddell, 1971) are large, pigmented stromal cells varying in  size from 3 to 12u. They are mostly found in the pupillary zone in the vicinity of the sphincter muscle.  They are spherical with numerous micro-  v i l l i on the cell surface but no cytoplasmic processes, as is characteristic of the normal melanocytes.  The c e l l cytoplasm is engorged with two types of  pigment granules (Tousimis,. 1963).  Some granules are small and are similar  to those of the other stromal melanocytes, while others are larger and are similar to those in the pigment epithelium. They may be displaced neuroectodermal cells or macrophages (Hogan, Alvarado and Weddell, 1971). In the anterior border of the human i r i s , there are sometimes nevuslike structures (Hogan, Alvarado and Weddell, 1971).  Dieterich and Franz  (1972) observed that the nevus c e l l morphologically resembles the melanocyte.  The cytoplasm is f i l l e d with pigment granules.  Mitochondria, ribo-  somes, filaments and a fairly well-developed Golgi complex are present. There may be pinocytotic vesicles at the c e l l membrane. Mast cells containing specific granules are sometimes found in the i r i s stroma. In electron microscopic studies, mention is sometimes made of ciliated cells in the i r i s stroma (Hogan, Alvarado and Weddell, 1971). The c i l i a may be seen on fibroblasts.  But i t is impossible to study the extent  of the occurrence of ciliated cells in electron microscopic studies because of the inherent limitations of the method, that is, the small size of the tissue sample under observation and the thinness of the tissue section. Vrabec (1971), using flat, frozen sections of rabbit and human irises, observed that almost a l l of the superficial cells from the anterior surface of the i r i s possess typical long c i l i a which might end in a spherule at the tip. C i l i a are rarely found on cells deeper in the stroma, but when present, they are short. The c i l i a are capable of beating synchronously. are probably embryonic remnants.  These c i l i a  It is interesting to speculate that since  the c i l i a are quite commonly found, they might be involved in the movement of aqueous humor across the surface of the i r i s . The ground substance of the stroma is a hyaluronidase-sensitive acid mucopolysaccharide.  Within this ground substance are different types of  f i b r i l s which comprise the connective tissue skeleton of the stroma (Tousimis and Fine, 1959; Ringvold, 1970b; Hogan, Alvarado and Weddell, 1971). Collagen f i b r i l s predominate in the stroma.  The collagen f i b r i l s form a  network which, in some mammals, is regularly arranged in interweaving arcs (Rohen, 1961).  In electron micrographs, they appear to be distributed at  random except around the blood vessels. The collagen f i b r i l s are between o o 200-700A in width and show an axial periodicity of about 600-640A. In addition, Ringvold (1970b) found two other types of f i b r i l s stroma.  Some appear in bundles which taper and s p l i t .  in the i r i s  These are unevenly  o wide along the length and also show cross-banding with a periodicity of 650A. o The third type of f i b r i l is about 130A in diameter, do not branch, associate in groups and also show cross-banding. may represent native collagen f i b r i l s 6.  These latter two types of f i b r i l s (Ringvold 1970b).  Iris Blood Vessels  The blood supply to the i r i s has been studied using various methods by different investigators.  The i r i s of albino rats or mice, two animals  commonly used in the laboratory, is thin and membranous, non-pigmented and highly vascularised.  The albino rat and mouse i r i s is then very suitable  for v i t a l microscopic examination of the i r i s vascular pattern and its blood flow behavior (Bensley, 1960; Castenholz, 1965, 1966, 1971).  Bensely (1960)  developed a method of trans illuminating the i r i s using a quartz rod. Castenholz (1965, 1966, 1971) developed another method for studying the living i r i s using the principle of the ophthalmoscope.  By means of a  series of prisms and mirrors, vertically directed light is reflected off the fundus of the eye so that the outlines of the blood vessels are quite distinctly set off against an illuminated background.  With this method the  eye is observed without being handled so that, possibly, comparative physiological studies can be made. Dyes and contrast media (Castenholz, 1965; Saari, 1971b, 1972), fluorescein (Craandijk and Aan d Kerk, 1970; Harris, Toyofuku and Shimmyo, 1972), and Neoprene latex (Wong and Macri, 1964; Saari, 1971b) are sometimes injected into the orbital blood vessels which supply the i r i s .  The passage  of the dyes or fluorescein through the i r i s is visualised by reflected light (Castenholz, 1965) or by fluorescein angiography (Craandijk and Aan de Kerk, 1970; Harris, Toyofuku and Shimmyo, 1972).  The velocity of the blood flow  in the different vessels of the i r i s can then be studied under different conditions (Castenholz, 1965; Craandijk and Aan de Kerk, 1970; Castenholz, 1971; Harris, Toyofuku and Shimmyo, 1972).  In the case of Neoprene injec-  tions of the eye (Wong and Macri, 1964), casts are made and studies are made on the casts. Saari (1970, 1971a, 1971b, 1972) has developed over the years a flat preparation method for studying the blood vessels of the pig i r i s .  The non-  vascular components of the i r i s are f i r s t digested away with trypsin so as to thin out the section of the i r i s .  The pigment granules are bleached in  solutions of potassium permanganate and oxalic acid.  The preparations are  floated onto a slide and stained with PAS-hematoxylin. pale purple in contrast to the dark purple myelinated  Blood vessels stain nerves.  From the above studies, there has emerged a pattern of the blood supply to the i r i s in man (Craandijk and Aan de Kerk, 1970), pig (Saari, 1970, 1971a, 1971b, 1972), cat (Wong and Macri, 1964), rabbit (Harris, Toyofuku and Shimmyo, 1972), and the albino rat (Castenholz, 1965, 1966,  1971).  The long posterior c i l i a r y artery and its branches enter the iris to  form the major arterial circle situated in the stroma near the iris root. Branches from the major circle course radially through the iris stroma towards the pupillary margin.  Capillaries branch off from these radial iris  vessels and anastomose to form a fine network in the iris stroma.  In man  (Hogan, Alvarado and Weddell, 1971), some of the larger branches of the radial iris vessels anastomose at the region of the collarette to form the minor circle.  From thence, capillaries course to the sphincter and pupillary  margin, where capillary arcades are seen.  The paths of the venous channels  for  the return of blood to the general circulation follow closely those of  the  arterial vessels.  There are some slight species differences from the  basic vascular pattern as seen in man.  In the albino mouse (Bensley, 1960),  there is a large arterial anastomosis midway in the i r i s . from here radiate limbus.  Branches arising  centrally towards the pupil and peripherally towards the  In the albino rat (Castenholz, 1965, 1966, 1971) and in the rabbit  (Harris, Toyofuku and Shimmyo, 1972), there is no minor arterial circle. Among the larger blood vessels, arterio-venous shunts are common. The minor circle is not purely arterial but is formed by arterio-venous anastomoses (Calmette, Lazorthes, Deodati, Bee and Bechac, 1959).  In disease, local  vascular changes may occur. Highly tortuous vessels are associated with pigmented i r i s tumors (Craandijk and Aan de Kerk, 1970). The rate of flow of blood through the i r i s vessels can be measured by observing the passage of a dye or fluorescein through the vessels. Castenholz (1965) found that 2.3 to 2.8 seconds after the injection of lissamine green into the femoral vein of the rat, dye is seen in the i r i s root vessels. iris.  It takes 5 to 11 seconds for the dye to be drained out of the  Harris, Toyofuku and Shimmyo (1972) injected 57. sodium fluorescein  into an ear vein of the albino rabbit and observed fluorescence in the  42.  major arterial circle 4 to 5 seconds later. In the rabbit, iris blood flow is responsive to stimulation of the cervical sympathetic nerve fibers (Cole and Rumble, 1970). Vasoconstriction occurs.  The alterations in the blood flow is measured in terms of the  changes in the amount of heat that is dissipated into the surrounding aqueous humor. This is done by implanting thermocouples in the anterior chamber just in front of the i r i s .  Thus the iris blood vessels are demon-  strated to be adrenergically innervated. Early light microscopic studies showed that arterioles, venules and capillaries make up most of the blood vessels of the human i r i s stroma, with capillaries being predominant.  In a l l of these vessels, i t is observed  that there are tubular tissue spaces surrounding the endothelial channels (Gregersen, 1959b; Lassman, 1964).  External to this clear space are cir-  cularly oriented collagen f i b r i l s .  Thus, the iris vessel is visualised as  two tubes separated by a space.  Such a unique configuration of the i r i s  blood vessels may be useful for maintaining a constant and unimpaired  cir-  culation of blood in the i r i s despite the extreme movements of the i r i s tissue in miosis and mydriasis.  Imbibition studies show that molecules  like Dextran, when introduced into the anterior chamber, find their way into this clear space around the endothelium (Gregersen, 1959b). These tissue spaces are not present in baby eyes but they slowly increase in width during development into adulthood.  In adults, variations do occur  between individuals with respect to the size and number of the tubular spaces around the i r i s blood vessels. Electron microscopic studies (Ikui, Mimatsu, Maeda and Tomita, 1960; Tomita, 1960; Purtscher, 1966; Ringvold, 1969; Tamura, 1969; Ringvold, 1970a; Vegge and Ringvold, 1969) have elucidated the nature of this clear perivascular space seen in light micrographs.  It is really a light collag-  enous zone (Hogan, Alvarado and Weddell, 1971). Sparse collagen f i b r i l s , o about 300A in diameter, embedded in a fine granular ground substance, are oriented longitudinally, parallel to the length of the vessels. o  External to  this is a thick layer of collagen f i b r i l s measuring 1000A in diameter. These f i b r i l s may be circularly (Hogan, Alvarado and Weddell, 1971) or obliquely and longitudinally (Ringvold, 1969) oriented around the vessel lumen. collagenous layer is visible with the light microscope.  This  In eyes with  exfoliation syndrome, changes occur in the light collagenous zone immediately adjacent to the endothelium (Ringvold, 1969; 1970a).  In normal eyes, this  zone appears clear in toluidine blue stained plastic sections (Ringvold, 1907a).  However, in eyes with exfoliation syndrome, a homogenous light blue  material is seen.  Electron microscopic examination of this blue zone shows  exfoliation material in the vessel wall (Ringvold, 1969). an abnormal amount and type of extracellular material. to oval granules make up the non-fibrillar elements.  It consists of  Membrane-bound round Besides the normal  f i b r i l s , f i b r i l s of different thicknesses with occasional cross-banding are found in groups. A single layer of endothelial cells surrounds the lumen of the i r i s blood vessels (Ringvold, 1969; Hogan, Alvarado and Weddell, 1971; Smith, 1971; Vegge, 1971, 1972).  The.endothelium is non-fenestrated in man  (Ringvold, 1969; Hogan, Alvarado and Weddell, 1971), monkey (Vegge, 1971b, 1972), rabbit (Harris, Toyofuku and Shimmyo, 1972) and mouse (Smith, 1971). There are conflicting reports on the i r i s capillaries of the rat. It is non-fenestrated according to some investigators (Saari, 1972) but Castenholz (1971) reports that there are fenestrations in the endothelium.  In the  cytoplasm are the usual organelles, such as, the nucleus, mitochondria, rough and smooth endoplasmic reticulum, ribosomes and microtubules.  In  addition, characteristic tubular bodies are found in the endothelial cyto-  plasm (Matsuda and Sugira, 1970),  They are rod-shaped, possess an internal  structure and are delimited by a unit membrane. 30 tubules.  Each rod consists of 10 to  They are postulated to be produced by the Golgi complex. The  functional significance of these bodies is obscure. present between adjacent endothelial cells.  Tight junctions are  A relatively thick basement  membrane is always present. Pericytes or typical smooth muscle cells may be present outside the endothelial layer. In fetal eyes, there is another vascular system associated with that of the i r i s , that is, the vessels of the pupillary membrane (Matsuo and Smelser, 1971).  However, these become non-functional and finally atrophy.  When functioning, the blood vessels consist of a layer of endothelial cells enclosing a lumen, a basement membrane external to i t and pericytes.  7.  Innervation of the Iris  Both myelinated and unmyelinated nerves are seen in the iris stroma. The nerves enter the iris at its root.  These nerves arise from the trigem-  inal, the superior cervical sympathetic and c i l i a r y ganglia (Schaeppi, 1966). There is some indication that there might be ganglion-like cells within the i r i s tissue i t s e l f .  Thus not a l l the ganglia which innervate the eye  are found extra-ocularly (Macri, 1971).  The fine unmyelinated branches of  the trigeminal are sensory and are diffusely distributed throughout the i r i s stroma.  They may also be vasomotor to the i r i s blood vessels.  The sympa-  thetic and parasympathetic nerves are motor nerves and they not only innervate the sphincter and dilator muscles but also the blood vessels (Cole and Rumble, 1970). The classical concept of the innervation of the i r i d i a l musculature is that the sphincter muscle is supplied by parasympathetic cholinergic  nerve fibers, while the dilator muscle is supplied by sympathetic adrenergic nerve fibers.  However, this concept of a simple and separate innervation of  the iris musculature now has to be modified.  There is much evidence to show  that cholinergic parasympathetic fibers also supply the dilator muscle cells and that adrenergic sympathetic fibers innervate the sphincter muscle as well.  These conclusions on the dual innervation of the iris sphincter and  dilator are based on studies employing a variety of techniques, anatomical, physiological and pharmacological.  For light microscopic observations, the  Falck-Hillarp fluorescence technique is used to demonstrate the cellular localisation of noradrenalin. Acetylcholinesterase is localised by Koelle's or Karnovsky's methods. Methylene blue is not a highly specific stain but is nevertheless used for demonstrating cholinergic nerve fibers.  For elec-  tron microscopic studies, i t is found that with potassium permanganate fixation, adrenergic sympathetic nerve endings can be differentiated from cholinergic parasympathetic nerve endings at the ultrastructural level. A l l these cytological studies are often coupled with selective denervations either of the sympathetic or parasympathetic.  From these anatomical studies,  a fairly good idea of the intricacies of the innervation of the i r i s is obtained.  Concomitant physiological and pharmacological studies help to  complete the picture. The adrenergic innervation of the iris has been studied extensively in the rat (Ehinger, 1964; C s i l l i k and Koelle, 1965; Ehinger and Falck, 1965; Eranko and Raisanen, 1965; Malmfors, 1965a, 1965b; Malmfors and Sachs, 1965a, 1965b; Ehinger, 1966b; Ehinger and Falck, 1966; Ehinger, Sporrong and Stenevi, 1967, 1968; Staflova, 1969b; Farnebo and Lidbrink, 1971).  The rat  i r i s is thin and has a dense network of adrenergic fibers associated with the dilator.  Because of the thinness of the albino rat i r i s , i t can be  stretched out flat and mounted whole on a microscope slide and the non-  terminal and terminal axons to be studied can be examined in their entirety. The adrenergic innervation of the i r i s of numerous other animals, including man, has also been studied (Ehinger, 1964; Malmfors, 1965b; Ehinger, 1966a, 1966b; Laties and Jacobowitz, Ehinger and Sjoberg, 1971).  1966; Ehinger, 1967; Staflova, 1969a, 1969b; The histochemical fluorescence method of Falck  and Hillarp is used to visualise the adrenergic transmitter, noradrenalin, at a cellular level.  The method is highly specific and sensitive and the  chemical bases of the reactions are known. The iris tissue is dried and exposed to an atmosphere of formaldehyde vapor. catecholamines,  Noradrenalin, like other  forms the highly fluorescent compound, 4, 6, 7-trihydroxy-  3, 4,- dihydroisoquinoline, with formaldehyde.  This fluorescence, indicat-  ing the presence of endogenous noradrenalin in the adrenergic nerves, is then examined with a fluorescence microscope. green and varies in intensity.  It appears green to yellow-  The methodology is discussed in detail by  Malmfors (1965a). In the rat i r i s , the adrenergic supply to the dilator is a dense, intermeshing plexus of main, preterminal and terminal axons.  The fibers  are so dense that i t is not possible to follow a l l of the ramifications of the system of terminals belonging to any one individual adrenergic neuron. However, Malmfors and Sachs (1965a) found that following destruction or extirpation of the superior cervical sympathetic ganglion, the adrenergic ground plexus of the i r i s is much reduced. remain and are s t i l l fluorescent.  Only a few postganglionic fibers  The other postganglionic fibers lose their  noradrenalin contents and cannot be seen with the fluorescence microscope. The morphology and pattern of distribution of the branches of a single postganglionic fiber can then be followed from when i t enters the i r i s to its terminals (Malmfors, 1965a; Malmfors and Sachs, 1965a).  The terminals  are easily visualised as they are the storage sites of noradrenalin. The  intensity of the fluorescence in the other non-terminal axons can be increased by treating the experimental animals with noradrenalin prior to sacrifice.  The main axon is smooth and is only weakly fluorescent in un-  treated animals.  At varying distances from its entry at the i r i s root, many  branches are given off the main axon. run to a l l parts of the i r i s .  These are the preterminal axons which  The preterminal axons are similar in appear-  ance and in the degree of fluorescence as the main axons, except at its distal extremities where a few enlargements may be seen. axons branch profusely to give the terminal axons.  The  The preterminal terminal axons  possess characteristic, strongly fluorescent enlargements or varicosities. A terminal axon may or may not end in a varicosity. in the terminal varicosities.  Noradrenalin is stored  If an isolated rat i r i s is stimulated by an  electric field, noradrenalin is released and there is a slight decrease in the fluorescence of the slightly less prominent varicosities (Farnebo and Lidbrink, 1971).  If noradrenalin synthesis is inhibited prior to sacrifice,  the effect is much enhanced.  Terminals from more than one neuron run to-  gether and converge to innervate the same group of dilator cells. Adrenergic terminals are not only found in the dilator but also in the sphincter region and in the walls of the small i r i s blood vessels.  In  the sphincter, the adrenergic terminals run alongside and in between the muscle cells.  Sometimes a single preterminal axon gives rise to one system  of terminal axons which end at the dilator and another set of terminal axons which end at the sphincter or blood vessel.  The adrenergic innervation to  the dilator is excitatory while, in a l l probability, that to the sphincter is inhibitory.  Recently i t has been shown that there appears to be function-  ally different types of sympathetic neurons in one and the same ganglion (Edvinsson, Owman, Rosengren and West, 1972). innervate the different components of the i r i s .  They may thus selectively  Comparative studies on the adrenergic innervation of the iris have been carried out using the Falck-Hillarp fluorescence technique (Ehinger, 1964; Malmfors, 1965a, 1965b; Ehinger, 1966a, 1966b; Laties and Jacobowitz, 1966; Staflova, 1969; Ehinger and Sjoberg, 1971). cal  Such comparative anatomi-  studies of the adrenergic innervation are useful and necessary when  attempts are being made to correlate physiological and studies done on one species to another species.  pharmacological  From denervation studies i t  is seen that almost a l l of the adrenergic innervation to the i r i s is from the ipsilateral superior cervical ganglion (Malmfors, 1965b; Ehinger, 1966b). Most of the adrenergic terminals go to supply the dilator muscle.  In the  rat and mouse, the adrenergic terminals are primarily on the anterior surface of the dilator (Malmfors, 1965a; Ehinger, 1966b). so thin in the rat  and mouse, i t is possible to get an overview of the  total adrenergic innervation to the i r i s . 1966b).  Since the i r i s is  The nerves form arcades (Ehinger,  In the cat, rabbit, dog and pig, the nerves are not only very  dense to the anterior dilator surface (as in the mouse and rat), but branches are seen in between the muscle cells themselves (Malmfors, 1965b; Ehinger, 1966b).  The latter configuration is similarly seen in man and the  Cynomologus monkey (Ehinger, 1966a).  In the rat and guinea pig, several  varicose nerve terminals run together in the same strand, whereas in the mouse the terminals are found singly (Malmfors, 1965b; Ehinger, 1966b). The sphincter muscle always receives an adrenergic supply but to differing degrees according to the species. in the rat sphincter.  There are few adrenergic fibers  The pig and dog possess more than do the rats.  however, have a very rich adrenergic innervation to the sphincter.  Cats,  This  shows up as a very intense fluorescence with the Falck-Hillarp technique (Ehinger, 1964).  In the monkey, cat and rabbit, the fibers to the sphincter  are always parallel to the long axis of the sphincter cells (Laties and  Jacobowitz, 1966)_  In pigmented animals, there are more adrenergic fibers  to the sphincter than in albinos (Malmfors, 1965b). In phytogeny, i t is seen that primates have overall fewer adrenergic fibers than the lower animals (Staflova, 1969). a l l (Ehinger, 1966a). the  Man has the least number of  Differences also exist in the rate of degeneration of  adrenergic network within the i r i s following ganglionectomy.  In the  rabbit i t only takes four to five days, whereas in the monkey i t might be as long as twenty days (Staflova, 1969). In developmental studies on man and on the guinea pig (Ehinger and Sjoberg, 1971) i t is observed that in young eyes the nerves have a smooth appearance.  The varicosities on the nerve terminals develop in time.  The  nerves do not have any inductive effect on the development of the dilator and sphincter muscles as these develop before the nerves appear on the scene. The adrenergic nerves do not enter the eye together with the blood vessels. Innervation to the blood vessels develops later. Noradrenalin is the adrenergic transmitter at the nerve terminals. Its presence is detected by means of the Falck-Hillarp fluorescence technique.  Acetylcholine is the cholinergic transmitter but there are no histo-  chemical methods to directly stain for acetylcholine.  Rather, the enzyme  which breaks down the transmitter, acetylcholinesterase, is stained by the thiocholine method of Koelle (1955).  A combination of the Falck-Hillarp  fluorescence technique for the localisation of noradrenalin and the Koelle technique for the histochemical localisation of acetylcholinesterase has been used to study the adrenergic and cholinergic network of nerves in the cat  (Ehinger, 1967) and rat (Csillik and Koelle, 1965; Eranko and Raisanen,  1965; Ehinger and Falck, 1965, 1966) i r i s .  Pairs of micrographs from  acetylcholinesterase staining and from fluorescence microscopy are superimposed on each other so as to be better able to compare the networks of  nerves demonstrated by the two methods (Eranko and Raisanen, 1965).  It is  found that the pattern of acetylcholinesterase staining is generally similar to the pattern of fluorescence, although there is a much greater density of acetylcholinesterase towards and in the sphincter region (Csillik and Koelle, 1965; Ehinger and Falck, 1966;  Ehinger, 1967).  Both noradrenalin and  acetylcholinesterase are contained within the terminal varicosities (Csillik and Koelle, 1965).  From these studies i t is observed that there are three  types of nerves (Eranko and Raisanen, 1965;  Ehinger and Falck, 1965,  1966).  Many fibers show both fluorescence and histochemical staining for acetylcholinesterase.  Some fibers are fluorescent but show no acetylcholinesterase  activity, while others show acetylcholinesterase activity but are not fluorescent.  The question then arises as to whether some nerve fibers are  both cholinergic and adrenergic at one and the same time, or whether the fibers that show both acetylcholinesterase activity and fluorescence at the light microscope level are in reality closely associated but distinctly separate fibers.  The former view is suggested by the work of Eranko and  Harkonen (1964) using fluorescence microscopy and acetylcholinesterase staining on the superior cervical ganglion cells.  It is shown that there are  some ganglion cells which show both fluorescence and also much acetylcholinesterase activity.  These neurons may  with dual characteristics. (1962, 1965)  then issue forth nerve fibers  This would support the theory of Burn and Rand  that there is a cholinergic component in the adrenergic trans-  mission of a nerve impulse.  It is postulated that the release of noradrena-  l i n is initiated or triggered by the release of acetylcholine.  The latter  view that adrenergic and cholinergic fibers only accompany each other very closely is favored (Ehinger, 1967).  This is based on electron microscopical  studies of the nerve endings themselves, as w i l l be discussed later, and on bioassays  for the acetylcholine content in the i r i s following selective  denervation (Ehinger, Falck, Persson, Rosengren and Sporrong, 1970). Essentially the same pattern of innervation is obtained using a combination of the Falck-Hillarp fluorescence technique and staining with methylene blue (Ehinger, Sporrong and Stenevi, 1967, 1968; Ehinger, 1971; Takkunen, 1971). Methylene blue is an axon stain but its r e l i a b i l i t y and selectivity is sometimes questionable (Richardson, 1969; Takkunen, 1971). Although both adrenergic and cholinergic nerves take up the stain, there is enough difference to distinguish between the two at the light microscopic level. From these light microscopic observations, i t is seen that both the sphincter and dilator muscles are dually innervated.  The sphincter muscle  receives predominantly a cholinergic nerve supply although there is a substantial degree of adrenergic innervation as well.  The reverse is true  of the dilator. The nerve terminals have also been examined with the electron microscope, which has a much greater magnification range and resolving power than the light microscope, to see i f there are any ultrastructural differences between the adrenergic and cholinergic nerve terminals (Richardson, 1964; Nilsson, 1964; Hokfelt, 1966; Richardson, 1966; Hokfelt, 1967; Richardson, 1968, 1969; Ochi, Konishi, Yoshikawa and Sano, 1968; Geltzer, 1969; Matsuda, 1969; Nishida and Sears, 1969a, 1969b; Roth and Richardson, 1969; Ehinger, Falck and Sporrong, 1970; Nishida and Sears, 1970).  The axon terminals may  come into very close contact with the effector cells, namely, the dilator o and sphincter, leaving only a gap of 170-220A (Nishida and Sears, 1969b), or the plasma membranes of the muscle and the axon terminal may be fused (Nilsson, 1964). Nerve terminals appear as enlargements or varicosities containing mitochondria and different types of vesicles.  The cholinergic  and adrenergic nerve terminals can be differentiated from each other by  their populations of vesicles.  Early studies on the rat i r i s revealed that  there were granular and agranular vesicles in the nerve terminals.  But they  were not correlatable as to the nature of the terminals, either sympathetic or parasympathetic  (Nilsson, 1964).  With the technique used, there are no  ultrastructural differences between adrenergic and cholinergic nerve terminals.  But in 1966, Richardson found that fixation of the i r i s in potassium  permanganate, instead of in glutaraldehyde or in osmium, produces consistent results in terms of the vesicular populations of the adrenergic and cholinergic nerve terminals.  There are two types of vesicles.  Adrenergic termi-  o nals contain predominantly 400-500A vesicles with dense cores (granular vesicles).  Cholinergic terminals contain small agranular vesicles.  Nor-  adrenalin is stored in the form of dense granules in the membrane-bound ves icles. With the light microscope,  i t is seen that methylene blue is an axon  stain relatively specific for cholinergic nerves (Ehinger, Sporrong and Stenevi, 1967, 1968; Ehinger, 1971; Takkunen, 1971). With the electron microscope, methylene blue is seen to be not highly specific for cholinergic axons except under well-controlled conditions which are pH dependent (Richardson, 1969).  The methylene blue is toxic to cholinergic terminals  and ultrastructural changes occur (Richardson, 1968, 1969).  The axon may  or may not swell and be deformed. Most often the varicosities are swollen and vacuolated.  Scattered membrane densities are present.  These swollen  varicosities contain a population of agranular vesicles, thus distinguishing  them as cholinergic terminals. The adrenergic and cholinergic nerve terminals run together in  bundles.  They are in close association but they are separate entities.  At  times, there are cellular axo-axonal contacts between two types of terminals (Ochi, Konishi, Yoshikawa and Sano, 1968; Ehinger, Falck and Sporrong, 1970).  Thus, i t is possible for the cholinergic and adrenergic nerves to mutually influence each other. From both light and electron microscopic studies, i t is evident that the sphincter (Richardson,  1964; Hokfelt and Nilsson, 1965; Ochi, Konishi,  Yoshikawa and Sano, 1968; Geltzer, 1969; Hirano, 1969; Nishida and Sears, 1969b) and dilator (Nilsson, 1964; Richardson, 1964;  Geltzer, 1969; Hirano,  1969; Nishida and Sears, 1969a, 1970) are dually innervated.  The extent of  dual innervation varies with the species, with either the adrenergic or cholinergic predominating. (Ehinger, 1971).  One system predominates while the other modulates  Early physiological studies had shown that this dual  innervation is present.  Joseph (1921) produced a relaxation of the sphinc-  ter of cats and dogs by stimulating the cervical sympathetic.  Thus the  sphincter has an excitatory parasympathetic nerve supply from the occulomotor via the c i l i a r y ganglion, as well as an inhibitory sympathetic nerve supply from the cervical ganglion.  Neuropharmacological studies show that  there are two types of adrenergic receptors, °^ and p  .  Activation of  the o( receptors results in muscle contraction while activation of the y3 receptors results in muscle relaxation. Whether i t is the c< or the y0 receptors which are activated depends on the type of stimulation applied (Schaeppi and Koella, 1964a, 1964b).  Both the sphincter and dilator are  dually innervated, by cholinergic and adrenergic  ( oi and y3 ) nerve fibers.  In pupillary constriction or dilation both systems work in harmony rather than as two discrete entities (Apter, 1956).  During pupillary constriction,  cholinergic parasympathetic nerve fibers stimulate the sphincter muscle cells to contract while at the same time inhibiting the action of the dilator muscle cells, thus resulting in the relaxation of the dilator (Ehinger, Falck and Persson, 1968).  During pupillary dilation, the  adrenergic sympathetic nerve fibers cause dilator muscle contraction while  the  terminals cause sphincter muscle relaxation. The dilator and the sphincter muscles are not the only components •  of the i r i s that are innervated.  As mentioned previously, the i r i s blood  vessels are also innervated almost exclusively by the sympathetic system (Cole and Rumble, 1970).  Melanophores of rats (Ehinger and Falck, 1970),  skates (Kuchnow and Martin, 1972), monkey and man (Ehinger, 1966a) also possess a nerve supply.  In the monkey and man some adrenergic fibers come  very close to the melanophores but not to the clump cells.  In rats, melano-  phores appear to have both a cholinergic and an adrenergic nerve supply (Ehinger and Falck, 1970). the  The functional significance of a nerve supply to  pigmented cells of higher animals is not readily perceptible.  In lower  vertebrates which can change their coloration, such a nerve supply would be useful.  8.  Miscellaneous Studies on the Iris  Some enzyme histochemical studies have been carried out on the i r i s , spurred on by the idea that perhaps the i r i s does not merely act as a shutter but may also be involved in other metabolic activities, for example, a contribution towards the production of aqueous humor (Berkow and Patz, 1964; Lessell and Kuwabara, 1964; Hvidberg-Hansen, 1971a).  Numerous oxi-  dative enzymes (Berkow and Patz, 1964; Hvidberg-Hansen, 1971a), alkaline and acid phosphatase (Lessell and Kuwabara, 1964; Hvidberg-Hansen, 1971a) and specific phosphatases (Lessell and Kuwabara, 1964) are localised to the i r i s and are indicative of the metabolic potential of the i r i s .  The histochemi-  cal pattern of the distribution of many of the dehydrogenases are essent i a l l y similar (Berkow and Patz, 1964).  The reaction precipitate is denser  in the posterior epithelium than in the stroma both in the adult and in the developing eyes. This enzymatic activity is detectable at 17 days fetal  and reaches the adult pattern by 10 days post-natal (Berkow and Patz, 1964). In the i r i d i c epithelium as a whole, there are some slight differences in • the distribution of the dehydrogenases in terms of the intensity i f the reaction (Hvidberg-Hansen, 1971a).  In numerous vertebrate species, both  alkaline and acid phosphatases are present in the i r i s epithelium (Lessell and Kuwabara, 1964).  Hvidberg-Hancen (1971a) demonstrated that in the  rabbit i r i s epithelium there are regional differences between the localisation of alkaline and acid phosphatase. Kuwabara (1964).  This is not shown by Lessell and  On the basis of alkaline and acid phosphatase staining,  the rabbit i r i s epithelium can be divided into two zones, a central zone concentric to the pupillary margin and of about the same extent as the sphincter, and a peripheral zone. and acid phosphatases.  The central zone is rich in both alkaline  Ultrastructurally, these enzymes are localised to  the membranes of the numerous pinocytotic vesicles associated with the lateral interdigitations of the i r i d i c epithelium. a lesser amount of both phosphatases. lysosomes.  The peripheral zone has  Acid phosphatase is found in the  The i r i s epithelium also shows intense adenosine triphosphatase  activity (Lessell and Kuwabara, 1964).  Thus the i r i s epithelium appears to  be a highly metabolic epithelial layer. The i r i s is also very active in l i p i d synthesis (Culp, Cunningham, Tucker, Jeter and Deiterman, 1970).  If C ^  sodium acetate is administered  into the anterior chamber of the rabbit eye, i t is taken up by the i r i s tissue and incorporated into fatty acids.  These lipogenic pathways may  be  important as a source of metabolic energy (for the sphincter and dilator muscles, perhaps), or for replenishing membrane constituents that are lost as a result of the wear and tear that might occur during the incessant movements of the i r i s . In lower vertebrates, Wolffian lens regneration occurs, that i s , i f  the lens is extirpated certain epithelial cells from the i r i s are able to be transformed in a series of steps into lens cells. adult newt (Dumont and Yamada, 1972).  This is seen in the'  This regenerative capacity of the  i r i s epithelium is lost as one ascends the evolutionary scale.  However, the  i r i s and the lens may s t i l l be similar in some ways, for example, in its immunological properties.  By using various immunochemical methods, Brahma,  Bours and van Doorenmaalen (1971) show that there are certain proteins (antigens) in the chick i r i s which are immunologically gens extracted from the lens.  similar to the anti-  The chick i r i s epithelium antigens,  present  in small but detectable amounts, have antigenic properties of lens o<.crystallin. Another aspect of i r i s research concerns the dynamics of pupillary movements (Mapstone, 1970; Loewenfeld and Newsome, 1971; Tucker, Davanger, 1972).  1971;  As is well known, changes in pupillary size occur in re-  sponse to changing light conditions, certain emotional and psychological phenomena and to direct or indirect stimulation of the associated ganglia and nerves (Mapstone, 1970; Pierau, Alexandridis, Spaan, Oksche and Klussmann, 1970; Borthne and Davanger, 1971; Davanger, 1971; Loewenfeld and Newsome, 1971).  The resting size of the pupil is dependent on the age of  the individual and on the color of the i r i s . Senile atrophic changes occur in the i r i s stroma. with age.  The sphincter and dilator muscles lose their tonus  Thus with increasing age, there is a concomitant decrease in the  resting pupil size (Borthne and Davanger, 1971).  The average resting pupil  size of dark irises is also smaller than that of light, blue irises.  This  may be related to the density of the stroma. Mydriatics and miotics, that is, drugs used for dilating or constricting the pupils respectively are often used in experimental  conditions  to study the mechanics of i r i s movements. When cyclopentolate or phenyle-  phrine, two mydriatics,  are applied to the eyes, it is seen that the pupils  dilate at a speed which is proportional  to the difference between the  resting and maximum pupillary size (Davanger, 1971).  However, the effects  of the mydriatics are not identical. Pupillary dilation occurs quicker with cyclopentolate than with phenylephrine (Borthne and Davanger, 1971).  Also  cyclopentolate results in greater dilation than does phenylephrine in young people, but the reverse is the case with old people.  Such age  differences  in drug effects is probably related to senile changes that occur in the i r i s tissue. The pupil has a linear range of movement in response to bright light or dim light conditions (Loewenfeld and Newsome, 1971).  When a bright light  is shone into the eye, the pupil constricts very promptly at a certain velocity.  However, at a particular pupil size, which is not the limit of  constriction, the rate of pupillary constriction slows down. The diameter of the pupil at which this happens from individual to individual.  is fixed for each individual but varies  In pupillary dilation, the same phenomenon  is observed, that is, the pupil would dilate linearly until a certain pupillary diameter is attained. velocity and in extent.  Then the dilation process slows down in  There seems to be a mechanical barrier at a partic-  ular pupil diameter which slows down pupillary dilation or constriction. Mathematical attempts are made to analyse the forces which determine pupil size (Mapstone, 1970;  Davanger, 1972).  According to Mapstone (1970),  the forces due to the pull of the sphincter and dilator muscles determine the pupil size.  The sum of these forces is greatest when the pupil is con-  stricted but diminishes when the pupil dilates.  The pull of the  sphincter  at its maximum is twice that of the dilator.  ^Davanger (1972) however,  starts with a different set of assumptions.  The size of the pupil is de-  termined by the tension of the sphincter and dilator muscles.  The result-  ant constricting force of the sphincter is centripetally directed and is equal to S/r, where S is the total force of the sphincter and r is the radius of the pupil.  On the other hand, the resultant dilating force of  the dilator is centrifugally directed and is equal  to 1/2 "fr • D/r, where  D is the total force of the dilator and r is the radius of the pupil. At equilibrium, that is, when the pupil is at rest, the constricting and dilating forces must be equal, so that S/r = D/2 r. The pupil can be at equilibrium at any pupil size, but the force of the dilator w i l l always inherently be 2lT(6.28) times the force of the sphincter.  This is also exemp-  l i f i e d i f there is a small change in the radius of the pupil, A r . The change in the dilator force is D x 4 r , while the change in the constrictor force is S x 2T>Av.  These changes balance each other at equilibrium so that  again D = 2HS. So i f the force of either the dilator or sphincter is changed, the pupil size is adjusted until equilibrium is reached, that is, when the force due to the dilator is 2TT that of the sphincter. According to current thinking on pupillary movements, pupillary constriction is a result of the contraction of the sphincter muscle and relaxation of the dilator muscle, while the opposite is true during pupillary dilation.  The active contraction of one muscle is accompanied by a concomi-  tant relaxation of the other muscle. support this point of view. (Tucker; 1971).  There appears to be much evidence to  However, this view is not shared by a l l  It is felt that the muscles do not act reciprocally.  Rather, when a l l the muscles contract, the i r i s shortens and results in pupillary dilation.  When the muscles relax, fluids from the aqueous humor  rush to occupy the tissue spaces in the i r i s stroma, thus, expanding the i r i s and constricting the pupil.  Here the sphincter and dilator muscles  only act as stabilisers, respectively, of the pupillary margin and the i r i s as a whole.  Such a view on pupillary dilation and constriction, however,  does not take into account the results obtained from many selective denervation, physiological and pharmacological studies.  G.  Thesis Proposal  1.  The histology of the i r i s in man as well as in other vertebrates has  been studied over the years.  However, in a l l the investigations,  except for  one (Alphen, 1963), the histology of the i r i s is described as i f i t were a static rather than a dynamic structure.  No attempts have been made to com-  pare the structure of the i r i s during different degrees of pupillary dilation and constriction.  The i r i s is capable of extensive excursions both in re-  sponse to changing light conditions and in response to miotics and mydriatics. The changes in the overall size of the i r i s control the changes in the size of the pupil.  These changes in pupillary diameter are highly precise and occur  very rapidly.  During extreme pupillary constriction, the pupil appears only  as a mere pin-hole.  On the other hand, during extreme pupillary dilation, the  pupil is enlarged many times over.  The  rim of tissue at the edge of the cornea.  i r i s appears as a thin, barely visible Obviously, this must entail struc-  tural changes within the i r i s tissue to accommodate for the great changes in total surface area from pupillary constriction to pupillary dilation.  Also,  the i r i s architecture must be structurally adapted to facilitate these movements so that they occur as smoothly and as efficiently as possible.  Thus,  the f i r s t major part of the present investigation deals with the alterations in i r i s structure during the extremes of pupillary size which occur in dilation and constriction.  The changes observed are pharmacologically induced.  The rat, a common laboratory experimental animal, is used in this study.  It has a typical mammalian i r i s with a round pupil.  The  basic  histological components of the i r i s is well known. The rat i r i s consists  of a posterior epithelial layer and a modified anterior epithelial layer. In the adult rat, the anterior epithelium has differentiated into two contractile masses, the sphincter muscle and the dilator muscle, which are readily detectable at the light and electron microscopic levels.  Anterior  to both the posterior epithelium and the dilator is the stroma consisting of blood vessels of varying sizes, myelinated and unmyelinated nerves of the sensory, sympathetic and parasympathetic systems, and numerous stromal cells which are a l l enmeshed within a connective tissue framework and a mucopolysaccharide ground substance.  With the aid of drugs, the rat iris is main-  tained in pupillary dilation and constriction.  The rat iris tissue, in the  dilated or constricted state, is examined with (a) the light microscope, (b) the transmission electron microscope, and (c) the scanning electron microscope.  Interest is focussed on:  (a) what are the structural changes that are observed in the i r i s when the pupil is constricted and when the pupil is dilated, (b) what structural features of the various components of the i r i s would facilitate, or at least not hinder, the excursions of the i r i s , and (c) what new information can be gleaned from scanning electron microscopic studies on the posterior and anterior surfaces of the rat i r i s .  (a)  Changes in the shape and orientation of the posterior  epithelium,  the dilator and the stromal elements are examined with the light microscope on toluidine blue stained plastic sections which have been prepared by the conventional methods for transmission electron microscopy.  The relationships  of the stromal elements to the epithelium and dilator are examined. It is known that there is a collagenous connective tissue framework in the iris stroma, although i t is by no means abundant (Tousimis and Fine, 1959b).  The overall organisation of both the cellular and intercellular  (collagen) components in the iris stroma during pupillary dilation and constriction are observed.  Particular emphasis is placed on the relationships  of the collagen network to the stromal cells during these rapid changes in pupillary size.  With ultrathin sections, the overall organisation of the  collagen network is not readily apparent since collagen is not abundantly present.  Thus, parafin sections (7-10u) are specifically stained for colla-  gen using a modified Mallory's Trichome Stain to reveal the general orientation of the collagen during miosis and mydriasis. (b)  The investigation is carried one step further. The rat i r i s ,  either dilated or constricted, is examined with the transmission electron microscope to see what ultrastructural changes, i f any, occur, especially in the posterior epithelium and the dilator. collagen to the surrounding cells is noted.  The intimate relationships of the Kelly and Arnold (1972) have  studied the sphincter muscle of the rat i r i s in the constricted and dilated state with the transmission electron microscope.  However, their main  interest is the demonstration of both thin and thick filaments within the sphincter muscle, which, according to them, is dependent on the means and mode of fixation used. (c)  In recent years, a new tool has been introduced for the study  of biological materials, the scanning electron microscope.  A recent review  article deals with the potential usefulness of the scanning electron microscope for biological research (Hollenberg and Erikson, 1973).  The main  advantage of the scanning electron microscope lies in its great depth of focus so that clear three dimensional pictures of the surface features of cells and tissues can be obtained.  The basic principle for the construction  and operation of the scanning electron microscope has been discussed in numerous investigations (Nixon, 1971; Oatley, Nixon and Pease, 1965).  The  scanning electron microscope also uses an electron gun, as does the trans-  mission electron microscope, which sends out a beam of electrons towards the specimen.  However, the electrons do not pass through an ultrathin section  but strike the surface of the specimen which has been previously coated with a metal.  Secondary electrons are emitted from the specimen surface and are  amplified and displayed on.a cathode ray screen. During pupillary dilation and constriction, changes must inevitably occur on the anterior and posterior surfaces of the i r i s .  To reconstruct  the surface configurations of the i r i s in these two pupillary conditions would entail an  inordinate number of serial sections.  Using the scanning  electron microscope drastically reduces the labor involved, gives us a view of the i r i s surface in its totality and in much greater detail.  New  infor-  mation is obtained on the anterior and posterior surface structures of the iris.  The ultrastructure of the rat i r i s surface has been examined with  the scanning electron microscope (Hansson, 1970). in isopentane-propane and freeze-dried.  The tissues were frozen  However, no attempts were made to  compare the ultrastructural images obtained in pupillary dilation and constriction.  These studies are presented here.  Both the camphene method  (Watters and Buck, 1971) and the c r i t i c a l point drying method (Boyde and Wood, 1969; Smith and Finke, 1972) are used for preparing the i r i s tissues for examination with the scanning electron microscope.  The specimens are  coated with a thin layer of gold.  2.  Structural alterations of the rat i r i s are not only associated with  its function but also with i t s growth and development.  This is the concern  of the second major part of this investigation. Embryological  studies show that the development of any tissue or  organ is a composite of processes, cell division, c e l l differentiation and often cell rearrangements.  The relationships of cells to each other alter  in the process of growth and development. unique in one aspect.  Developmentally, the i r i s is  In the i r i s , as in many tissues, both mesodermal and  ectodermal components come together and are closely inter-related. i r i s , the mesoderm gives rise to the stromal elements.  In the  However, the poster-  ior epithelium, the dilator muscle and the sphincter muscle are neuroectodermal derivatives.  The posterior epithelium retains the epithelial charac-  ter of the neuroectoderm.  The anterior neuroectodermal layer differentiates  in an unusual direction and gives rise to a myoepithelium, the dilator, and to smooth muscle, the sphincter.  Over the years, there has been much  controversy over the embryological origin of the sphincter, as to whether i t is derived from mesoderm, as is the usual case with smooth muscle, or whether i t is indeed derived from neuroectoderm. of  Reports on the development  the rat i r i s observed with the transmission electron microscope have  recently appeared (Imaizumi and Kuwabara, 1971; L a i , 1972a, 1972b; Tamura and Smelser, 1973).  In these papers special emphasis is placed on the  development of the sphincter and dilator muscles. rather than mesodermal origin is affirmed.  Their neuroectodermal  Information, however, is scarce  on the overall development of the i r i s , that is, the changes that take place with respect to the histology of the various components of the iris and their relationships to each other.  Such developmental studies would be  useful when attempting to correlate the development of both the structure and function of the i r i s .  The second part of this study deals with the  following aspects of development of the i r i s in terms of its histology and funct ion: (a) To observe, with the light microscope on toluidine blue stained plastic sections, the structural alterations taking place in the i r i s as i t develops from the rim of the optic cup in fetal stages to the adult form. (b) To observe the changes in the configuration and arrangement of  the posterior epithelial cells of the developing rat i r i s , and the architecture of the peri-natal vascular system and its regression with development, using the scanning electron microscope. (c) To observe, with the light microscope, the changes in the permeability of the iris capillaries to an intravenously injected tracer substance, Horse-radish Peroxidase (HRP), with development.  These tracer  studies would also give some indication as to the role of the i r i s vascular system in the production of aqueous humor. produced in the c i l i a r y body.  In adult eyes, aqueous humor is  The path of the aqueous humor can be followed  by an intravenous injection of a tracer substance.  It is found that in the  Vervet monkey (Vegge, 1971a) and in the mouse (Smith, 1971), the capillaries of the c i l i a r y processes are readily permeable to the tracer HRP.  The  c i l i a r y capillaries are fenestrated (Pappas and Smelser, 1961; Smith, 1971; Vegge, 1971a).  However, the i r i s capillaries are non-fenestrated  present a barrier to the HRP  (Smith, 1971; Vegge, 1971b, 1972).  and The HRP is  only seen within the lumens of the i r i s vessels but not external to the endothelium.  In the rat i r i s , there are no known HRP tracer studies.  There  are also conflicting reports as to the nature of the rat iris capillaries, whether they be fenestrated or non-fenestrated present study, HRP  (Saari, 1972).  In the  is injected intravenously into fetal, young post-natal  and adult rats and the localisation of the reaction precipitate is observed on toluidine blue stained plastic sections.  II.  A.  MATERIALS AND METHODS  A Light Microscopic Study of the Rat Iris in Pupillary Dilation and Constriction Adult Wistar rats were used.  Their pupils were dilated with a ,few  drops of a mixture of 57. phenylephrine hydrochloride and 0.57. cyclopentolate, or constricted with a few drops of 0.1257. echothiophate iodide. The rats were anesthetised with ether and the eyes were removed immediately. A small meridional s l i t was made in the eyes extending from the cornea anteriorly to the ora serrata posteriorly. This was done to facilitate the inflow of fixative into the interior of the eye to ensure rapid fixation. The eyes were fixed in 47. glutaraldehyde  buffered with 0.1M sodium cacody-  late at pH 7.2, or in formalin, held at room temperature.  After 30 minutes  in the respective fixatives, the eyes were cut in half, the lenses were removed and fixation was continued or 5 hours in formalin.  for a total of 4 hours in glutaraldehyde  The tissues were rinsed with and stored in 0.1M  sodium cacodylate buffer at 4°C. The tissues were then processed as follows:  (1) for demonstrating the overall histological characteristics  of the i r i s in pupillary dilation and constriction, and (2) for demonstrating the collagen network in the stroma of the rat i r i s in pupillary dilation and constriction.  1.  For Demonstrating the Overall Histology of the Iris  The anterior halves of the eyes were trimmed into smaller pieces while in the cacodylate buffer wash.  The tissues were post-fixed in 17.  osmium tetroxide in 0.1M cacodylate buffer for 1 hour, stained en bloc in uranyl acetate for 1 hour, dehydrated through a graded series of alcohols, infiltrated and embedded in epon-araldite.  Thick (about lji) epon-araldite  sections were cut on a Porter-Blum MT-2 microtome and stained with toluidine blue.  The sections were examined with and light micrographs were taken on  a Zeiss Photomicroscope II.  2.  For Demonstrating the Collagen Network in the Iris Stroma  The glutaraldehyde- or formal in-fixed tissues were dehydrated through a graded series of alcohols, cleared in chloroform and embedded in parafin.  7-10u sections were stained with a modified Mallory's Trichrome  Stain (Culling, 1963).  The collagen network within the stroma was examined.  B. A Transmission Electron Microscopic Study of the Rat Iris in Pupillary Dilation and Constriction Adult albino rats of the Wistar strain were used.  Their pupils  were dilated or constricted with a few drops of a mixture of 57» phenylephrine hydrochloride and 0.57 cyclopentolate, 0.1257. echothiophate iodide, o  respectively.  Various methods were tried to obtain good preservation of  the tissues for transmission electron microscopy. 1. immediately.  The rats were anesthetised with ether and the eyes were removed A small meridional s l i t was made extending from the cornea  anteriorly to the ora serrata posteriorly and into the vitreous humor. The eyes were immersed in the fixatives within 30 seconds from the moment that they were enucleated.  The following fixatives were used:  (a) 47. glutaraldehyde in 0.1M cacodylate buffer (Sabatini, Bensch and Barnnett, 1963). (b) Full strength Karnovsky's fixative (Karnovsky, 1965). (c) Karnovsky's fixative diluted to 507, with 0.1M cacodylate buffer (Graham and Karnovsky, 1966). (d) Millonig's fixative (Millonig, 1961).  After about 30 minutes in the respective fixatives, the eyes were cut in half, the lenses were removed and fixation was continued for a total of 4 hours at room temperature. 2.  The rats were anesthetised with ether.  A s l i t was made in the  cornea and half strength Karnovsky's fixative, was dripped on to the eye in situ for 5 minutes.  Then the eye was rapidly removed, immersed in the  fixative and treated as in (1). 3.  The rats were anesthetised by an intraperitoneal injection of  sodium pentabarbitol and a subcutaneous.inject ion of sodium phenobarbitone. The left common carotid artery was exposed and cannulated.  The cannula  was connected to a 20 ml syringe containing one of the following fixatives: (a) 47. glutaraldehyde in 0.1M cacodylate buffer. (b) 47. glutaraldehyde  in 0.2M cacodylate buffer.  (c) Full strength Karnovsky's fixative. (d) 1:1 Karnovsky's fixative - diluted with 0.1M cacodylate buffer. The inferior vena cava was also exposed.  The fixative was perfused through  the carotid artery at a pressure of 100mm Hg.  As soon as perfusion started,  the inferior vena cava was cut just below the renal vessels for drainage to take place.  The perfused eyes were then immersed in fixative and treated  as in (1). The i r i s tissues were stored in the respective buffers at 4°C.  The  anterior portions of the eyes, with the attached i r i s , were trimmed into smaller pieces while in the buffer wash.  The tissues were post-fixed for 1  hour in 17. osmium tetroxide in 0.1M cacodylate buffer, stained en bloc in uranyl acetate for 1 hour, dehydrated through a graded series of alcohols, infiltrated and embedded in epon araldite.  Thin sections of well-preserved  tissues, as determined light microscopically on thick sections, were cut on a Porter-Blum MT-2 microtome.  The sections were stained with lead citrate  for 2 to 2\ minutes and examined on a Philips EM 200 operating at 60kV.  C. A Scanning Electron Microscopic  Study of the Rat Iris in Pupillary  Dilation and Constriction METHOD: Adult rats of the Wistar strain were used in these studies.  Their  pupils were dilated with a few drops of a mixture of 57, phenylephrine hydrochloride  and 0.57, cyclopentolate,  0.1257, echothiophate iodide.  or constricted with a few drops of  These drugs were allowed to act for 15 to 20  minutes at which time the pupils were widely dilated or much constricted, as the case may be. The rats were etherised and the eyes were removed immediately and immersed into the fixative of 47, glutaraldehyde in 0.1M cacodylate buffer at pH 7.2 at room temperature.  An antero-posterior  slit  was made through the corneal-scleral junction extending from the anterior and posterior chambers into the vitreous humor to facilitate the penetration of the fixative into the i r i s tissue.  This step was very c r i t i c a l  when dealing with eyes in pupillary constriction. Almost as soon as the eye is detached from the rat, "death dilation" occurs.  This seems to be some-  what arrested i f fixation is almost instantaneous.  After about 30 minutes  in the fixative, the posterior halves of the eyes and the lenses were removed and discarded.  The eyes were fixed for a total of 4 hours, washed o  with and stored in 0.1M cacodylate buffer at pH 7.2 at 4 C. The tissues were prepared for examination on the scanning electron microscope in the following way: 1.  Camphene Method (Watters and Beck, 1971)  From the buffer the tissues were transferred through a graded alcohol series to be dehydrated.  While in absolute alcohol, the tissues  were cut into pieces suitable for examination on the scanning electron  microscope.  In order to view the anterior surface of the i r i s , the cornea  was cut away as close to the corneal-scleral junction as possible.  It was  found that i t was better to remove the cornea while the tissue was in absolute alcohol rather than when i t was in buffer.  The i r i s , being very  delicate, was slightly stiffer at this point and therefore held its shape much better even though the cornea was absent.  From absolute alcohol, the  tissues were transferred into propylene oxide.  They were then infiltrated  with a mixture of equal volumes of propylene oxide and camphene for _ hour, followed by another _ hour in pure camphene.  Both the propylene oxideo  camphene mixture and camphene were kept in a water bath at 45 C to prevent recrystallisation from occurring.  The tissues were removed from the  camphene, mounted and oriented on tissue stubs and left in the vacuum evaporator (Edwards) overnight to evaporate the camphene from the tissues. These were then coated with a thin layer of gold. 2.  Critical Point Drying Method (Boyde and Wood, 1969; Smith and Finite, 1972)  The eyes, stored in 0.1M cacodylate buffer, were either post-fixed in 17. osmium tetroxide in 0.1M cacodylate buffer at pH 7.2 for 1 hour and then dehydrated, or they were dehydrated immediately without previous post-fixation in osmium.  Dehydration was done in a graded alcohol series.  The tissues not treated with osmium were appropriately trimmed down while in absolute alcohol, whereas those tissues to be post-fixed in osmium were trimmed down while in the buffer wash. after osmium tetroxide fixation.  The latter would be too b r i t t l e  Thus any manipulation might be liable to  cause mechanical damage to the tissues.  The absolute alcohol was then  substituted by iso-amyl acetate (Fisher) in a stepwise manner.  The iso-  amyl acetate is a polar solvent miscible with carbon dioxide and i t is  also a protective agent against ice crystal formation during the c r i t i c a l point drying process. Carbon dioxide was used as the transitional fluid in our Critical Point Drying Apparatus (Parr).  When the tissues were dried,  they were mounted on tissue stubs, and coated with gold in a vacuum evaporator. A l l the specimens were examined on a Cambridge Stereoscan (Model S4) microscope.  Scanning electron micrographs were taken on Agfa Isopan ISS  film.  D. A Light Microscopic Study of the Development of the Rat Iris Using Toluidine Blue Stained Epon Sections and Horse-radish Peroxidase (HRP) Studies of the Iris in Fetal, Post-natal and Adult Rats METHOD: Fetal and post-natal rats of different ages, as well as adult rats, of the Wistar strain, were used in these studies.  1.  Fetal Rats  The ages of the fetal rats were determined by the method of Christie (1964).  Pregnant female rats were anesthetised by an intraperitoneal in-  jection of sodium pentobarbitol (0.4-0.7 ml of a solution of 200 mg sodium pentabarbitol dissolved in 6 ml saline) and a subcutaneous injection of sodium phenobarbitone (0.3-0.6 ml of a solution of 240 mg sodium phenobarbitone dissolved in 12 ml saline).  Small doses of sodium phenobarbitone,  given subcutaneously, were administered during the course of the experiment whenever warranted, that is, when the female rat became a l i t t l e sensitive  to manipulations within the abdominal cavity. onto a dissecting board. the abdominal cavity.  The rat was held down firmly  As small as possible a s l i t was made to expose  Each fetus was exposed individually beginning with  the fetus at the very tip of the uterine horns.  Only that part of the  uterus pertaining to the fetus being used was cut open to reveal the fetus s t i l l held within its own amniotic sac and attached to its placenta. The rest of the uterus containing the other fetuses was kept within the abdominal cavity at a l l times.  Whenever possible the amniotic sac was left in-  tact during the experiment.  In several instances the umbilical vessels  were too small and i t was technically very d i f f i c u l t to infuse the Horseradish Peroxidate amniotic sac.  (HRP) into the umbilical veins without breaking the  Small amounts of HRP dissolved in saline (Table 1) were in-  jected into the fetuses via the umbilical vein which was in general recognisably larger and lighter in color than the umbilical artery.  After  the HRP injection, the fetus was put back into the abdominal cavity to keep warm. The incision in the abdominal cavity was closed with a hemostat. This method was found to be very effective. The fetuses felt warm to the touch and looked pink and healthy.  The fetuses were sacrificed by decapi-  tation at various times from 1 minute to 45 minutes after injection of the HRP. The eyes were removed and fixed whole in 4% glutaraldehyde cacodylate buffer at pH 7.2 held at room temperature.  in 0.1M  After _ to 1 hour  in the fixative, the eyes were cut in half, the lenses were removed and fixation was continued  for a total of 4 hours at room temperature. The  eyes were washed with and stored in 0.1M cacodylate buffer at pH 7.2 at o 4 C.  2.  Post-natal and Adult Rats  Rats, aged 1 to 10 days after birth, were anesthetised by being  placed in the freezer for 10 minutes.  This was sufficient to lower their  metabolism to such a low level that the rats were easily manageable. The rats were placed on a bed of ice cubes during injection of the HRP. Various doses of HRP (Table 1) were injected into the most readily accessible vein, the retro-orbital, jugular and saphenous veins.  These post-  natal rats were easily aroused from anesthesia by warming them up inbetween the palms of the hands.  At various times from 1 to 45 minutes  after injection of HRP the rats were sacrificed by decapitation and the eyes removed. Rats older than 10 days after birth and adult rats were lightly anesthetised with ether.  HRP was injected into the saphenous veins and  allowed to circulate in the body from 5 to 45 minutes.  The rats were re-  anesthetised and the eyes were removed. In a l l adult and post-natal rat eyes, an antero-posterior s l i t was made through the ora serrata. The eyes were fixed by immersion in a solution of 4% glutaraldehyde in 0.1M cacodylate buffer at pH 7.2 for _ to 1 hour.  Then the eyes were cut into smaller pieces and fixation continued  for a total of 4 hours at room temperature. o  The eyes were washed overnight  in a 0.1M cacodylate buffer at pH 7.2 at 4 C.  3.  Processing of the Tissues  A l l fetal, post-natal and adult rat eyes were incubated in open petri dishes for 40 to 60 minutes at room temperature in a solution made up of 5 mg 3,3' diaminobenzidine tetrahydrochloride (DAB - Reagent grade, Nutritional Biochemicals Corp.) dissolved in 0.05M Tham buffer, pH 7.6, containing 0.017o W-2®2 (Karnovsky, 1967).  The tissues were washed in dis-  t i l l e d water and post-fixed for 1 hour in VL osmium tetroxide in 0.1M cacodylate buffer at pH 7.2.  The tissues were then dehydrated in a graded  TABLE 1 HRP TRACER STUDIES Age  Anesthes ia  Route of Intervenous Injection  19 df  None  Umbilical vein  0.08  20 df  None  Umbilical vein  0.10  21 df  None  Umbilical vein  0.10  21 df  None  Umbilical vein  0.10  1 dpn  Ice  Retro-orbital vein  0.10  3 dpn  Ice  Jugular vein  0.10  4 dpn  Ice  Jugular vein  0.11  5 dpn  Ice  Jugular vein  0.11  7 dpn  Ice  Saphenous vein  0.15  10 dpn  Ice  Saphenous vein  0.20  12 dpn  Ether  Saphenous vein  0.20  15 dpn  Ether  Saphenous vein  0.14  22 dpn  Ether  Saphenous vein  0.25  Adult  Ether  Saphenous vein  0.50  N.B.  Amount of HRP (ml) (40 mg/ml saline)  The fetal rats were not directly anesthetised.  However, they must,  in effect, have been anesthetised by the sodium pentobarbitol and sodium phenobarbitone administered to the mother rat crossing the placenta. were relatively quiescent.  They  If a pregnant female rat is etherised and the  abdominal cavity opened, i t w i l l be found that the fetuses are extremely active as compared to the fetal rats we were working with. HRP = Horse-radish Peroxidase, Type II - salt free powder (Sigma) df = days fetal dpn = days post-natal  alcohol series, infiltrated and embedded in an epon-araldite mixture. The o tissue blocks were polymerised in a 65 C oven overnight. Thick sections were cut on a Porter-Blum MT-2 ultramicrotome and stained with toluidine blue. 4.  Examination of the Tissues  The sections were examined on a Zeiss Photomicroscope II. It was found that the toluidine blue stain on the sections often obscured the sites of deposition of the brownish-black HRP reaction product. come this, a sliding monochromator was used.  To over-  By altering the wavelengths  of the light falling onto the sections on the slides, i t was then possible to study both the histology of the i r i s tissue as well as the sites of deposition of the HRP reaction product.  Light micrographs were made on  Kodak Plus-X Pan film.  E.  A Scanning Electron Microscopic Study of the Posterior Surface of the Developing Rat Iris  METHOD: Fetal and post-natal rats of the Wistar strain were used.  The ages  of the fetal rats were determined by the method of Christie (1964). The fetal rats were removed individually from the uterus while the mother rat was kept under anesthesia either with ether, or with injections of sodium pentobarbital(administered intraperitoneally) and sodium phenobarbitone (administered  subcutaneously).  The fetuses were sacrificed by decapitation.  The eyes were removed immediately and fixed whole in the fixative of 4% glutaraldehyde in 0.1M sodium cacodylate buffer at pH 7.2 at room temperature for a total of 4 hours.  Rats, aged up to six days after birth, were anesthetised by being placed in the freezer t i l l they were quiescent and then they were placed on ice cubes.  Older rats were anesthetised by ether.  The eyes were removed  and placed in the fixative of 47. glutaraldehyde in 0.1M sodium cacodylate buffer at pH 7.2.  After % to _ hour in the fixative, the posterior halves  of the eyes and the lenses were removed and fixation was continued for a total of 4 hours. The fetal and post-natal eyes were stored in a 0.1M cacodylate o buffer at 4 C.  The eyes were dehydrated through a graded series of alcohols.  While in 10% alcohol, the eyes were trimmed as much as possible so as to best display the posterior surface of the developing i r i s .  The absolute  alcohol in the tissues was then substituted by iso-amyl acetate (Fisher) in a stepwise fashion.  The tissues were dried using the C r i t i c a l Point Drying  method, as has been previously described.  The tissues were then mounted  on tissue stubs, coated with a thin layer of gold and examined on a Cambridge Stereoscan (Model S4).  76.  III.  A.  RESULTS  A Light Microscopic Study of the Rat Iris in Pupillary Dilation and Constriction  1.  The Iris in Pupillary Dilation (Figures  2-7)  When the pupil is well dilated, the i r i s is short in terms of its dimensions from the root to the tip of the i r i s , as seen in meridional sections.  It may only be about 1mm  or less across.  The i r i s tends to be  disposed in a slight convex arch towards the surface of the cornea. to buckle in the middle (Figure 2).  It seems  This is in part due to the fact that the  stroma in the middle one third of the i r i s is always thicker than that at the tip or at the root of the i r i s (Figure 2). In meridional sections, the posterior epithelial cells form a layer covering the posterior surface of the i r i s from the pupillary edge to the root of the i r i s where i t is continuous with the epithelium of the c i l i a r y processes.  Each individual c e l l may be discretely separated from its  neighboring c e l l thus giving the posterior surface a deeply appearance (Figures 2-3, 5-7).  convoluted  The peg-like epithelial cells then appear  like the myriad legs of a millipede (Figures 2-3, 5-7).  On the other hand,  the posterior epithelial cells may be so closely abutted on each other that there are no gaps in between the cells.  This is most prominently seen in  the middle one third of the i r i s (Figure 4), whereas the epithelial cells at the root of the i r i s may be separated one from another. The posterior epithelial cells are most often large, columnar in shape and they have a highly irregular c e l l outline (Figures 3, 5-7).  The  height of the epithelial cells may vary from being high cuboidal to low columnar to high columnar depending on the degree of pupillary dilation.  The anterior border of these cells is in apposition with the dilator muscle cells but the posterior and lateral surfaces are generally free and not touching other cells except in the instances mentioned above. surface of the cells show large or major c e l l processes.  The posterior  Oftentimes such a  major cell process imparts to the cell a bifurcate appearance.  The major  c e l l processes may in turn show minor processes (Figures 5 and 6). In the center of each cell is the nucleus.  The nuclear structure  and staining characteristics with toluidine blue seem to be in some way related to the configuration of the posterior epithelial cells.  If the epi-  thelial cells are discretely separated from each other, the nuclei stain uniformly intensely with toluidine blue. nuclei of the dilator muscle layer.  They stain much darker than the  Not much nuclear detail is visible at  a l l . These elongated nuclei have a very irregular outline. They are oriented such that the long axis of the nucleus lies along the long axis of the c e l l . On the other hand, i f the posterior epithelial cells are in close apposition, the nuclei are round to oval in shape and only sometimes elongated (Figure 4).  The nuclei stain lightly and consequently they show a l i t t l e  more nuclear detail.  The nuclear envelope stains intensely.  Specks of  darkly staining chromatin material are found scattered within the nuclear substance. These nuclei generally stain lighter than those in the adjacent dilator. The nuclear outlines are relatively smooth with a few clefts and indentations. If the nuclei are larger and slightly elongated, then they are situated parallel to the long axis of the cells (Figures 3, 5-7). The cytoplasm of the posterior epithelial ceils appears vesicular, especially so at the anterior poles of the cells where they come in contact with the dilator muscle cells.  Whether these vesicles are a consequence of  fixation or whether they serve as a membrane pool to accommodate to changes in c e l l shape and size during pupillary constriction and dilation is  speculative. At the junction of the dilator and sphincter, there is an abrupt change from a columnar to a squamous epithelium lining the whole posterior aspect of the sphincter region up to the pupillary border (Figures 2, 7). The nuclei stain similarly to those of the epithelial cells. are oriented radially with respect to the whole i r i s . irregular and there are many cell processes.  However, they  The cell outlines are  The cytoplasm is thin and  appears vesicular too. The dilator muscle layer is relatively thick and quite readily discernible with the light microscope (Figures 2-7).  The width of the dilator  may range from one half to two thirds the height of the columnar cells of the posterior epithelium. thelial cells are high.  Sometimes the dilator may be as wide as the epiThe posterior surface of the dilator is apposed onto  the anterior surface of the posterior epithelium.  There does not seem to be  a tight line of fusion between the two layers of cells. looks vesicular (Figures 3, 5, 6).  The junction region  It could be that here cell processes of  the two layers interdigitate loosely leaving relatively large intercellular spaces.  This would be deemed desirable in view of the fact that rapid  changes in relationships between the two cell layers might have to take place during pupillary constriction and dilation.  Unlike the posterior epi-  thelial cells, the boundaries between individual dilator muscle cells are not readily apparent (Figures 3, 5, 6). The nuclei, placed more or less in the center of the cells, vary both in shape and size. Also, the nuclei are not as regularly placed within the entirety of the muscle layer as in the posterior epithelium (Figure 6). These observations material.  are a result probably of the plane of sectioning of the  The nuclei are irregular in shape, sometimes somewhat rounded,  with grooves and indentations so that they may even appear lobulated.  These  usually exhibit a slightly more intense staining along the nuclear envelope with some fine dense particulate chromatin within the rest of the nucleoplasm. On the whole, the cytoplasm of the dilator muscle cells appear less vesicular than that of the posterior epithelial cells.  It is especially  noticeable in the anterior two thirds of the cells where the cytoplasm has a smooth and more solid appearance. located.  This is where a l l the myofilaments are  At the.boundary between the dilator and the stroma, the cytoplasm  stains much more intensely probably owing to an accumulation of myofilaments in this region (Figures 3, 4, 6).  At intervals a l l along this boundary  zone, the dilator muscle cells send out a series of arborescent processes (Figures 3, 6).  These cell processes, simple or complex, also stain darkly.  Large or major processes with their many minor processes which radiate and spread out in a fan-like manner, protrude into the stroma (Figure 6). At times i t seems that not only dilator cell processes but whole groups of dilator muscle cells encroach on the stroma (Figures 5, 7).  One receives  the impression that during extreme pupillary dilation there is just not enough room within the dilator muscle layer to accommodate a l l of the cells so that some of the cells are squeezed out of position, as i t were, by partial buckling of specific areas of the dilator muscle layer (Figures 5, 7).  Such muscle spurs, when present, are primarily seen in the medial half  of the iris (Figure 2). At the junction between the dilator and sphincter, some of the cells of the dilator seem to intrude into sphincter muscle territory.  Sometimes  they appear to spread out and envelope the laterial extents of the sphincter muscle fibers (Figure 7 ) . Thus there does not seem to be a very sharp clear-cut demarcation between the dilator and sphincter, as seen by light microscopy.  Most probably the myofilaments are disposed in their respective  directions within the muscle cells despite their proximity. The sphincter muscle is a distinct, but not a compact, bundle found at  the pupillary tip of the i r i s .  It is bounded anteriorly by a small  amount of stroma and posteriorly by a double-layered (Figures 2, 7).  squamous epithelium  The posterior epithelium lining the sphincter region seems  to be a continuation of the dilator and the columnar posterior epithelial cells which have abruptly changed their form. sphincter, there are few stromal cells.  In the stroma overlying the  This part of the stroma is primar-  ily occupied by numerous capillaries, presumably for efficient exchange of materials between the sphincter cells and blood.  Small capillaries are als  found in among the sphincter muscle cells themselves.  Being contractile  elements, the energy requirements must be high and thus they need to be wel vascularised.  The individual muscle cells may be closely associated with  each other or they may be far apart.  In meridional sections, the uniformly  darkly staining muscle fibers are cut in cross-section and appear round to polygonal.  The nuclei are small, round or e l l i p t i c a l in shape and are  irregularly distributed. There are very many more muscle cells seen than '• there are nuclei owing to the plane of sectioning. The stroma extends from the root to the tip of the i r i s .  In pupil-  lary dilation, its antero-posterior dimensions vary depending on the partic ular region of the i r i s .  There is usually just a small amount of stromal  tissue over the anterior face of the sphincter. i r i s the stroma is thick and highly cellular. towards the root of the i r i s .  In the middle third of the The stroma slowly tapers off  This uneven distribution of stroma over the  extent of the i r i s during pupillary dilation imparts to the iris a convexly curved appearance. No attempt is made to stringently identify the various c e l l types within the stroma.  This has been adequately done in numerous other works.  The main object of concern within this study is to examine the overall arrangement of the cellular elements within the stroma rather than the interrelationships of individual types of cells.  Suffice i t to mention that in  the stroma there are blood vessels of various sizes, both myelinated and unmyelinated nerves and stromal cells. The stroma over the sphincter is loose and not very cellular (Figures 2, 7).  Large intercellular spaces are present.  This area is primarily  occupied by numerous blood vessels. Most of these are capillaries; some consist of a single endothelial c e l l lining the lumen and thus large enough to accommodate only one erythrocyte (Figure 7), while others may be a l i t t l e larger and may have in addition a single layer of pericytes encircling the endothelial lining.  The stromal cells, when present, appear haphazardly  arranged. In the middle of the i r i s , the stromal cells are very closely packed together.  It is d i f f i c u l t or nearly impossible to trace the continuity of  each individual c e l l .  However, the numerous cytoplasmic cell processes seem  to be disposed perpendicularly with respect to the posterior layers of the i r i s , that is the posterior epithelium and dilator.  The spaces in between  the c e l l nuclei and their processes give the impression of vertical linearity (Figures 3,6).  The stromal cells then appear to be arranged in vertical  columns streaming down from the anterior to the posterior surface of the iris.  Also, the nuclei of the stromal cells seem to be kept away at a dis-  tance from the dilator muscle cell processes.  Only the stromal cell cyto-  plasmic processes come into close proximity to the dilator muscle layer. The nuclei, having more bulk and perhaps being less deformable, seem to be held aloof from the dilator almost so as not to impede the folding up of the dilator during pupillary dilation (Figures 3, 5, 6).  Occasionally a nucleus  might come fairly close to the dilator especially where muscle spurs are  82.  present. Nerves are found singly or in bundles of various sizes within the stroma.  They may be myelinated  with blood vessels (Figure 5).  or unmyelinated.  They are often associated  However, i t is not possible to t e l l i f there  are nerve terminals in close association specifically with the dilator or sphincter since toluidine blue is not a preferential stain for nervous tissue. Most of the blood vessels are large. f i l l e d with erythrocytes (Figures 2, 7). shape.  Their lumina are open and  The lumina may be round or oval in  When the lumina are oval, the length of the oval is oriented simil-  arly to the cells, that is, perpendicular to the posterior surface of the i r i s (Figures 3, 5, 6).  These blood vessels thus appear to be squeezed in  from side to side, but not sufficiently to occlude the lumina.  This is  important for the maintenance of v i t a l c e l l functions even in extreme pupillary dilation.  The walls of the i r i d i a l blood vessels are not very thick in  relationship to their size.  This is probably for greater f l e x i b i l i t y during  movements of the i r i s in pupillary dilation and constriction. The stroma at the root of the i r i s is in most respects similar to the stroma in the middle of the i r i s except for the fact that the width of the stroma is decreased.  Oftentimes the division of the stroma into vertical  columns of cells is much more evident (Figure 3).  A fair sized number of  blood vessels make up the bulk of the stroma here. The anterior surface of the i r i s usually shows a scalloped appearance. Sometimes i t is more pronounced (Figures 2-4, 7) than at other times (Figure 5).  This could be due to the plane of sectioning.  The anterior surface of  the stroma directly over the sphincter is in general smooth.  The scalloped  appearance over the rest of the stroma is the result of columns of cells protruding outwards or dipping inwards.  The protrusions are most often due  to the bulging out of large blood vessels at the anterior surface. occur in between the bulging blood vessels.  The di  These dips may be quite deep.  The blood vessels do not ever abut directly onto the anterior stromal surface.  The cytoplasm of other stromal cells are always interposed between  the blood vessels and the anterior chamber.  Oftentimes small  blebs may be seen off the anterior surface.  These may be a fixation  artifact.  cytoplasmic  84.  Figure 2 The Iris in Pupillary Dilation (LM) This low magnification light micrograph of the i r i s in pupillary dilation shows that the i r i s is short. Both the posterior epithelium (pe) and dilator (d) are very distinctly visible. The stroma (s) is thick, especially in the middle one third of the i r i s , so that the i r i s appears to buckle anteriorly. Dilator muscle spurs (arrows) are seen in the pupillary half of the i r i s . The sphincter (sph) is a mass of small cells at the pupillary tip of the i r i s . x 90  Figure 3 The Iris in Pupillary Dilation (LM) Each posterior epithelial c e l l (pe) is discretely separated from the next. The epithelial cells are peg-like giving the posterior surface of the i r i s a deeply convoluted appearance. The nuclei are large and are situated in the middle of the cells surrounded by a thin vesiculated rim of cytoplasm. The dilator (d) is a relatively thick layer in apposition with the posterior epithelium (pe) and with the stroma (s). At the stromal surface of the dilator, there is a dense line where a l l the myofilaments are concentrated. Fine, dark-staining processes from the dilator are seen a l l along the dilator-stroma boundary (arrows). The stroma (s) is f i l l e d with blood vessels containing masses of red blood cells. The lumens of the blood vessels may be round to oval. When oval, they are perpendicular to the dilator and the posterior epithelium. Some of the blood vessels appear to have only an endothelial lining (bvl) while others possess in addition a layer of pericytes (bv2). The stromal cells are arranged in columns extending from the anterior surface of the iris to the dilator-stroma boundary. The spaces in between the stromal cells emphasise the vertical linearity. The nuclei of the stromal cells are kept at a distance from the dilator. The anterior surface of the iris facing the anterior chamber (AC) is highly scalloped in outline. This is mainly caused by the bulging out of the i r i s blood vessels. x 240  85.  86.  Figure 4 The Iris in Pupillary Dilation (LM) Unlike in Figure 2, the posterior epithelial cells (pe) are quite close together. The posterior surface of the i r i s is not deeply convoluted but slightly wavy in appearance. The contractile portion of the dilator (d) layer is seen as a very distinct, smooth, dense line (arrows) while the epithelial portion is lighter staining. The cells in the stroma (s) are packed together. The anterior surface of the i r i s shows a scalloped outline. x 280  Figure 5 The Iris in Pupillary Dilation (LM) The posterior epithelial cells (pe) have a highly irregular outline. The cells may bifurcate and show numerous c e l l processes which are devoid of nuclei (arrows). The cytoplasm of the posterior epithelial cells appears vesiculated. The most distinctive feature of this light micrograph are the groups of dilator cells or muscle spurs (msl, ms2) which encroach on the stroma. They may also show fine dilator processes (ms2). Blood vessels of different sizes (bv), nerves (n), and stromal cells make up the stroma (s). The nerves are often found in bundles near to blood vessels. The anterior surface of the i r i s shows a relatively undulating outline. x 380  Figure 6  The Iris in Pupillary Dilation  (LM)  The posterior epithelial cells (pe) are large, high columnar with an irregular cell outline. The nuclei, with irregular nuclear envelopes, are elongated along the long axes of the cells. The cytoplasm has a lace-like appearance. The cells show processes of different sizes (p). The dilator (d) layer is quite thick. The nuclei are irregularly shaped. Small densely staining hillocks of dilator material (arrows) are distributed a l l along the anterior stromal surface of the dilator. From these hillocks arise numerous, highly branched dilator processes which spread out in a fan-like manner. The stromal cells, the spaces in between the cells and the lumens of the blood vessels are a l l perpendicular to the posterior surface of the i r i s . x 1400  Figure 7  The Iris in Pupillary Dilation  (LM)  The sphincter (sph) is a distinct bundle of small, darkly staining cells. It is bounded anteriorly by a thin strip of stroma containing few cells and numerous capillaries (bv). Posteriorly, i t is bounded by a barely perceptible squamous epithelium. At the peripheral extent of the sphincter (arrow), some of. the dilator cell processes seem to spread out and envelope the sphincter. Here, too (arroxi?), there is an abrupt transition between the high columnar epithelium lining the rest of the posterior surface of the iris and the squamous epithelium lining the posterior surface of the sphincter. The stroma (s) is cellular and vascular. As in Figures 2-4, and 6, the stromal elements are arranged perpendicular to the posterior surface of the i r i s .  88*  2.  The Iris in Pupillary Constriction (Figures  8-11)  When the pupil is constricted the iris is a thin, delicate and tenuous structure (Figure 8) covering the anterior surface of the lens.  In  meridional sections, the antero-posterior width of the i r i s varies in direct relationship to the degree of constriction of the pupil.  The iris may  be  evenly thin from the pupillary border to the root of the i r i s (Figure 8). At times, the root of the i r i s is slightly more attenuated than the rest of the i r i s .  Or, at other times, there may  be some unevenness along the  whole length of the i r i s and in which the root of the i r i s is extremely stretched out.  The configuration of the posterior epithelium, dilator,  sphincter and stroma a l l contribute  to the overall width of the i r i s .  differing changes in c e l l shape, size and configuration,  Thus  and changes in  inter-cell relationships occur depending on the extent of pupillary constriction. In extreme pupillary constriction the posterior epithelium is in general a relatively thin layer lining a l l of the posterior surface of the iris.  It has the appearance of a thick squamous epithelium (Figure 9).  The nuclei are flattened and there is l i t t l e discernible cytoplasm which may  be vacuolar.  Sometimes in amongst the thick squamous epithelium there  may  be low cuboidal cells.  These may  present singly (Figure 10).  be  The nuclei are thicker, with an irregular  outline and more prominently seen. to each other or they may  be present in groups or they may  The cuboidal cells may be pushed close  be set apart with the cytoplasm of one cell  extending out towards the cytoplasm of an adjacent c e l l (Figure 10). c e l l outlines are irregular.  The  In less extreme states of pupillary constric-  tion, the posterior epithelial cells are squamous only at the root of the iris.  Along the rest of the i r i s the posterior epithelial cells are thicker  with an associated  relative increase in nuclear size.  The cytoplasm may  bulge out over the nucleus giving the posterior surface a very slight scalloped appearance. The epithelium lining the posterior surface of the sphincter is not easily seen.  Since i t is such a thin squamous epithelium there is not a  sharp transition between this epithelium and that lining the rest of the i r i s , as is observed when the pupil is dilated. The dilator is an extremely thin squamous layer which is barely visible (Figures 9-11). ior epithelium.  It is only about half the thickness of the poster-  Essentially the dilator muscle layer is seen as a row of  flattened nuclei. The c e l l cytoplasm is attenuated and discernible only in certain areas.  The dilator may appear to be closely applied onto the  posterior epithelium or there may be spaces in between the two layers. Presumably, the cytoplasmic processes of both the posterior epithelial cells and the dilator muscle cells interdigitate loosely in this region. The anterior surface of the dilator most often appears as a smooth dense line.  There are no signs of dilator c e l l processes  when the pupil is dilated. pushed into the stroma.  (Figures 9, 10) seen  Neither are there clumps of dilator cells being  There is no necessity for this to occur.  With an  increase in total area of the i r i s in pupillary constriction a l l of the dilator cells can be accommodated within one layer (Figures 9-11) unlike the condition that is present when the pupil is dilated. The sphincter is a thick bundle of cells found at the pupillary edge of the i r i s (Figures 8, 11). It is just possible to make out the squamous epithelium lining its posterior surface. by a l i t t l e bit of stroma. together.  Anteriorly i t is covered  The sphincter muscle cells are closely packed  These muscle cells stain uniformly darkly in comparison to the  stromal cells.  The numerous small nuclei of the muscle cells do not stain  much darker than their cytoplasm.  The stroma is thin (Figures 9-11) as compared to its width when the pupil is in the dilated state.  The width of the stroma is in essence a  qualitative indicator of the extent of constriction or dilation of the pupil.  In pupillary constriction, the stromal cells and the associated  intercellular spaces are arranged parallel to the posterior surface (Figures 9, 10). This arrangement is more evident in certain parts of the i r i s than in others.  Likewise, the larger blood vessels, open and f i l l e d with red  blood cells, are stretched out lengthwise parallel to the posterior surface (Figures 9, 10). Large bundles of myelinated nerves are often found next to the blood vessels.  Only a few stromal cells and capillaries are present  in the stroma overlying the sphincter. In pupillary constriction, the anterior surface of the i r i s is quite smooth {Figure 8). the sphincter.  There may be a hint of some scalloping in the region of  Slight undulations along the rest of the anterior surface  of the i r i s may at times be observed but these are by no means a prominent or constant feature.  In the dilated pupil, i t is seen that the scalloped  appearance of the anterior surface of the i r i s is in large measure created by the bulging anteriorly of the blood vessels in the stroma.  However, in  the constricted state, the blood vessels are stretched out parallel to the posterior surface thus rendering the anterior i r i d i a l surface relatively smooth.(Figure 10).  92.  Figure 8  The Iris in Pupillary Constriction  (LM)  In pupillary constriction, the i r i s is long and thin. The posterior epithelium(ep) and dilator (d) are barely visible but as two rows of flattened nulei. The stroma (s) is thin. The sphincter (sph) is a darkly staining compact bundle at the pupillary tip of the i r i s . x 90  Figure 9  The Iris in Pupillary Constriction  (LM)  The posterior epithelium (pe) is a squamous layer of cells with the nuclei flattened parallel to the i r i s length. The dilator (d) is an even thinner layer. In most instances, i t is seen as a dense line with a few scattered elongated nucl e i . The stroma (s) appears relatively loose. There are many large spaces. The blood vessels and stromal cells are generally oriented parallel to the i r i s length. The anterior surface of the i r i s is smooth with openings (arrows) leading into the anterior chamber (AC). x 260  93.  8  p<z+d  sph  94.  Figure 10  The Iris in Pupillary Constriction (LM) Some of the posterior epithelial cells (ep), singly or in groups, are cuboidal or thick squamous in shape and they bulge slightly into the posterior chamber (PC). This gives the posterior surface of the i r i s a slightly scalloped appearance. The dilator (d) is a thin layer of dense cytoplasm and extremely attenuated nuclei. The stromal surface of the dilator is smooth. The blood vessels and stromal cells are packed close together parallel to the posterior surface of the i r i s . x 260  Figure 11  The Iris in Pupillary Constriction (LM) The sphincter (sph) is a compact mass of darkly staining cells. Spaces are found in between the cells, giving the sphincter as a whole a cracked glass effect. The anterior surface of the i r i s is relatively smooth except over the sphincter where i t is convoluted. Crypts (arrows) open into the anterior chamber (AC). x 260  95.  96.  B.  A Light Microscopic Study of the Collagen Network in the Stroma of the Rat Iris in Pupillary Dilation and Constriction Using a modified Mallory's Trichrome Stain, the collagen network  in the stroma of the i r i s is stained blue.  The disposition of the collagen  in the stroma follows closely the changes in arrangement of the stromal cells during pupillary dilation and constriction. When the pupil is d i lated, the stromal cells are arranged perpendicular to the posterior epithelium and dilator muscle.  Likewise, the collagen appear as bundles  streaming down from the anterior surface of the i r i s stroma towards the boundary zone between the dilator and stroma (Figure 12).  There seems to be  a condensation of collagen, as indicated by the relative increase in the intensity of staining, at this boundary zone (Figure 12). When the pupil is constricted, the i r i s is long and thin and a l l of the stromal cells are oriented parallel to the posterior epithelium and dilator muscle.  Because of the paucity of the collagen elements in the rat  i r i s stroma and the extreme thinness of the i r i s as a whole, i t is not often easy to visualise the collagen network.  However, where visible, i t is  found that the collagen is indeed arranged parallel to the posterior epithelium and dilator muscle, as are the stromal cells (Figure 13).  97.  Figure 12  The Collagen Network in Pupillary Dilation  (LM)  In pupillary dilation, the i r i s is short. The stromal (s) cells are arranged perpendicular to the posterior epithelium (pe) and the dilator (d). The collagen network, stained blue, are seen as bundles streaming from the anterior surface of the i r i s to the boundary zone with the dilator muscle. There is a condensation of collagen a l l along this boundary zone. x 250  98.  5)9.  Figure 13  The Collagen Network in Pupillary Constriction  (LM)  In pupillary constriction, the i r i s is long and thin. The posterior epithelium (pe) and dilator (d) cannot be easily distinguished from each other. The stromal cells and the collagen bundles are oriented parallel to the length of the iris. x 400  99i.  100.  C. A Transmission Electron Microscopic Study of the Rat Iris in Pupillary Dilation and Constriction  1.  General (Figure 14)  Various chemical fixatives and methods of fixation are used, as outlined in the method.  The eye, in pupillary dilation or constriction, as the  result of the topical application of mydriatics or miotics, is removed immediately from the rat. A s l i t is made through the cornea to facilitate the inflow of fixatives into the anterior chamber and thus to the i r i s . eye is then immersed in the respective fixatives. in the constricted or dilated state.  The  The i r i s is maintained  The so called "death dilation" which  is normally observed in the pupil when death ensues, does not occur i f the i r i s is fixed right away after being removed from the animal.  Light micro-  scopically, the i r i s usually appears adequately fixed when the immersion method is used. distended.  The lumens of the blood vessels are patent although not  However, with the transmission electron microscope, i t is found  that there is some tissue destruction, especially to the mitochondria of the posterior epithelial cells.  The mitochondria are broken up.  The  cristae disintegrate within the mitochondrial membranes leaving a large vesicle with cellular debris.  The extent of mitochondrial disruption varies  from specimen to specimen and cannot be correlated with either the speed of transfer of the tissue from the animal to the fixative, or with the type of fixatives used. As an alternate method, the i r i s is f i r s t fixed in situ prior to being immersed in the fixative.  A s l i t is made in the cornea and fixative  is dripped onto the eye for 5 minutes.  In situ fixation prior to immersion  fixation does not seem to improve the quality of fixation as compared to that obtained with immersion fixation alone.  The best fixation of the i r i s tissue is obtained by perfusion fixation.  The animal stiffens as the fixative circulates through a l l parts of  the body.  This is one indication that the whole animal has been thoroughly  perfusedo  Also the pupils remain in the same degree of miosis or mydriasis  as perfusion occurs, and when the eyes are enucleated.  This is another  gross evidence that the i n i t i a l fixation has been almost instantaneous. Light microscopically, the tissues usually appear well-fixed.  The blood  vessels are relatively distended and have a smooth outline to their lumens. Observations made with the transmission electron microscope on such specimens reveal that the i r i s is usually well-fixed.  The mitochondria are pre-  served whole and there are no vesicles with cellular debris, although other types of vesicles are present.  The posterior epithelium of the i r i s is  most capricious and d i f f i c u l t to fix well.  Even though a l l other parts of  the Iris may be adequately fixed, the posterior epithelium may appear slightly broken up.  The ultrastructural features of the posterior epithel-  i a l cells differ slightly from one part of the i r i s to the other, suggesting that the posterior epithelial cells are not similarly susceptible to fixation conditions.  It is extremely d i f f i c u l t to obtain good fixation  throughout a l l of the posterior epithelium at any one instance.  When the  fixation is good, both thick and thin myofilaments are observed in the sphincter and dilator muscles (Figure 14). present in tissues fixed by immersion.  The thick filaments are not  Kelly and Arnold (1972) found that  thick filaments can only be demonstrated i f good fixation is achieved. This is so in our studies.  However, unlike Kelly and Arnold's investiga-  tions, the rat does not have to be perfused with a salt solution prior to fixation to maintain a l l of the ultrastructural features of the i r i s . In our study, attention is focussed only on the posterior epithelial and dilator layers, as i t is here that the ultrastructural changes are  observed between pupillary dilation and constriction.  2.  The Iris in Pupillary Dilation (Figures 15-23)  In a low magnification transmission electron micrograph, the posterior surface of the i r i s is seen to be highly convoluted or scalloped (Figures 15, 16). Towards the root or periphery of the i r i s , the grooves in between the individual posterior epithelial cells and their processes are deep and quite wide (Figure 15).  Usually, towards the mid-portion of  the i r i s , the posterior epithelial cells are separated by deep but narrow grooves (Figure 16).  The cells may appear to be in apposition in light  micrographs, but in electron micrographs, a narrow space and the basement membrane of the posterior epithelial cells always separates out the individual cells. Following the contours of the posterior surface of the i r i s at the bases of the posterior epithelial cells there is a basement membrane (Figures 15-19).  The basal c e l l membranes of the posterior epithelial  cells show numerous complicated  infoldings which extend relatively deeply  into the cytoplasm (Figures 15-19). cytoplasm around the nucleus.  In fact, there is only a thin strip of  The cell infoldings are found along the  posterior and lateral walls of the cells. other in a three dimensional  They interdigitate with each  fashion so that these small, fine, basal c e l l  membrane infoldings are always sectioned in different planes.  Besides  these basal c e l l infoldings, the posterior epithelial cells in pupillary dilation also show large or major cytoplasmic processes  (Figures 15, 19).  These processes do not contain a nucleus and consist almost exclusively of the basal c e l l infoldings and some c e l l organelles (Figure 15). plasmic cell processes are of different sizes (Figure 15).  The cyto-  The thin base-  ment membrane follows the outlines of the cytoplasmic processes but i t does  103.  not follow the outlines of the basal c e l l infoldings. The basement membrane is only loosely associated with the surface of the posterior epithelial cells.  There is always a space of varying widths between the electron  dense basement membrane and the cell membrane'. This is very clearly shown in some of the electron micrographs. The cytoplasm contains the usual organelles (Figures 15-19).  There  are quite a number of mitochondria,  rough and smooth endoplasmic reticulum,  golgi apparatus and free ribosomes.  There are a number of vesicles in the  cell cytoplasm,(Figure epithelial cells.  16).  The number of vesicles vary for different  Occasionally, there are some dense bodies which are  membrane bound (Figures 16, 17, 19).  Another component of the c e l l cyto-  plasm which has not been observed previously is the masses of fine filaments (Figures 15, 17-19).  The filaments are sometimes very clearly seen in  bundles (Figures 15, 17-19). nucleus (Figure 18).  The filaments are usually found around the  They seem to cascade down to surround the nucleus of  the posterior epithelial cells.  These filaments are also found in the more  apical or anterior part of the cell cytoplasm (Figure 17). bundles which curve around the cell organelles. of filaments cut in cross-section (Figure 19).  They form  Very rarely is a bundle Here, i t is clearly seen  that the filaments do form a distinct bundle devoid of cell organelles situated in the middle of a large c e l l process. The nuclei of the posterior epithelial cells assume different shapes but in a l l instances, they are highly indented (Figures 15-19).  The nuclei  are situated in the posterior portions of the cells surrounded by a thin layer of cytoplasm, and occasionally, filaments, and external to that, a layer of cell infoldings. Dense heterochromatin is usually associated with the nuclear envelope but there are also patches of heterochromatin throughout the nuclear substance.  The nuclear material is enclosed within a  double layer of nuclear membranes. However, there always appears to be a gap between the inner and outer nuclear membranes.  The nuclear outline is  not smooth but is indented to varying degrees, depending on the plane of sectioning.  Tongue-like processes of cytoplasm, devoid of c e l l organelles,  occupy the indentations of the nuclear envelope (Figures 15-18). It is impossible to delineate the boundaries of one c e l l from the next, as adjacent posterior epithelial cells also interdigitate with each other.  Cell junctions are not seen between the individual posterior epi-  thelial cells.  The cells appear to associate loosely with each other. In  the dilated condition, the boundaries between the posterior epithelium and dilator are also not distinct.  The junctions between the posterior epi-  thelium and the dilator are better seen when the i r i s is observed in the constricted state and w i l l be described then. The cytoplasm of the epithelial portion of the dilator layer is relatively dense.  There are numerous delicate-looking mitochondria  scattered throughout the cytoplasm (Figures 20-23).  Vesicles and vacuoles  of different sizes, rough and smooth endoplasmic reticulum and free ribosomes are found in the cytoplasm.  The nuclear outline is much more convo-  luted than that of the posterior epithelium. Heterochromatin is associated with the nuclear envelope and is present as patches in the center of the nucleus.  Usually, but not always, the nuclear membranes appear to be  closely adherent to the nuclear material. Except for a small amount of cytoplasm around the nucleus, most of the cell cytoplasm consists of an immense number of microvillous, finger-like cell processes (Figures 20-23). The cytoplasmic processes from adjacent cells interdigitate loosely with each other in a l l planes. ses.  There are large spaces between the c e l l proces-  Occasionally, neighboring dilator cells are joined together by cell  junctions (Figures 22, 23). It appears that the membranes are fused to-  gether but this may be due to the plane of sectioning. tight junctions.  Probably these are  Tight junctions have been observed by other investigators  (Hogan, Alvarado and Weddell, 1971). The contractile portion is confined to the stromal poles of the dilator cells.  The sarcoplasm is very dense so that the myofilamentous  nature of this layer is not always distinguishable. ent (Figure 21).  It is sometimes appar-  There is one constant and interesting feature of the di-  lator that is always observed in irises in pupillary dilation. Numerous arborescent protrusions are regularly found a l l along the length of the dilator-stroma boundary (Figures 20-23).  These dilator processes may be  relatively simple in configuration where they consist of single projections of the dilator into the stroma (Figure 21).  More often, the dilator proces-  ses are quite complex and branch profusely (Figures 20-23).  Small hillocks  of dilator muscle are disposed along the length of the dilator at its boundary with the stroma.  From these hillocks arise long, delicate, dila-  tor processes which branch extensively in a l l planes.  Pinocytotic vesicles  are present both in the dilator processes and in the underlying layer. A l l of the dilator processes are covered by a basement membrane. Much like the basement membrane of the posterior epithelium, the basement membrane of the dilator cells does not adhere to the c e l l membranes. There is alwasy a distinctly visible clear region between the c e l l membrane and the basement membrane (Figures 20, 21). The collagen fibers in the stroma seem quite closely associated with the basement membrane (Figure 20).  The basement  membrane and the neighboring collagen fibers give to the dilator processes a halo effect.  106.  Figure 14 The Iris in Pupillary Dilation (TEM) The sphincter muscle cells contain a nucleus (N), numerous mitochondria (m) and both thick and thin myofilaments„ The presence of the thick filaments, cut in cross-section, is an indication of adequate fixation of the tissue. The muscle cells come relatively close to each other. There may be some condensation of the adjacent cytoplasm (arrow). x 28,400  14  108.  Figure 15 The Iris in Pupillary Dilation (TEM) In a low magnification electron micrograph, the posterior epithelial cells (pe) are discretely separated from each other by deep grooves (g). The posterior epithelial cells often show ~ large cytoplasmic processes (cp) devoid of a nucleus. The basement membrane (bm) loosely follows the outlines of the posterior epithelial cells and the cytoplasmic processes. Within the posterior epithelial cells, there is a nucleus (Npe) and numerous mitochondria (m). Bundles of intracellular f i l a ments (if) surround the nucleus. The cell membrane is highly infolded to give numerous c e l l infoldings (ci) which interdigitate with each other. The cytoplasmic processes consist almost exclusively of c e l l infoldings. The posterior epithelium is closely related to the dilator layer (d). The nucleus of the dilator (Nd) is highly indented. x 8,900  109.  110.  Figure 16 The Iris in Pupillary Dilation (TEM) The posterior epithelial cells (pe) are very close together. The grooves (g) in between the posterior epithelial cells are very deep and narrow. However, the cells are always separated by the basement membrane (bm). The nuclei of the posterior epithelial cells (Npe) show deep indentations which are occupied by the cytoplasm devoid of c e l l organelles (*). In the cytoplasm, there are numerous mitochondria (m), vesicles (v) and dense bodies (db). There are also many cell infoldings ( c i ) . There are large spaces in between the posterior epithelium and the dilator (d). x 8,900  111.  112.  Figure 17 The Iris in Pupillary Dilation (TEM) Each posterior epithelial c e l l is separated from the next by a deep groove (g). There is always a clear space* between the basement membrane (bm) of the posterior epithelial cells and the cell membrane. The c e l l membrane is deeply and complexly infolded (ci). The cell infoldings interdigitate in various planes. Right around the nucleus (Npe) there is a thin rim of cytoplasm containing mainly mitochondria (m) and a few dense bodies (db). In the anterior cytoplasm, there are some bundles of intracellular filaments (if) cascading down. The nucleus of the posterior epithelial cells is located in the middle of the c e l l . Dense heterochromatin is mainly associated with the nuclear envelope. There is a clear peri-nuclear space present (arrow). The nucleus is indented and the indentation is occupied by c e l l cytoplasm (*). x 21,300  114.  Figure 18 The Iris in Pupillary Dilation (TEM) The basement membrane (bm) of the posterior epithelial cell does not follow the contours of the cell infoldings (ci). The cell infoldings are extensive. Where the cell infoldings are absent, there are many mitochondria (m) with delicate cristae. Some free ribosomes (r) are seen. A large bundle of intracellular filaments (if) forms a hammock around the nucleus of the posterior epithelial c e l l (Npe). The indentations of the nucleus are f i l l e d with tongues of cytoplasm (*). x 26,600  115.  116.  Figure 19  The Iris in Pupillary Dilation (TEM) A large (cp2) and small (cpl) cytoplasmic process of the posterior epithelium are shown. A golgi apparatus (go), and endoplasmic reticulum (er), free ribosomes (r), mitochondria (m), vesicles (v) and dense bodies (db) are seen in the c e l l cytoplasm. Intracellular filaments sectioned longitudinally ( i f l ) and in cross-section (if2) are present. The intracellular filaments form a large discrete bundle (if2). The basement membrane (bm) is loosely adherent to the posterior surface of the cells. A very clear peri-nuclear space (arrow) is distinctly seen. x 21,300  118.  Figure 20 The Iris in Pupillary Dilation (TEM) The dilator cells (d) are so complexly interdigitated and interrelated with each other that the c e l l boundaries are not observable. The nucleus (Nd) has a highly irregular and indented outline. Dense heterochromatin is mainly associated with the nuclear envelope. The cytoplasm is very electron dense, especially in the region of the dilator hillocks (dh) and dilator processes (dp). A l l along the stromal boundary of the dilator, hillocks of dilator material are seen. From these hillocks arise a number of complex and profusely branching series of dilator processes which protrude into the stroma. The basement membrane (bm) of the dilator follows the contour of the dilator processes loosely. There is a clear space between the basement membrane and the cell membrane of the dilator cells. Small pinocytotic vesicles (pv) are found in the stromal poles of the dilator cells. Larger vesicles are found deeper in the cell cytoplasm. The i r i s stroma consists of stromal cells (sc), blood vessels (bv) and a network of collagen fibers (co). The collagen fibers are sectioned in a l l planes. Occasionally, the collagen fibers appear to be closely associated with the basement membrane (arrow). The stromal blood vessel shows microvillous processes of the endothelium (mi). x 8,900  119.  120.  Figure 21 The Iris in Pupillary Dilation (TEM) The dilator cytoplasm is very dense. There are few organelles in the stromal parts of the cells except for some mitochondria (m) and vesicles (v). Some myofilaments are seen in one of the dilator hillocks (*). The dilator interdigitations (di) are complex and i t is d i f f i c u l t to decipher whether the interdigitations belong to two different cells or to parts of the same c e l l . The dilator processes may be simple protrusions of the dilator into the stroma (dpi), or they may arise from a dilator hillock (dh) and branch (dp2). The basement membrane (bm) loosely follows the outlines of the dilator processes. The endothelial cells of the blood vessels have numerous microvilli (mi). Stromal cells and collagen fibers (co) make up part of the stroma. x 21,300  .122.  Figure 22 The Iris in Pupillary Dilation (TEM) There are numerous small pinocytotic vesicles in the dilator processes (pvl) as well as in the dilator hillock (pv2). The characteristic features of the dilator processes (dp) and the dilator hillocks (dh) have been described. The nuclei (Nd) show irregular outlines. Apparent fusions of the c e l l membranes of two adjacent dilator cells are seen (arrow). The well-fixed mitochondria (m) look delicate. x 21,300  123.  124.  Figure 23  The Iris in Pupillary Dilation (TEM) The interdigitations of the dilator cells (di) are clearly shown. Microvillous cytoplasmic c e l l processes interdigitate loosely in a complicated three dimensional network. The nucleus of the dilator (Nd), mitochondria (m) and pinocytotic vesicles are present in the cell cytoplasm. The characteristic branching of the dilator processes (dp) is clearly shown. x 21,300  125.  3.  The Iris in Pupillary Constriction (Figures 24-30)  In pupillary constriction, the posterior epithelial and dilator layers are much thinned out. more clearly seen.  The relationships between the two layers are  Certain changes from that present in pupillary dilation  are observed. The posterior surface of the i r i s is relatively smooth (Figure 24), except for an occasional posteriorly directed bulge (Figures 25-28).  There  are no grooves in between the posterior epithelial cells so that one cell cannot be separated from the next.  The basal cell membranes appear to show  as many infoldings as in pupillary dilation.  The basement membrane forms  a straight covering for the basal surfaces of the posterior epithelial cells.  Within the epithelial cells themselves, the usual organelles are  present, including a large number of vesicles which are distributed through out the c e l l .  The orientation of the organelles within the cells do not  appear to be organised in any particular fashion.  However, the filaments  within the c e l l cytoplasm seem to be aligned parallel to the length of the cells (Figures 24-25, 27-28).  The filaments are longitudinally sectioned.  They run in bundles which may branch (Figures 24, 27) and criss-cross (Figure 27).  These filaments are quite prominently seen in some cells and  less so in others.  The filaments are usually located in the posterior  portions of the cells (Figures 24, 25, 27, 28).  Rarely are filaments seen  in the anterior apical portions of the cells near to the boundary with the dilator.  Sometimes, densities of the cell membranes are seen (Figure 28).  Occasionally, a bundle of filaments appear to attach to the densities of the c e l l membrane (Figure 28). Remarkable changes are seen with respect to the shapes of the nucle of the posterior epithelium and dilator when compared to that observed during pupillary dilation.  The nuclei of the posterior epithelial cells  are oval or elongated  (Figures 25, 26, 29). The length of the nuclei are  oriented parallel to the length of the cells and the posterior surface of the i r i s .  The nuclear outline is relatively smooth. There are no indenta-  tions of the nuclear envelope. At the boundary between the posterior epithelium and the dilator, there are a number of c e l l junctions.  The cell membranes may only come  close together (Figure 25), or the membranes may appear to fuse (Figure 24, 26) without any specialisation of the adjacent cytoplasm.  Occasionally,  the c e l l membranes are in close apposition and there is an apparent increased density in the surrounding cytoplasm (Figure 24). somes are not observed in the rat i r i s .  However, desmo-  Oftentimes, in this boundary region  there are a number of vesicles (Figures 24, 26, 29). Also, microvillous cytoplasmic processes from both the posterior epithelial and dilator layers interdigitate with each other (Figures 25, 29). In many instances, i t seems as i f the interdigitations occur in a plane parallel to the posterior surface of the i r i s . The dilator is also thin (Figures 24-26, 29). There is l i t t l e cytoplasm in comparison to the size of the nucleus. are present.  The usual organelles  The nucleus of the dilator becomes long and thin and the  length of the nucleus is also parallel to the length of the cells.  The  nuclear outline is smooth and usually shows no indentations (Figures 24, 30) Occasionally, the nuclear envelope may be indented and a spit of cytoplasm is seen occupying the space (Figure 25).  But when this occurs, the nuclear  indentation is parallel to the posterior surface of the i r i s (Figure 25). The muscular portion of the dilator cells is confined to a small region to the basal poles of the cells (Figures 24-26, 29, 30). The sarcoplasm is not as dense as in pupillary dilation.  The myofilaments of the  dilator are seen running along the length of the stromal poles of the cells  (Figures 24-26, 29, 30).  Sometimes, bundles of filaments are seen towards  the apical poles of the cells (Figure 29).  The dilator may consist of one  layer (Figures 25, 26, 29), or i t may consist of stacks of a few layers which show interdigitations with each other (Figures 24, 30).  The nucleus  of the dilator layer is usually surrounded by cell cytoplasm and cell organelles (Figures 24-26). in myofilaments  (Figure 30).  myofilamentous region.  Sometimes, however, the nucleus is embedded Numerous mitochondria are often present in the  In addition, there are lots of pinocytotic vesicles  within the sarcoplasm and along the cell membranes (Figure 30).  Some of  the pinocytotic vesicles may be open to the outside. The anterior stromal surface of the dilator is normally quite smooth (Figures 24-26).  Right at the boundary with the stroma, the cell  membrane sometimes appears dense (Figures 24-26). processes are very rarely seen (Figure 29).  They are only present in iso-  lated spots and are very simple in configuration. processes jut into the stroma.  Arborescent dilator  Small and simple dilator  They do not show the complex branching  that is observed in pupillary dilation.  If there is a pile up of muscle  spurs (Figure 30), the anterior surface of the dilator is no longer smooth. The basement membrane of the dilator is not as easily visible (Figures 24-26) except where the small dilator processes protrude into the stroma (Figure 29).  Collagen fibers are also not as apparent as in  pupillary dilation.  When present, they tend to be oriented parallel to  the length of the i r i s .  129.  Figure 24 The Iris, in Pupillary Constriction (TEM) Both the posterior epithelial (pe) and dilator (d) layers are thin. The posterior surface of the i r i s is smooth and is covered by a loosely adherent basement membrane (bm). The c e l l infoldings (ci) of the posterior epithelium are present. In the cytoplasm of the posterior epithelial cells, there are mitochondria (m), vesicles and intracellular filaments ( i f ) . The posterior epithelium and dilator are joined by apparent fusions of the cell membranes (double arrows). Occasionally, the cell membranes are close together and there is some modification of the immediately adjacent cytoplasm (single arrow). Almost a l l of the dilator c e l l is occupied by the cigar-shaped nucleus (Nd). The anterior surface of the dilator is a smooth line. x 21,600  131.  Figure 25 i ]  The Iris in Pupillary Constriction (TEM) There is an occasional bulge of the posterior surface of the i r i s . Cell infoldings (ci) are numerous. Intracellular filaments (if) are found in the posterior portion of the cells and are closely associated with the nucleus (Npe). The nucleus of the posterior epithelium is oval in shape and has a smooth outline. The nucleus of the dilator (Nd) shows a deep indentation parallel to the length of the i r i s . Microvillous cytoplasmic processes from both layers interdigitate with each other (*). The contractile myofilamentous portion of the dilator (arrow) is only a thin strip at the stromal pole of the c e l l . There may be slight condensation of the cell membrane a t the stromal boundary. The basement membrane (bm) is perceptible. It is straight, as is the stromal surface of the dilator. x 21,600  132.  133.  Figure 26  The Iris in Pupillary Constriction (TEM) The nucleus of the posterior epithelium (Npe) is smooth and oval in shape. There is a clear peri-nuclear space (arrow). Mitochondria (m) and c e l l infoldings (ci) are confined to the posterior portions of the cells. The cell membranes of the posterior epithelium (pe) and of the dilator (d) fuse. Large vesicles (v) are present in this boundary zone. The stromal surface of the dilator is a smooth dense line. Myofilaments in diverging bundles are present in the stromal cytoplasm. x 21,600  Figure 27  The Iris in Pupillary Constriction (TEM) Cell infoldings (ci) occupy most of the superficial parts of the posterior aspect of the posterior epithelium (pe). In the posterior cytoplasm, there are large bundles of intracellular filaments (if) which run parallel to the posterior surface of the i r i s . The filaments may branch or they may come together and intermesh. x 27,000  135.  Figure 28 The Iris in Pupillary Constriction (TEM) The intracellular filaments (if) run in bundles parallel to the posterior surface of the i r i s . They sometimes appear to attach to dense areas on the c e l l membrane of the posterior epithelium (arrow). A golgi apparatus (go) and c e l l infoldings (ci) are also seen. x 21,600  Figure 29 The Iris in Pupillary Constriction (TEM) The anterior surface of the dilator (d) is not smooth. There are, instead, a few dilator processes (dp). These are relatively simple protrusions of the dilator into the stroma. The myofilaments of the dilator are seen as longitudinal bundles running along the length of the cells (*). The myofilaments are found in the stromal as well as in the deeper parts of the c e l l . The basement membrane (bm) is loosely adherent to the dilator layer. There appears to be a wider space between the basement membrane and the dilator processes than between the basement membrane and the smooth part of the dilator. x 21,600  136.  137.  Figure 30  The Iris in Pupillary Constriction (TEM) Occasionally, there are stacks of dilator muscle spurs f i l l e d with myofilaments, mitochondria (m) and pinocytotic vesicles (pv) „ Some of the pinocytotic vesicles are open to the intercellular space (arrows). The elongated nucleus of the dilator (Nd) is embedded in myofilaments. x 21,600  138.  139.  D.  A Scanning; Electron Microscopic Study of the Rat Iris in Pupillary Dilation and Constriction  1.  General  The iris tissues are either only fixed in glutaraldehyde, or in glutaraldehyde followed by post-fixation in osmium tetroxide.  Then, both  the camphene method (Watters and Buck, 1971) and the c r i t i c a l point drying method (Boyde and Wood, 1969; Smith and Finke, 1972) are used to prepare the specimens for examination with the scanning electron microscope.  It is  found that these differing methods of preparing the i r i s tissues do not result in different images obtained with the scanning electron microscope.  2.  The Posterior Surface of the Iris in Pupillary Dilation (Figures 31-38)  In a low magnification scanning electron micrograph, the posterior surface of the iris is seen as a circumferentially grooved surface (Figure 31).  Peripherally, the c i l i a r y processes overhang the root of the i r i s .  At times, zonular fibers attaching the c i l i a r y processes to the lens capsule are observed on the posterior iris surface (Figure 32).  At the pupil-  lary margin, the posterior surface is relatively flat and smooth (Figure 31).  Occasionally, there may be some hints of a few shallow ridges and  grooves which are not distinctly visible (Figure 31). though, is deeply grooved. ial cells (Figure 32). around the pupil.  The rest of the i r i s ,  The ridges represent rows of posterior epithel-  The ridges are not uniformly concentrically arranged  Rather, a ridge may bifurcate, taper down, or blend with  another ridge of cells (Figures 33, 34).  These epithelial ridges appear  rounded, bulging into the posterior chamber.  An amorphous basement membrane  layer covers a l l of the posterior surface of the epithelial cells so that  the cell boundaries are obscured. thelial cells are spindle-shaped  According to Hansson (1970), the epior polyhedral.  However, with our prepara-  tions of the i r i s in pupillary dilation, the margins between individual cells in an epithelial ridge are not discernible. In man,  the cells within  any one ridge are arranged in a staggered fashion (Fine and Yanoff, 1972). The basement membrane covering the posterior epithelial cells is not smooth but shows bumps and large and small corrugations.  This unevenness of the  basement membrane may give a suggestion of the numerous infoldings, interdigitations and grooves of the posterior surface of the individual epithelial cells, as is readily verified in meridional sections of the rat i r i s in pupillary dilation observed with the light microscope. However, the basement membrane does not extend deeply into the row of cells but covers them superficially.  Occasionally a much larger ridge is seen ex-  tending from the base of the c i l i a r y processes to midway in the i r i s . This ridge is radially rather than circumferentially oriented (Figure 34) and i t is larger than the circumferential ridges.  The circumferential  ridges do not extend over this bump but skirt around i t , or there might be a break in the continuity of the epithelial ridges (Figure 34). The grooves in between the epithelial ridges are deep and of varying widths and depths.  Like the epithelial ridges, the grooves also bifur-  cate or blend into one another (Figures 33, 34).  At the large c i l i a r y - i r i s  ridge or process (as i t w i l l be referred to here), both the epithelial ridges and the intervening grooves are absent. The epithelial ridges are not always rounded posteriorly. Sometimes they are a l i t t l e flattened and cordlike (Figures 35, 36).  In lesser  degrees of dilation, the distinctions between the epithelial ridges and grooves are not as clear-cut although they are s t i l l visible (Figures 37, 38).  The ridges are not as high and the grooves not as deep.  The  141.  cr ink 1 i rigs of the basement membrane always follow the circumferential direction of the ridges and grooves. Occasional spindle shaped bulges are seen along the ridges.  These probably represent the nuclei of the posterior  epithelial cells (Figure 37). Sometimes wandering cells, presumably white blood cells, are seen attached to the posterior surface of the i r i s (Figure 38). They are large and may span one or two ridges of epithelial cells.  142.  Figure 31  The Posterior Surface of the Iris in Pupillary Dilation (SEM) In a low magnification scanning electron micrograph, most of the posterior surface of the i r i s , except for the pupillary margin, is circumferentially grooved. Around the pupillary margin, the surface is relatively smooth but for a few shallow, circumferentially disposed striations. The c i l i a r y processes overhang the periphery of the i r i s . x 280  Figure 32 The Posterior Surface of the Iris in Pupillary Dilation (SEM) Zonular fibers are sometimes encountered. The epithelial ridges represent rows of posterior epithelial cells covered by a basement membrane. These ridges are rounded. The ridges may bifurcate, taper down or join with adjacent ridges of epithelial cells. The ridges are separated by grooves. x 500  144.  Figure 33 The Posterior Surface of the Iris in Pupillary Dilation (SEM) At a higher magnification, the.epithelial ridges are found to be separated by deep grooves. The basement membrane covering the epithelial ridges dips deep into the grooves. There are bumps and corrugations of the basement membrane giving a suggestion of the infoldings and interdigitations of the posterior epithelial cells. x 2,100  Figure 34 The Posterior Surface of the Iris in Pupillary Dilation (SEM) A c i l i a r y - i r i s process is seen here as a radial ridge perpendicular to the circumferential direction of the posterior epithelial ridges. The radial ridge is much larger and higher than the posterior epithelial ridges. The continuity of the rows of posterior epithelial cells is broken or they may skirt around the edge of the radial ridge. x 1,000  145  146.  Figure 35 The Posterior Surface of the Iris in Pupillary Dilation (SEM) The circumferential epithelial ridges are more cord-like rather than rounded (compare with Figures 32-34). x 620  Figure 36 The Posterior Surface of the Iris in Pupillary Dilation (SEM) The grooves in between the posterior epithelial ridges are deep and narrow. The epithelial ridges have a more cord-like appearance. The basement membrane covering the epithelial ridges is.relatively smooth. Occasional bulges along the row of cells may be due to the outward bulging of the nuclei. x 1,260  148.  Figure 37  The Posterior Surface of the Iris in Pupillary Dilation  (SEM)  The epithelial ridges are not as high nor are the intervening grooves as deep. The continuity of the epithelial ridges is not as obvious. There are occasional spindle bulges of the nuclei of the posterior epithelial cells. The crinklings of the basement membrane covering the epithelial ridges are also circumferentially oriented. x 780  Figure 38  The Posterior Surface of the Iris in Pupillary Dilation  (SEM)  Large wandering cells, presumably white blood cells, are seen on the posterior surface of the i r i s . The epithelial ridges are of varying heights. The ridges divide and rejoin. x 1,560  149.  150.  3.  The Posterior Surface of the Iris in Pupillary Constriction (Figures 39-56)  The posterior surface of the i r i s is observed in two degrees of pupillary constriction.  This is only a qualitative distinction based on  some of the morphological  characteristics that are present.  In one condi-  tion, referred to here simply as pupillary constriction, the pupil is constricted such that the pupillary diameter is about 2/7 of the total i r i s diameter (Figures 39, 40).  In another condition, referred to as extreme  pupillary constriction, the pupil is very small and makes up only about of the total i r i s diameter (Figures 41, 42).  1/14  These two aspects of the  posterior surface of the i r i s will be treated separately. In pupillary constriction, the posterior i r i d i a l surface is a flat, smooth, circular structure with an aperture, the pupil, situated in the center (Figures 39, 40).  In a low magnification scanning electron micro-  graph, the pupillary margin is smooth (Figure 39), but at a slighter higher magnification, a few blebs may be seen along parts of the pupillary margin (Figure 40).  Not much detail of the rest of the i r i s surface is discernible.  There are some tube-like structures which radiate from the pupillary margin to the region of the c i l i a r y processes  (Figure 39).  At a higher magnifica-  tion, i t is seen that these are actually capillaries running along the posterior surface of the i r i s . observed or reported.  These vessels have not been previously  The beginning and termination of these vessels are  not well defined (Figure 40).  These vessels could presumably be remnants  of the peri-natal circulatory system. present varies from i r i s to i r i s .  The number of these vessels that are  At the periphery of the i r i s , in the  region of the c i l i a r y body, there are numerous c i l i a r y - i r i s (Figures 39, 43, 44). the rim of the i r i s .  connections  They seem to be quite regularly spaced out along The lengths of these processes may differ very  151.  slightly.  They are tissue processes which, perhaps, anchor the c i l i a r y  processes to the i r i s .  Near the c i l i a r y body, i t is superficial to the rest  of the i r i s surface but a l i t t l e centrally, the process goes into the depths of the iris tissue i t s e l f .  Its presence is only detected as a minor radial  ridge as one moves centrally (Figures 43, 44). here, the c i l i a r y - i r i s  If an analogy is warranted  process is likened to the buttress root of a tree.  It slants up near the trunk but dives into the ground further away. These ciliary-iris  processes perhaps act as stabilisers of the root of the i r i s  during the continuous excursions of the pupil. At a higher magnification, the posterior surface of the i r i s shows slight corrugations and bulges which are generally oriented circumferentially  (Figure 43).  There are no distinct grooves or ridges (Figure 45)  although the posterior epithelial cells appear to be arranged in circumferential rows in some parts of the i r i s (Figure 45) but are more haphazardly arranged in other parts of the i r i s (Figure 46).  Each posteriorly  directed bulge probably represents a single epithelial c e l l .  They are  slightly spindle shaped or polygonal, as has been reported (Hansson, 1970), although the shapes of the cells do vary quite a bit. The posterior basement membrane is probably s t i l l present over most of the posterior surface of the epithelial cells.  However, i t may be thinned out during pupillary  constriction or i t may be removed during the processing of the material. An enormous number of processes are seen radiating out from the individual cells and intermingling with those of the adjacent cells (Figures 46, 47). In extreme pupillary constriction, the pupil appears as a mere pinhole (Figures 41, 42).  From our present observations, we cannot say  whether this degree of pupillary constriction is normally attainable in the usual responses to intense light, or whether i t is only attainable with the aid  of drugs.  Most of the i r i s appears as a smooth sheet except for a  small area around the pupil (Figure 41).  The pupillary edge is not smooth  but presents a highly irregular outline (Figure 42) which is clearly seen in higher magnification scanning electron micrographs (Figures 48, 49).  It  is a purse-string effect, where the string (equivalent to the sphincter) is drawn so tightly that a l l the tissue around the pupillary edge is gathered together.  Numerous ridges and grooves radiate out from the pupillary margin  like the rays of the sun.  These peter out before the mid-iris is reached.  The ridges and grooves are of varying lengths. Right around the rim of the pupil, are big humps of tissue.  This  is probably in the sphincter region and the humps represent bundles of much constricted sphincter muscle cells.  The surface of these humps is rela-  tively smooth with only a small amount of furrowing (Figures 48, 49).  The  pupillary margin is not a single layer but consists of a pile-up of these sphincteric humps (Figures 48, 49).  A capillary is seen extending from  the pupillary edge (Figures 41, 49) to the c i l i a r y region (Figure 41). The capillary lies superficially on the posterior i r i d i a l surface. pupillary margin, i t appears to make a turn anteriorly.  At the  It may perhaps  continue anteriorly to become one of the blood vessels of the i r i s stroma. At a l i t t l e distance from the pupillary edge, the posterior i r i s surface shows a series of grooves and ridges which are radially oriented (Figures 48-50).  The ridges are higher, rounder and closer together cen-  trally but they become broader and lower peripherally. Eventually, the ridges flatten out so that the rest of the i r i s posterior surface is smooth. The grooves in between the ridges are deep and narrow centrally but they become shallower peripherally. These ridges are probably rows of epithelial cells covered by a basement membrane (Figure 50).  Unlike the epithelial  ridges seen in pupillary dilation, these ridges are radially rather than circumferentially oriented.  The ridges bifurcate as well as merge.  153.  Within this zone of epithelial ridges and grooves, are some grotesquely large bulbous structures (Figure 42).  They are very many times  larger than the surrounding epithelial ridges (Figures 51-53).  They may be  round (Figure 51), or they may be drawn out at both ends (Figure 52).  They  look bloated at times (Figures 51, 52), but at times they look collapsed (Figures 51-53). When puffed-up, the surface is relatively smooth (Figures 51, 52), but when collapsed the surface shows some irregularities (Figure 53).  The basement membrane covering the rest of the posterior i r i s surface  also covers these bulbous structures.  The crinkles and folds of the base-  ment membrane over the epithelial ridges are also seen over these bulbous structures.  Thus they are indeed part of the posterior i r i s surface.  Mapstone (1970) suggests that past a certain pupillary size, the pursestring effect of the pull of the sphincter no longer produces further miosis but instead produces "eversion of the pigment epithelium".  These bulbous  structures may be the posterior pigment epithelium of the i r i s which has been crowded out of its normal position with extreme pupillary constriction. The rest of the posterior i r i s surface outside of the central ridged area is smooth and quite non-descript  (Figures 54-56).  The c i l i a r y -  i r i s process may cause a slight, low, wide ridge to be seen at the periphery (Figure 54).  Some of the polygonal epithelial cells are distinguishable at  times (Figure 56). pattern.  They are not arranged in any particular order or  Processes of the epithelial cells form a fine meshwork (Figure 55).  At other parts of the i r i s , the surface is very smooth (Figure 56).  Cell  boundaries cannot be traced but slight variations in the contour of the surface suggest where the outlines of the cells may be (Figure 56). cipitated proteinaceous  Pre-  material is oftentimes present on the i r i s surface.  154.  Figure 39  The Posterior Surface of the Iris in Pupillary Constriction (SEM) At a low magnification, the posterior surface of the i r i s in pupillary constriction is smooth. The pupil is round. There are a few tube-like structures spanning the posterior surface of the i r i s from the pupillary edge to the c i l i a r y region. A l l along the c i l i a r y region at the periphery of the i r i s are a series of c i l i a r y - i r i s processes. x 20  Figure 40  The Posterior Surface of the Iris in Pupillary Constriction (SEM) A high magnification of the pupillary margin shows that the margin is relatively smooth except for a few blebs. The tubelike structures seen in Figure 39 are capillaries which seem to arise from around the pupillary margin. x 50  155.  156.  Figure 41 The Posterior Surface of the Iris in Extreme Pupillary tion (SEM)  Constric-  In extreme pupillary constriction, the posterior surface of the i r i s is smooth except for a small portion around the pin-hole pupil. x 20  Figure 42 The Posterior Surface of the Iris in Extreme Pupillary tion (SEM)  Constric-  A high magnification electron micrograph of the pupillary region shows that the pupillary margin is irregular in outline. There are some humps right around the pupillary rim. Epithelial ridges and grooves and a capillary radiate outwards from the pupil. In amongst the epithelial ridges and grooves are numerous bulbous structures of different sizes. x 170  157.  158.  Figure 43  The Posterior Surface of the Iris in Pupillary Constriction (SEM) In partial pupillary constriction, the posterior surface of the i r i s shows corrugations and bulges which are generally oriented circumferentially. At the c i l i a r y region, there are c i l i a r y iris processes of differing lengths. x 200  Figure 44  The Posterior Surface of the Iris in Pupillary Constriction (SEM) A high magnification electron micrograph of the c i l i a r y - i r i s process shows that i t stands high above the posterior surface of the i r i s at the ciliary region. It then dives deep into the i r i s tissue i t s e l f more centrally. x 500  159.  160.  Figure 45 The Posterior Surface of the Iris in Pupillary Constriction (SEM) The posterior epithelial cells are irregularly shaped bulges which are organised in some semblance of circumferential rows. x 960  Figure 46 The Posterior Surface of the Iris in Pupillary Constriction (SEM) The posterior epithelial cells are haphazardly arranged. Occasionally some fine interdigitations are seen, x 960  161.  162.  Figure 47 The Posterior Surface of the Iris in Pupillary Constriction (SEM) Where the basement membrane has been thinned out or has been removed, a large number of c e l l processes are observed. x 960  Figure 48 The Posterior Surface of the Iris in Extreme Pupillary Constriction (SEM) In extreme pupillary constriction, the pupillary edge of the i r i s consists of many layers of humps of tissue. These probably represent bundles of sphincter muscle cells. The surface of the humps is smooth. Numerous epithelial ridges radiate from the sphincter region. The ridges are high and rounded near the sphincter but they decrease in height peripherally. x 430  163.  164.  Figure 49 The Posterior Surface of the Iris in Extreme Pupillary Constriction (SEM) The sphincter humps at the pupillary margin have a smooth posterior surface with only a few fine striations. The capillary lies superficial to the posterior surface of the i r i s . At the pupillary edge, i t appears to turn anteriorly. x 760  Figure 50 The Posterior Surface of the Iris in Extreme Pupillary Constriction (SEM) The radial epithelial ridges show numerous branchings. The basement membrane covering the posterior surface of the epithelial cells is highly wrinkled. x 3,900  166.  Figure 51  The Posterior Surface of the Iris in Extreme Pupillary Constriction (SEM) In amongst the radial epithelial ridges are bulbous structures of different shapes and sizes. They are large and bloated or small and collapsed. x 760  Figure 52  The Posterior Surface of the Iris in Extreme Pupillary Constriction (SEM) The bulbous structures are sometimes drawn out at both ends. It is puffed-up. The posterior surface of the structure is relatively smooth. x 760  167.  168.  Figure 53 The Posterior Surface of the Iris in Extreme Pupillary Constriction (SEM) The bulbous structures are sometimes collapsed. The basement membrane over the bulbous structures is continuous with the basement membrane covering the rest of the epithelial cells. x 1,570  Figure 54 The Posterior Surface of the Iris in Extreme Pupillary Constriction (SEM) The posterior surface outside of the pupillary region is smooth. A few low ridges are caused by the c i l i a r y - i r i s processes, x 90  170.  Figure 55 The Posterior Surface of the Iris in Extreme Pupillary Constriction (SEM) In parts of the posterior surface of the i r i s , there are numerous, haphazardly arranged polygonal bulges of the posterior epithelial cells. Two delicate blood vessels traverse the posterior surface of the i r i s . x 430  .Figure 56 The Posterior Surface of the Iris in Extreme Pupillary Constriction (SEM) In extreme pupillary constriction, many parts of the posterior surface of the i r i s is smooth and non-descript. An occasional bulge suggests the location of the underlying nuclei of the epithelial cells. x 3,900  172.  4.  The Anterior Surface of the Iris in Pupillary Dilation (Figures 57-65)  The most distinctive feature of the anterior surface of the i r i s in pupillary dilation is the large number of blood vessels which bulge out anteriorly from the i r i s surface (Figures 57-59).  Usually, the anterior  surface can be divided into two zones, the central half consists of a series of small-sized blood vessels with intervening crypts, while the peripheral half consists of a mass of large blood vessels of varying c a l i bers (Figures 58, 59). (Figure 59).  The crypts in the medial half may be quite distinct  However, with a greater degree of pupillary dilation, this  zone is telescoped into the peripheral zone (Figure 58) until eventually the anterior i r i s surface only appears as a mass of large blood vessels (Figure 57).  The pupillary margin, i f observed edge on, is quite smooth as  compared to the rugged contours of the rest of the i r i s surface (Figure 60). The i r i s blood vessels assume different configurations although the overall general orientation is circumferential. Occasionally, a large blood vessel is seen entering the i r i s at its periphery (Figure 57). has a radial course.  It  But shortly after entering the i r i s , i t gives rise to  a large straight vessel at right angles to i t , that is, oriented circumferentially.  This would most probably be the vessel of the major arterial  circle situated at the i r i s root (Figure 57).  The rest of the i r i s vascu-  lature forms an interwoven network of large and small blood vessels.  The  blood vessels may be in the form of an 'X' bifurcating at both ends (Figure 57).  Another vessel may be inserted through the arms of a bifurcation and  join up with one of the branch blood vessels (Figure 58).  At times, a small  branch blood vessel curls around its parent vessel (Figure 59). common feature is exemplified in Figure 61. furcates.  A quite  Here a large blood vessel bi-  Shortly afterwards, one of its branches bifurcates again while  the other branch dives deep into the i r i s stroma. figuration is also quite common (Figure 62).  The 'tuning fork' con-  The two branch blood vessels  are just as large, i f not larger, than the originating blood vessel. The junction of these three blood vessels usually bulges out prominently anteriorly.  A row of these may be seen from the periphery medially, giving the  appearance of a row of sand-dunes. zags its way across the i r i s . (Figure 63).  It is very rare that a blood vessel zig-  But when i t does, i t is very distinctive  The path of the zig-zag is not directly perpendicular to the  pupillary margin but rather, i t is slightly askew. From light microscopic histological studies, i t is known that the i r i s blood vessels are not freely exposed to the aqueous humor in the anterior chamber. surface.  There is an endothelial lining covering the anterior  The endothelial cells are not easily visible, but occasionally,  an endothelial c e l l nucleus is seen to bulge anteriorly (Figure 64). blebs and microvillus-like processes are seen on the c e l l surface. outlines of the cells are not clear but can be faintly made out. boundary with an adjacent c e l l is not a tight adhesive zone. consists of an intermingling of cytoplasmic c e l l processes.  Little  The  The  It merely These features  are observed in much better detail when the anterior surface of the i r i s is examined in pupillary constriction. At times, a sieve-like structure is observed over one of the bulging blood vessels (Figure 64).  It is d i f f i c u l t  to say whether this is formed at the junctions of a few endothelial cells, or whether this sieve is within the confines of the ctyoplasm of a single endothelial c e l l .  Whatever i t may be, i t certainly exposes the underlying  blood vessel more directly to the circulating aqueous humor. The blood vessels in the central half of the i r i s are small and are presumably capillaries (Figures 58, 59). They do not bulge out as much anteriorly although their course can s t i l l be charted.  Most of them form  an interconnecting series of X's.  Towards the pupillary margin, most of  these smaller blood vessels sometimes drain into a larger cord-like blood vessel which follows the pupillary margin (Figures 58, 59).  It is not as  large as the vessels in the lateral extent of the i r i s but i t is s t i l l of a considerable size.  In this capillary zone, there are a large number of  relatively large crypts (Figures 59, 65).  The crypts are usually found in  between the arms of a bifurcating set of capillaries (Figure 65). crypt is not just one large orifice.  A series of c e l l processes divide up  the opening into a series of smaller apertures. mesh over the crypt opening.  The  It appears like a wire  Deeper down, other cell processes, belonging  to the underlying stromal cells, are seen.  175.  Figure 57  The Anterior Surface of the Iris in Pupillary Dilation (SEM) The anterior surface of the i r i s consists of a mass of large blood vessels bulging anteriorly. The blood vessels are generally circumferentially oriented. A large blood vessel is seen entering the i r i s at the periphery and immediately dividing. Other blood vessels may form an X which bifurcates at both ends. x 190  Figure 58  The Anterior Surface of the Iris in Pupillary Dilation (SEM) The anterior surface of the i r i s can be divided into two regions, the pupillary region with a series of small, blood vessels, and the peripheral region with a series of larger blood vessels interwoven in a complicated network. In general, the blood vessels are circumferentially disposed. Along the pupillary edge, there is a medium-sized, cord-like smooth blood vessel. x 250  176  177.  Figure 59  The Anterior Surface of the Iris in Pupillary Dilation  (SEM)  The cord-like blood vessel along the pupillary edge is connected to a series of smaller blood vessels which form X's with each other. Sometimes a small branch blood vessel may curl around its parent vessel. In the pupillary region there are many crypts. x 260  Figure 60  The Anterior Surface of the Iris in Pupillary Dilation  (SEM)  The pupillary margin viewed edge on is smooth. The blood vessels near the pupillary margin are small and are generally circumferentially arranged. The blood vessels in the other parts of the i r i s protrude quite prominently anteriorly. They appear highly tortuous. x 280  179.  Figure 61  The Anterior Surface of the Iris in Pupillary Dilation  (SEM)  A blood vessel bifurcates. One of its branches dives deep into the i r i s tissue while the other branch continues on to form another bifurcation. x 530  Figure 62  The Anterior Surface of the Iris in Pupillary Dilation  (SEM)  The blood vessels are shaped like a tuning fork where each of the branch blood vessels is as large as the parent vessel. x 800  181.  Figure 63  The Anterior Surface of the Iris in Pupillary Dilation  (SEM)  One of the blood vessels protruding out anteriorly is zigzagging obliquely across the anterior surface of the i r i s . x 260  Figure 64  The Anterior Surface of the Iris in Pupillary Dilation  (SEM)  A sieve-like structure covers the surface of a blood vessel which is bulging out anteriorly. x 1,780  182.  183.  Figure 65  The Anterior Surface of the Iris in Pupillary Dilation (SEM) The i r i d i c crypts in the pupillary region are often located between the arms of a bifurcating blood vessel. A series of c e l l processes form a wide mesh over the crypt opening. x 670  184.  185.  5.  The Anterior Surface of the Iris in Pupillary Constriction (Figures 66-76)  In a low magnification scanning electron micrograph, the anterior surface of the i r i s is large, relatively smooth and slightly pock-marked, especially towards the pupillary zone (Figure 66).  Some of the blood  vessels, presumably only the larger ones, are readily seen. erally oriented radially.  The blood vessels wind their way  fashion, from the periphery centrally.  They are genin a serpentine  At the periphery, the zig-zag course  of the blood vessels is very prominent (Figures 66, 67).  Often, i t appears  that branches are given off at each external bend of the parent vessel and these plunge into the deeper parts of the i r i s stroma. (Figure 67).  The  smaller blood vessels are not easily seen although they too seem to have a twisting course.  However, they tend to go deep and disappear.  They may  perhaps reappear again some distance away (Figures 66, 67). In the pupillary region, the blood vessels are much more in evidence (Figures 68, 69).  They are tortuous and form a complicated pattern.  twist and turn and divide. vessel.  They  It is d i f f i c u l t to follow the course of any one  Sometimes, they may appear to form arcades which go over and  brace the pupillary margin i t s e l f (Figure 69). there are always holes and spaces.  em-  In between the blood vessels,  These are the i r i d i c crypts, which are  of varying sizes (Figure 68). The detailed surface structure of the i r i s can only be studied at higher magnifications.  The anterior surface is covered by a thin endo-  thelial layer (Figure 70). in shape.  The endothelial cells are variously polygonal  The cell size differs between the individual cells, but the  nuclear size is relatively constant.  The nucleus is oval in shape.  It is  usually situated in the centre of the cell mass and bulges a l i t t l e anteriorly into the anterior chamber.  Thus, the whole anterior i r i d i a l surface  has a slightly undulating aspect to i t . cells are quite well-defined. studies (1970).  The boundaries of the endothelial  This is not so clearly shown in Hansson's  This difference could be the result of the different  methods used in preparing the tissues for examination with the scanning electron microscope.  Each endothelial cell may be associated with its  neighbor by various means.  But in a l l cases, there is no tight adhesive  boundary between adjacent endothelial cells. 70-72), cytoplasmic processes gap (Figures 70-72).  In certain instances (Figures  from adjacent cells bridge the intervening  The intercellular gap may be large (Figures 70, 71),  or i t may be quite small (Figure 72).  The number of cytoplasmic  processes  spanning the intercellular gap may be numerous (Figure 71), or i t may be few in number (Figure 72).  Sometimes tiny holes are present within the endo-  thelial c e l l cytoplasm (Figure 71), but at other times the c e l l surface is relatively smooth and non-pitted (Figures 72, 73).  Another configuration  often seen at the boundary zone between the endothelial cells is the series of large holes or crypts (Figures 72-74).  The crypts are of varying sizes.  Their relative sizes can be determined by u t i l i s i n g the adhering red blood cells as a comparative means of measure (Figure 74).  The crypts are rela-  tively well-defined structures, as compared to the holes seen by Hansson (1970).  They are usually irregularly round or oval in shape.  The borders  of the crypts are quite smooth although occasionally a few small blebs be present.  Sometimes cytoplasmic processes  may  from the endothelial cells  making up the crypt border span the openings of the crypts.  A network of  the cytoplasmic prolongations of the underlying stromal cells are seen at a slightly deeper level. entry.  These are the cellular components beneath the crypt  A fine f i b r i l l a r material is sometimes seen in addition to the  stromal c e l l processes  (Figure 73).  At times, the f i b r i l l a r material may  occupy the totality of the crypt opening (Figure 75).  The i r i d i c crypts  are often found quite closely associated with blood vessels (Figure 76). The proximity of the crypts to the blood vessels, may facilitate any inter' change of materials that might ensue between the i r i s blood vessels and th surrounding aqueous humor.  188.  Figure 66 The Anterior Surface of the Iris in Pupillary Constriction (SEM) A low magnification scanning electron micrograph shows that the anterior surface of the i r i s is pock-marked. Numerous blood vessels zig-zag from the periphery to the pupillary edge. x 90  Figure 67  The Anterior Surface of the Iris in Pupillary Constriction (SEM) At a higher magnification, the zig-zagging-blood vessels are seem to be of different sizes and they are found at different depths in the i r i s tissue. At each external bend of the parent blood vessel, branches appear to be given off, which then immediately dive deep into the i r i s tissue. x 180  189.  190.  Figure 68  The Anterior Surface of the Iris in Pupillary Constriction (SEM) The blood vessels at the pupillary region form a complicated pattern. In between the blood vessels, there are holes and crypts of various sizes. x 460  Figure 69  The Anterior Surface of the Iris in Pupillary Constriction (SEM) Some of the blood vessels at the pupillary region form arcades which embrace the pupillary margin i t s e l f . In between the vascular arcades, i r i d i c crypts and pores are found. x 460  192.  Figure 70 The Anterior Surface of the Iris in Pupillary Constriction (SEM) The nuclei of the anterior endothelial cells are oval in shape and they bulge slightly anteriorly. The nuclei are located in the middle of the polygonal cells. The anterior endothelial cells do not form a continuous covering for the anterior surface of the i r i s . Spaces and pores are present in between the individual cells. x 1,800  Figure 71 The Anterior Surface of the Iris in Pupillary Constriction (SEM) A single anterior endothelial c e l l is shown in this micrograph. The oval nucleus, in the middle of the c e l l , bulges slightly. The cytoplasm shows other small bulges, presumably, of the underlying c e l l organelles. There are some fine holes present in the c e l l cytoplasm. A gap of varying widths separates the anterior endothelial c e l l from its neighbors. A large number of cytoplasmic processes from this and adjacent cells span the gap. x 4,600  193.  194.  Figure 72  The Anterior Surface of the Iris in Pupillary Constriction (SEM) The intercellular gaps between the anterior endothelial cells are sometimes small so that the endothelial cells appear to be joined together by loose seams. Crypts, with relatively welldefined margins, are numerous. They are quite often close to a blood vessel. Below the crypt opening, cytoplasmic processes of the stromal cells are present. x 1,800  Figure 73  The Anterior Surface of the Iris in Pupillary Constriction (SEM) A single endothelial c e l l , as identified by its nucleus, is almost totally surrounded by a series of crypts. The crypts are round. The margins are smooth and well-defined. Cytoplasmic processes of the underlying stromal cells and a dense network of f i b r i l l a r material is seen through the crypt openings. x 4,100  196.  Figure 74  The Anterior Surface of the Iris in Pupillary Constriction  (SEM)  The iridic crypts are of different sizes. The erythrocyte can be used as an indication of the relative sizes of the crypts. x 1,600  Figure 75  The Anterior Surface of the Iris in Pupillary Constriction (SEM) A very large crypt opening is completely f i l l e d with f i b r i l l a r material. x 4,100  197.  198.  Figure 76  The Anterior Surface of the Iris in Pupillary Constriction Iridic crypts are very often associated with blood vessels. x 1,800  f  (SEM)  200.  E.  A Light Microscopic Study of the Development of the Rat Iris Using Toluidine Blue Stained Plastic Sections  1.  19 Days Fetal (Figures 77-79)  At 19 days fetal, the c i l i a r y body and i r i s are not too well developed as yet.  In meridional sections the i r i s and the c i l i a r y body to-  gether form a cone-shaped structure with its broad base capping the end of the retina (Figures 77-79).  The c i l i a r y body may be seen as a slight bulge  interposed between the retina and the developing i r i s , or, at times there may be the faintest hint of a dip between the retina and c i l i a r y body, and between the c i l i a r y body and the i r i s , thus demarcating the three regions (Figure 78).  However, most often one region just flows onto the next  (Figure 77).  Therefore, posteriorly there is a smooth line from the retina  through the would-be c i l i a r y body and onto the i r i s . The c i l i a r y body may not be grossly but i t is cytologically delineated from the retina.  There is a definite line of demarcation between  the cells which are differentiating into the retina and those which are differentiating into the c i l i a r y epithelium of the c i l i a r y body (Figure 78). This is apparent in toluidine blue stained epon sections but not in hematoxylin and eosin stained paraffin sections. Viewed as a whole in meridional sections, the c i l i a r y epithelium is conical shaped with a wide base at the retinal junction and the apex directed towards the i r i s (Figures 77-79).  At the ciliary-retinal  there is a thick stratified mass of cells.  This tapers down centrally to  become a simple columnar epithelium at the c i l i a r y - i r i s junction. at the ciliary-retinal  junction  Unlike  junction where the cells of the developing retina  and those of the developing c i l i a r y epithelium are cytologically different, there is not a marked difference between the cells of the c i l i a r y epithelium  and those of the posterior epithelium of the i r i s .  Thus the epithelium is  arbitrarily assigned as being part of the c i l i a r y body or part of the i r i s . At the retinal junction, the c i l i a r y epithelial cells are closely packed together in long vertical rows (Figures 77, 78). The nuclei are generally small, elongated and tapered at both ends. chromatin specks and nucleoli. whole of the cells.  They are darkly staining and contain fine  These elongated nuclei occupy practically the  Oftentimes mitotic figures are seen.  More centrally to-  wards the i r i s , the nuclei are larger and are angularly elongated or. polygonal in shape (Figure 79).  They contain nucleoli as well.  regularly disposed within the cells.  The nuclei are i r -  The cells of the c i l i a r y epithelium  when compared to those of the immediately adjacent retina, are particularly sensitive to the fixation and dehydration procedures used in preparing the tissues.  The cytoplasm is highly vacuolated.  Smaller vesicles are present  in the apical (anterior) and basal (posterior) poles of the cells. cytoplasm then appears much darker.  The  Anteriorly next to the pigment epi-  thelium, the c i l i a r y epithelial cells seem to send out cytoplasmic  proces-  ses which invaginate in between each of the pigment epithelial cells, thus giving the anterior surface of the c i l i a r y epithelium a scalloped appearance (Figure 78).  Posteriorly, the surface of the c i l i a r y body has a  slightly frayed look.  Blood vessels, presumably from the hyaloid system,  may be found along the posterior c i l i a r y surface (Figure 78).  Similar  blood vessels are also seen along the posterior surface of the i r i s . The pigment epithelium of the c i l i a r y body, a continuation of the retinal pigment epithelium, may be seen as a long straight columnar epithelium (Figure 77), or the epithelium may be bent into a widely open U or V with the shallow trough of the U or V facing the c i l i a r y stroma anteriorly (Figures 78, 79). At the points where the epithelium bends, there is a piling up of cells and thus the simple epithelial nature of this layer is  lost (Figure 79).  When the c i l i a r y pigment epithelium is an almost straight  columnar epithelium, blood vessels and cells of the c i l i a r y stroma are seen beginning to push into the epithelium (Figure 77).  The slight anterior  concavity of the epithelium is f i l l e d with blood vessels and stromal cells. In practically a l l of the specimens that were studied, the anterior (basal, stromal) surface of the pigment epithelium is very intimately associated with the endothelium of the blood vessels of the c i l i a r y stroma.  The endo-  thelium of the incoming blood vessel closely lines the stromal surface of the pigment epithelium.  There is hardly any visible intercellular gap  between the two components, as seen light microscopically (Figures 78, 79). Neither are there any stromal cells interposed between the endothelium and the pigment epithelium.  In some instances, the endothelium of a large  blood vessel not only lines the anterior surface of the c i l i a r y pigment epithelium but also then extends centrally to line the stromal aspect of the anterior epithelium of the i r i s (Figure 78).  In comparison with the  c i l i a r y epithelium, the pigment epithelium is lightly staining.  Generally,  the nuclei are quite large, oval in shape, pale with fine specks of chromatin and contain nucleoli.  The nuclei do vary in size and shape but this  may be due to the plane of sectioning of the tissue. They are usually placed in the center of the cells but may also be slightly staggered from one cell to the next.  In a few cells the nuclei are seen in mitosis.  The  cytoplasm is uniformly pale with very fine vesicles which are mainly concentrated towards the posterior apical poles of the cells (Figures 78, 79). The stroma of the c i l i a r y body is not very thick.  Half its bulk  consists of incoming blood vessels and the other half of stromal cells which appear to stream into the c i l i a r y body from the cornea-scleral junction.  The blood vessels, although large, have only a lining endothelium  which abuts on the pigment epithelium (Figures 78, 79).  The lumen is  widely patent and may be f i l l e d with blood cells. vessels are also seen within the stroma.  Smaller caliber  blood  Sometimes the c i l i a r y stromal  blood vessels are observed to continue centrally into the stroma of the iris (Figure 78). and cytoplasm.  The stromal cells are small with dark staining nuclei  They are closely packed parallel to each other.  a few intercellular spaces between the stromal cells. fibroblast-like with cigar-shaped nuclei.  There are  These cells are  Some of the stromal cells are  dividing. The surface of the stroma facing the anterior chamber is smooth. It seems to be lined by a layer of squamous cells.  Occasionally a cell  bubbles outwards into the anterior chamber (Figure 78).  This might be just  an artifact of fixation. The i r i s is not always clearly marked off from the c i l i a r y body. It is short.  The developing sphincter, which is a relatively well-defined  egg-shaped bulge capping the extreme pupillary tip of the i r i s , makes up half to one third of the i r i s length (Figures 77-79). The posterior epithelium of the i r i s (Figures 77-79) consists of high columnar cells peripherally near to the c i l i a r y junction. There is a gradual transition to low columnar or high cuboidal cells at the peripheral extent of the developing sphincter. The nuclei are darkly stained and large, occupying most of the c e l l height and width.  Near the c i l i a r y body  where the cells are columnar, the nuclei are long and thin with irregular nuclear outlines and they are stretched out between the poles of the cells. Towards the peripheral edge of the sphincter where the cells are lower, the nuclei are more or less angulated or diamond-shaped.  The cytoplasm is  broken up and lacy throughout the cells except for thin strips of dark staining cytoplasm along the anterior and posterior poles of the epithelium. It also appears that the cytoplasm is more vacuolated in the cells nearer  to the c i l i a r y epithelium.  The posterior epithelial cells form a relatively  smooth lining for the posterior i r i d i a l surface. Unlike the posterior epithelium, the anterior epithelium is an evenly wide, uniformly pale, simple high columnar epithelium, except in parts where there may appear to be two layers of cells (Figures 78, 79). The nuclei are large, pale, oval to round in shape with prominent nucleoli. They are situated in the center or a l i t t l e towards the posterior half of the cells. tions.  The nuclear envelope is smooth or there may be some indenta-  The cytoplasm is pale with some fine vesicles which may be randomly  distributed but are generally slightly concentrated towards the posterior poles of the cells at the boundary zone with the posterior epithelium (Figures 78, 79).  Anteriorly, the stromal surface of the epithelium is  molded and contoured by large stromal blood vessels which also extend centrally over the anterior surface of the developing sphincter.(Figures 78, 79). The developing sphincter is a distinct knob-like or egg-shaped mass of light staining, elongated and centrally tapering cells (Figures 77-79). It is the pupillary (central) expansion of the anterior epithelium of the iris.  The cells may be randomly arranged or they may be oriented towards  the center of the c e l l mass. The developing muscle cells are closely packed together.  Individual cell outlines are not visible.  The nuclei,  like the cells, are elongated and smaller than those of the adjacent anterior epithelium.  The cytoplasm is sometimes slightly finely vesiculated.  The central and anterior surfaces of the developing sphincter presents a smooth outline to the overlying and overhanging stroma.  A small but quite  easily discernible intercellular space separates the smooth surface of the developing muscle tissue from the i r i s stroma (Figure 79).  Over the rest  of the i r i s there is usually no intercellular space between the stroma and  the anterior epithelium (Figure 77). The iris stroma is highly vascular.  In fact, sometimes one large  blood vessel with its patent lumen oriented parallel to the length of the iris occupies most of the extent of the stroma (Figures 78, 79). Unlike other large blood vessels in other locations in the body, or even some of the blood vessels in the adult i r i s stroma, these blood vessels have only an endothelial lining and no other supporting structures.  As before men-  tioned, the endothelium closely lines the stromal surface of the anterior epithelium.  The few stromal cells that are present are small, fibroblast-  like with dark nuclei.  They are usually arranged parallel to each other  and to the posterior i r i s surface.  These stromal cells can also come into  very close contact with the anterior epithelium, where the latter is not lined by endothelium.  The stroma overhangs the extreme pupillary edge of  the developing sphincter and the stromal elements and the blood vessels seem to stream centrally presumably to contribute to the make-up of the pupillary membrane (Figures 77-79). The anterior surface of the i r i s is convex anteriorly so that there is more stromal tissue in the middle of the i r i s .  This surface is rela-  tively smooth except for an occasional bubble-like cell (Figures 73, 79).  206.  Figure 77  19 Days Fetal (LM) The retina, developing c i l i a r y body and i r i s are not grossly well separated from each other. Cytologically, there is a distinct line of demarcation between the cells differentiating into the retina and those differentiating into c i l i a r y epithelium (double arrow). The c i l i a r y epithelium (ce) consists of a stratified mass of cells. The cytoplasm is highly broken up except for a thin strip along the boundary with the c i l i a r y pigment epithelium (cpe) . The c i l i a r y pigment epithelium is a relatively straight, light staining, high columnar layer. A cell is seen in a mitosis (arrow). The posterior surface of the c i l i a r y pigment epithelium is closely adherent to the c i l i a r y epithelium. The contour of the stromal surface is being carved out by the blood vessels (bv). The posterior epithelium (pe) of the i r i s decreases in height from the c i l i a r y junction towards the pupillary margin. As with the ciliary epithelium, the cytoplasm is broken up except for a thin strip between the anterior (ae) and posterior epithelium. The anterior epithelium is a uniformly high columnar pale staining layer. The developing sphincter (sph), a knob-like structure, consists of closely packed cells oriented centripetally. Nucleoli are seen in many of the cells of the c i l i a r y pigment epithelium, the anterior epithelium of the i r i s and the developing sphincter. The c i l i a r y stroma (cs) and i r i s stroma (s) are in continuity. It consists of small blood vessels and stromal cells. The stromal cells are closely adherent to the stromal surface of the anterior epithelium but there is a visible gap between the stroma and the anterior boundary of the sphincter. The anterior and posterior surfaces of the i r i s and c i l i a r y body is relatively smooth. x 220  Figure 78  19 Days Fetal (LM) The developing c i l i a r y body forms a slightly more prominent bulge between the i r i s and the retina. The appearance of the ciliary epithelium (ce), posterior epithelium (pe), anterior epithelium (ae) and developing sphincter (sph) is similar to that in Figure 77. However, the c i l i a r y pigment epithelium (cpe) is bent into a shallow V with the trough of the V being f i l l e d with blood vessels. The endothelium of the blood vessel (bv) in the stroma (cs) is closely apposed to the c i l i a r y pigment epithelium (arrow). The blood vessel of the c i l i a r y stroma extends forwards as the blood vessel lining the anterior'surface of the anterior epithelium of the i r i s (ae). Blood vessels are seen along the posterior surface of the c i l i a r y body (bvl) and past the pupillary tip of the sphincter (bv2) x 220  208.  Figure 79  19 Days Fetal (LM) The histological features of the c i l i a r y epithelium (ce), posterior epithelium (pe), anterior epithelium (ae) and sphincter (sph) are similar to those in Figure 77. The c i l i a r y epithelium (cpe) is bent into a V with a blood vessel occupying the trough (bv). At the crest of the fold of the c i l i a r y pigment epithelium, there is a pile-up of cells. One of the cells of the c i l i a r y pigment epithelium is dividing (double arrow). The cells of the c i l i a r y stroma (cs) stream past into the i r i s stroma (s). Large blood vessels (bv) with the lumens parallel to the length of the i r i s are present. A large blood vessel lines the whole of the stromal surface of the anterior epithelium and of the sphincter. The endothelium (arrow) is adherent to the stromal surface of the anterior epithelium but there is a space between i t and the sphincter (*). x 220  209.  2.  20-21  Days Fetal (Figures 80-82)  At 20 and 21 days fetal the appearance of the c i l i a r y body and iris is not too different from that at 19 days.  Grossly, the c i l i a r y body  may be seen as a slightly larger bulge posteriorly better delineated from the retina peripherally and the i r i s centrally (Figures 80, 82).  Fetal  rats from the same l i t t e r do show differences in the stage of development of the tissues under study.  Thus in some instances what is observed at 20  days fetal is very similar to what is observed at 19 days fetal. Histologically, some features which are not as apparent at 19 days fetal are slightly accentuated at 20 days fetal.  In the c i l i a r y body the  appearance of the posterior c i l i a r y surface and of the ciliary.epithelium is much like that at 19 days fetal.  However, the anterior (apical) out-  line of the c i l i a r y epithelium may bulge anteriorly so that the c i l i a r y epithelium looks like l i t t l e hillocks directed towards the pigment epithelium.  A thin strip of darkly staining cytoplasm of the c i l i a r y epi-  thelium contrasts remarkably with the lighter staining vesiculated apical cytoplasm of the c i l i a r y pigment epithelium. The pigment epithelium is beginning to buckle and shows the earliest signs of folding of the whole epithelium (Figure 80).  The  stromal  surface of the epithelium may be deeply folded so that only a small cleft separates the epithelial surfaces (Figure 82).  The cleft is normally  entirely occupied by capillaries with thin walled squamous endothelium. On the other hand, the epithelium may show a couple of shallow folds (Figure 80).  Blood vessels and stromal cells from the c i l i a r y stroma  tumble down to f i l l the troughs of the folds (Figures 80, 82). the capillaries are found in the depths of the folds.  Most often,  The darker staining  endothelial c e l l nuclei can be seen protruding into the lumen.  The peaks  of these folds are usually intimately associated with the stromal cells.  The pigment epithelium may be uneven in its thickness. is a light  In most places i t  staining, simple, high columnar epithelium with oval to poly-  gonal nuclei containing nucleoli.  However, particularly at the crests of  the folds there may be instead a thick mass of cells (Figure 80). In the anterior half of the c i l i a r y stroma the cells seem to stream past the c i l i a r y body on to the i r i s , whereas, in the posterior half of the stroma the cells cascade down into the folds of the pigment epithelium (Figures 80, 82). The i r i s has not grown considerably in length as compared to that at 19 days fetal. 81).  The sphincter appears slightly more pronounced (Figure  The developing muscle cell mass is an egg-shaped structure.  cells are elongated, centrally tapering and centripetally oriented.  The In  terms of its stainability with toluidine blue, the developing sphincter muscle cells stain similar to, or at the very most, very slightly different from that of the anterior epithelium.  The developing sphincter is essen-  t i a l l y the pupillary expansion of the anterior epithelium. At this stage many of the cells are actively dividing. Mitotic figures are not confined to any particular region of the i r i s or c i l i a r y body so that i t is not possible to speak of a zone of growth. Many of the mitotic figures are found in the c i l i a r y stroma (Figure 80), c i l i a r y epithelium (especially near the retinal junction) and the i r i s anterior .epithelium (Figure 82).  Some of the developing sphincter muscle cells are  also dividing (Figure 82).  Since no chemical agents, like colchicine, are  used to arrest mitosis at metaphase, the number of mitotic figures present is only a mere indication of the extent of c e l l division that might be taking place.  212.  Figure 80  20-21 Days Fetal (LM) The histological characteristics of the posterior epithelium (pe), anterior epithelium (ae) and c i l i a r y epithelium (ce) are much like those at 19 days fetal (Figures 77-79). The sphincter muscle cells are closely packed together (sph). The c i l i a r y pigment epithelium (cpe) is beginning to buckle giving rise to a series of shallow folds, the depths of which are f i l l e d with capillaries containing red blood cells (bv). Mitoses are seen in a l l parts of the c i l i a r y (cs) and i r i s (s) stroma and in the c i l i a r y pigment epithelium (arrows). The cells and blood vessels (bvl) of the i r i s stroma stream past the pupillary tip of the sphincter. x 220  Figure 81  20-21 Days Fetal (LM) The developing sphincter (sph) is clearly seen as the pupillary expansion of the anterior epithelium (ae) of the i r i s . The nuclei are large, pale and irregularly shaped. The developing muscle cells are packed close together. A few vesicles are present. There is a small but perceptible gap (arrow) between the anterior surface of the sphincter and the overlying stromal (s) blood vessel (bv). The posterior epithelium (pe) decreases in height t i l l the peripheral extent of the sphincter where the cells become trapezoidal as they line the posterior surface of the sphincter. The stroma consists of a mass of small cells mainly oriented parallel to the i r i s posterior surface. A large blood vessel of the i r i s stroma is seen following the contours of the stromal surface of the anterior epithelium (ae), the anterior surface of the sphincter, turning round at the pupillary tip of the sphincter and appear to be headed posteriorly. The anterior and posterior surfaces of the i r i s is smooth. x 350  Figure 82  20-21 Days Fetal (LM) Mitotic figures are seen in the c i l i a r y epithelium (ce), anterior epithelium (ae) and in the developing sphincter (sph) (arrows). The i r i s stroma (s) continues centrally as part of the pupillary membrane (pm). x 220  3.  1-4 Days Post-natal (Figures 83-86)  In the first few days after birth, certain changes are seen in the c i l i a r y body as well as in the i r i s .  In the first day, the c i l i a r y body  forms a prominent bulge into the posterior chamber (Figure 83). tion between retina, c i l i a r y body and i r i s is quite distinct.  The demarcaThe c i l i a r y  body is seen as one large bulky process, unlike the delicate c i l i a r y processes in the adult eye.  However, by the third and fourth days, the beginn-  ings of the division of the c i l i a r y body into c i l i a r y processes (Figure 84).  is observed  At birth, the c i l i a r y pigment epithelium is folded, with  blood vessels occupying the clefts of the folds.  Now,  thelium follows suit and begins to fold (Figure 84). iary body bifurcates.  the c i l i a r y epiThe tip of the c i l -  Initially, the bifurcation is only seen in the pig-  ment epithelium, but gradually the c i l i a r y epithelium also bifurcates.  In  certain specimens, the c i l i a r y body is divided into two sets of processes, one being directed centrally and the other more laterally. processes are short and bulky.  These c i l i a r y  Or, there may be more than two c i l i a r y  processes.  Differences are quite common, and is the rule rather than the  exception.  As a whole, the c i l i a r y body is s t i l l attached to the retina  by a broad base, but i t is not as broad as in younger eyes. The i r i s grows in length although not to a considerable extent. The ratio of the bulk of the sphincter to the total length of the i r i s is an indication of the rate of growth. relatively constant. iris length.  The bulk of the sphincter remains  Initially, the sphincter makes up about half of the  By.four days after birth, the sphincter may comprise one  third to one f i f t h of the i r i s length (Figure 83). The c i l i a r y epithelium (Figures 83, 84) shows different configurations according to its location. Near the ciliary-retinal junction, the cells are elongated and are closely packed together.  The darkly staining  nuclei are long and thin and tapering at both ends. specks of heterochromatin.  There may be fine  Since the cells are narrow, one gets the im-  pression of a series of closely aligned dark nuclei. The c i l i a r y epithelial cells over most of the c i l i a r y body are columnar, especially at the tip of the c i l i a r y body. larly oval.  The nuclei are elongated, polygonal or irregu-  They may show indentations of the nuclear envelope.  If c i l -  iary processes are distinguishable, then the cells at the tip of the c i l iary process are slightly higher than those at the base of the c i l i a r y process.  The change is gradual.  Towards the root of the i r i s , the c i l i a r y  epithelial cells alter in form from columnar to cuboidal. dark and polygonal. sistent.  The nuclei are  The overall appearance of the ctyoplasm is not con-  It is usually vesiculated, more so posteriorly. The cytoplasm  is a l i t t l e denser at the boundary zone with the pigment epithelium (Figure 83).  The cytoplasm may even acquire a lace-like effect at the tip of the  developing c i l i a r y processes seen in mitotic division.  (Figure 84).  Some of the epithelial cells are  The posterior surface of the epithelium is  relatively smooth, or at times, i t might have a frayed look to i t (Figures 83, 84). The pigment epithelium of the c i l i a r y body is always more deeply and more extensively folded than the c i l i a r y epithelium.  The posterior  surface follows the contour of the c i l i a r y epithelium but the anterior surface outline is much more exaggerated than the contour of the posterior surface.  The cells are columnar, in differing degrees (Figure 84),  The  epithelium consists of a single layer of columnar cells except where the folds occur.  Here, there is a pile up of cells.  It appears that some re-  arrangement of the cells in this layer is taking place. large, polygonal and light staining (Figure 83). in the center of the cells.  The nuclei are  They are usually placed  Nucleoli are often present.  The cytoplasm  is finely vesiculated in most cells. culated while its neighbor is not.  Sometimes, though, one cell is vesiOverall, the cytoplasm appears pale.  There are fewer vesicles towards the stromal poles of the cells, so that the cytoplasm is darker, whereas towards the apical poles of the cells, the cytoplasm at times appears frothy.  The boundary between the pigment  epithelium and the stroma is barely visible except in isolated spots. The c i l i a r y stroma is not wide (Figure 83). closely-packed stromal cells and some blood vessels.  It consists of small, The stromal elements  are closely associated with the anterior surface of the pigment epithelium. They squeeze in to occupy the narrow grooves formed by the pigment epithelial folds (Figure 83).  In general, blood vessels (mainly capillaries)  invaginate themselves into these grooves accompanied only by a few stromal cells.  The stromal cells towards the anterior and more superficial sur-  face of the c i l i a r y stroma are oriented differently from those deeper in the stroma.  They are parallel to the i r i s , whereas the deeper cells change  direction by ninety degrees to enter the clefts of the developing c i l i a r y processes  (Figure 83).  Dividing cells are not only encountered in the c i l i a r y epithelium but also in the pigment epithelium and in the stroma.  The formation of  the c i l i a r y processes would entail not only a shifting and repositioning of cells but also an increase in the number of cells.  Hence, the number of  mitoses that are present. The i r i s is short and stubby at f i r s t but grows a l i t t l e by 4 days post-natal.  The pupillary membrane is s t i l l seen extending centrally past  the developing sphincter. The posterior epithelium as a whole is diminishing in height (Figures 83-86).  The posterior epithelial cells are cuboidal or brick-shaped  cells over most of the i r i s posterior surface.  At the junction with the  217.  c i l i a r y body, they may be slightly higher (Figure 83).  Towards the pupillary  margin, the posterior epithelium becomes squamous or very low cuboidal where it lines the posterior surface of the sphincter (Figures 85, 86).  The  nuclei are small, irregularly polygonal and occupy most of the c e l l volume. Where the cells are squamous, the nuclei are flattened parallel to the c e l l length.  The cytoplasm stains darkly and is vesiculated.  The posterior i r i s  surface is relatively smooth (Figure 83) or may be slightly wavy or frayed (Figure 86). The anterior epithelium is a high columnar epithelium throughout its extent.  In most instances, the anterior epithelium is two to three  times the height of the posterior epithelium.  Towards the i r i s root, the  epithelium may appear stratified with two rows of nuclei being observed (Figure 84). epithelium.  The epithelium, as a whole, stains lighter than the posterior The nuclei are pale and are usually round or oval in shape,  possessing an occasional nucleolus.  When the nucleus is oval, the long  axis of the oval is perpendicular to the length of the i r i s .  The nuclei  are quite regularly spaced out along the epithelium, indicating the number of cells in the epithelium. are not easily seen.  However, the boundaries between adjacent cells  The cytoplasm is light staining. Tiny vesicles are  seen in the cytoplasm usually towards the posterior halves of the cells at the boundary zone with the i r i s posterior epithelium (Figure 83). I n i t i a l l y , the anterior half of the cytoplasm stains uniformly.  However, by the  third or fourth day after birth, patches of uniformly darker staining areas are seen along the sides of the nuclei and especially more so at the anterior stromal parts of the anterior epithelium.  Oftentimes, there are more  such dense patches towards the periphery of the i r i s .  These dense patches,  visible at the light microscopic level at this stage in the development of the i r i s , are possibly patches of myofibrils of the future dilator muscle.  The anterior surface of the anterior epithelium is very closely associated with the stromal elements (Figures 83, 85).  It is d i f f i c u l t to separate  out the two layers since the cytoplasm of the stromal cells stains almost the same intensity as the cytoplasm of the anterior epithelial cells. almost seems as i f the cytoplasm of both layers merge together.  It  The out-  line of the anterior surface of the epithelium is fashioned out by the stromal elements.  It may have a scalloped or undulating appearance (Fig-  ure 83). The anterior epithelium decreases in height and becomes flattened at the peripheral edge of the developing sphincter (Figure 85).  It invagi-  nates itself between the posterior epithelium and the sphincter cells at the peripheral half of the sphincter mass (Figure 85).  Then, centrally,  the anterior epithelium seems to flare out, as i t were, to give rise to the sphincter cells.  There is no intercellular space between the anterior  surface of the squamous epithelial cells and the sphincter cells. Rarely, there is a space towards the tip of the i r i s between the anterior and posterior epithelia. (Mann, 1964).  It is the once so-called "margin sinus".  However, recent data suggest that the marginal sinus is  only- an artifact of fixation in the human (Hvidberg-Hansen, 1970) monkey (Tamura and Smelser, 1973) developing i r i s .  and  Hence, in the rat, any  such space, which is rarely encountered, is probably also an artifact. The sphincter, in meridional sections, is variously shaped (Figures 83, 85, 86).  It may be a large oval (Figure 83) or sausage-shaped mass of  cells (Figure 85).  It is sometimes slipper-shaped like a Paramecium.  The  pupillary and peripheral border of the sphincter is smooth and rounded. Sometimes, though, a bit of the sphincter may extend peripherally much like a c l i f f over the anterior epithelium, with stromal tissue occupying the intervening space (Figure 86).  On the f i r s t day after birth, the  anterior outline of the developing sphincter is relatively smooth (Figure 83) But by the third or fourth day post-natal, i t has acquired a scalloped appearance, as i f the whole sphincter muscle is being divided into a number of large bundles of fibers (Figures 85, 86).  The sphincter, unlike the  rest of the anterior epithelium, is distinctly separated off from the stroma by an intercellular space (Figures 83, 85, 86). On the f i r s t post-natal day, the developing sphincter cells are usually elongated or wedge-shaped and they are arranged centripetally.  The  cytoplasm is uniformly staining, much like that of the anterior epithelium. The cells are packed closely together so that the c e l l boundaries are not apparent.  The nuclei are small, elongated or irregularly shaped.  From  the first to the fourth post-natal day, a few changes are observed. are more sphincter cells. cells.  There  This is a result of mitosis among the sphincter  An occasional mitotic figure is seen.  s t i l l quite close together.  The cells are small and are  However, some small intercellular spaces are  beginning to appear in between the developing sphincter cells, thus giving the sphincter a crackled glass effect (Figure 86).  The nuclei are small,  have irregular nuclear outlines and are unevenly placed among the cells. The cytoplasm may be uniformly densely staining for a l l of the cells. Most commonly, though, the individual cells show varying staining intensities.  This perhaps indicates that the muscle cells do not a l l attain the  same degree of differentiation at the same time.  This is logically to be  expected i f some of the cells are s t i l l dividing. The i r i s stroma is both highly cellular and highly vascularised (Figures 83, 85, 86).  Overall the stroma is not very thick.  At the i r i s  root, the stroma is thin but its width increases slightly as one moves towards the pupillary margin.  At the i r i s root, the stromal cells are small  and are closely packed so that there are no, or few, intercellular spaces.  At the root, unmyelinated nerves are sometimes discernible. of the stroma, the cells are more loosely spaced.  In other parts  The cells may be hap-  hazardly arranged although in general they are oriented parallel to the posterior i r i s surface.  There are many blood vessels of various sizes in  the stroma.  Capillaries are seen throughout the stroma but are especially  concentrated  in the sparse stroma over the sphincter.  These capillaries  and some stromal cells extend beyond the pupillary margin of the sphincter muscle and overhang i t (Figure 83).  The capillaries continue on as, pre-  sumably, part of the pupillary membrane.  The capillaries of the pupillary  membrane are interconnected to the i r i s stroma proper by fine connective tissue strands.  Large or small blood vessels usually follow the curve of  the anterior epithelium and of the sphincter (Figure 83).  Sometimes a  single large blood vessel enters the root of the i r i s and extends a l l the way to the pupillary margin with its lumen parallel to the i r i s length. These blood vessels, though large, consist only of a simple endothelial lining.  The endothelium often abuts directly onto the anterior epithelium.  Sometimes, a stromal c e l l or two may be interposed between the endothelium and the anterior epithelium.  As mentioned previously, the stroma is  almost adhesive to the anterior surface of the anterior epithelium (Figures 83, 85), but is distinctly separated  from the sphincter (Figure 83).  Whether this is of any functional significance is not known. The anterior surface of the i r i s stroma is smooth (Figures 83, 85, 86).  It almost seems to be lined by a continuous endothelium.  some of the anterior endothelial cells show fine microvilli  Occasionally,  projecting  into the anterior chamber. Mitoses are seen among these endothelial cells as well as in the stroma.  221.  Figure 83  1-4 Days Post-natal  (LM)  At 1 day post-natal, the i r i s is s t i l l short. The c i l i a r y body forms a prominent bulge into the posterior chamber (PC). The ciliary epithelium (ce) is a dark-staining layer. The c i l i a r y pigment epithelium (cpe) is light staining and deeply folded. Blood vessels and stromal cells tumble in from the c i l i a r y stroma (cs) to occupy the narrow clefts of the c i l i a r y pigment epithelium. At the boundary with the c i l i a r y epithelium, the cytoplasm of the c i l i a r y pigment epithelium is vesiculated. The posterior epithelium of the i r i s (pe) is not as high as in younger eyes. The cells are more or less cuboidal or brickshaped with very dark staining nuclei. At the posterior surface of the sphincter (sph), the posterior epithelial cells are more flattened with lighter staining nuclei. The anterior epithelium (ae) is a pale, high columnar layer. The boundary between the anterior epithelium and the stroma (s) is not distinct. The stromal surface of the anterior epithelium is being molded by the stromal elements. The stroma of the c i l i a r y body and i r i s is f i l l e d with small stromal cells and numerous thin walled capillaries. The anterior outline of the sphincter is relatively smooth. The sphincter is oval in shape and consists of a mass of small cells which appear to stain darker than the rest of the anterior epithelium. Small intercellular spaces are seen in between some of the sphincter muscle cells. x 220  Figure 84  1-4 Days Post-natal (LM) At 4 days post-natal, a number of large, bulky c i l i a r y processes are formed by the foldings of both the c i l i a r y pigment (cpe) and the c i l i a r y epithelium (ce). Stromal cells and blood vessels insinuate in between the spaces formed by the folds of the epithelia. The cytoplasm of the c i l i a r y epithelial cells at the tip of the developing c i l i a r y process has a lace-like effect. . x 220  223.  Figure 85  1-4 Days Post-natal  (LM)  The posterior epithelium (pe) of the i r i s is a layer of cuboidal cells which become squamous along the posterior surface of the sphincter (sph)„ One of the posterior epithelial cells is dividing (arrow). The anterior epithelium (ae) is just very slightly higher than the posterior epithelium. At the peripheral posterior half of the sphincter, the anterior epithelial cells become squamous and invaginate themselves in between the posterior epithelium and the sphincter (double arrows). The sausage-shaped sphincter has acquired a slightly scalloped appearance anteriorly. Small intercellular spaces are seen in between some of the cells. x 220  Figure 86  1-4 Days Post-natal (LM) The anterior (ae) and'posterior (pe) epithelium of the i r i s are as in Figure 85. The sphincter (sph) consists of groups of muscle cells separated by intercellular spaces giving the sphincter a crackled glass effect. The anterior surface of the sphincter is highly scalloped. At the peripheral extent of the sphincter, a portion of the sphincter extends over the anterior and posterior epithelium and the intervening gap is f i l l e d with stromal elements (arrow). x 220  4.  5-10 Days Post-natal. (Figures 87-90)  Between the f i f t h and tenth days after birth, the iris and c i l i a r y body are slowly acquiring characteristics typical of the adult form.  The  c i l i a r y body at first consists of one or two large, bulky and short processes.  But by the tenth day . they have grown a l i t t l e (Figure 87). They are  s t i l l relatively short and are by no means anywhere like the delicate finger-like processes of the adult.  The c i l i a r y processes are not held  close to the retina but are positioned more towards the i r i s .  The i r i s  has grown in length so that by the tenth day, the sphincter forms only one f i f t h or less of the length of the i r i s .  Overall, the i r i s appears quite  long and thin and has somewhat the configuration of the adult i r i s in pupillary constriction. When the c i l i a r y processes are short and bulky, as at 5 days postnatal, the c i l i a r y epithelium is much like that at 4 days post-natal.  By  the seventh to tenth days after birth, the c i l i a r y processes are a l i t t l e longer.  Differences are seen among the cells of the c i l i a r y epithelium  according to their location (Figure 87). The c i l i a r y epithelium near the retina consists of columnar cells which may have a stratified appearance. The nuclei are darkly staining and elongated.  The cytoplasm is highly  vesiculated except for a small strip of cytoplasm adjacent to the pigment epithelium which is devoid of vesicles. The c i l i a r y epithelium along the sides of the developing c i l i a r y processes is low columnar or cuboidal.  The nucleus, which is variously  shaped, is in the center of the c e l l and occupies most of the c e l l volume. The cytoplasm is quite dark staining with a few vesicles towards the posterior surface.  At the apical poles of the c i l i a r y epithelial cells the  cytoplasm is uniformly darkly staining in contrast to the lighter staining, generally vesiculated cytoplasm of the pigment epithelium.  The  posterior surface of the c i l i a r y epithelium lining the sides of the c i l i a r y processes  is relatively smooth or i t may be just very slightly irregular.  The c i l i a r y epithelial cells at the tip of the developing c i l i a r y processes are high columnar cells (Figure 87). are oriented along the length of the cells.  The nuclei are longer and  They have a tendency to be  placed towards the apical poles of the cells, that is, towards the pigment epithelium.  The apical cytoplasm is uniformly darkly staining whereas the  basal cytoplasm appears finely vesiculated a l l over and has a lacy appearance.  There seems to be many invaginations and interdigitations, thus  imparting to the posterior surface of the tips of the c i l i a r y processes a highly irregular outline. As the c i l i a r y epithelium continues towards the posterior epithelium of the i r i s , there is a gradual transition from low columnar to low cuboidal cells to almost squamous cells (Figure 87). and dense and f i l l up almost a l l of the c e l l .  The nuclei are large  The posterior surface of the  epithelium facing the posterior chamber is smooth. No significant changes are seen in the pigment epithelium or stroma of the c i l i a r y body. and described.  Both are similar to what has been previously observed  As before, dividing cells are present in the c i l i a r y epi-  thelium, pigment epithelium and in the stroma. By the tenth day after birth, the i r i s is quite long and distinct. The posterior epithelium consists of a single row of very low cells. may be trapezoidal in shape.  They  At other times, the whole epithelium consists  of a row of brick-like cells, or the cells may be flattened (Figures 87-90). In a l l cases, the posterior epithelium becomes flattened when i t lines the posterior surface of the developing sphincter (Figure 90). small and are usually flattened antero-posteriorly.  The nuclei are  They may occur regu-  larly along the whole epithelium or there may be large gaps between the  nuclei.  This is probably due to the plane of sectioning. The posterior surface of the i r i s is usually smooth although  occasionally i t may be a l i t t l e wavy (Figure 90). birth, there are s t i l l  By the tenth day after  some posterior epithelial cells which are dividing  to form more epithelial cells. The anterior epithelium is of a uniform width from the root to the lateral edge of the sphincter.  The shape of the cells range from columnar  (Figures 88, 90) to rectangular (Figure 89).  I n i t i a l l y , the anterior epi-  thelial cells are two to three times the height of the posterior epithelial cells.  But gradually, i t almost seems as i f the anterior epithelium is  being stretched out such that by the tenth day post-natal the anterior epithelium is oftentimes the same height as the posterior epithelium (Figure 89).  When the anterior epithelium is columnar, the nuclei are large  and round or oval with some nuclear indentations and are placed in the center of the cells. poles (Figure 88).  The cytoplasm is slightly vesiculated at its apical In the cytoplasm at the basal (or stromal) parts of  the anterior epithelium there are dark patches of the developing muscle.  dilator  When the anterior epithelium is about the same height as the  posterior epithelium, the cells are cuboidal or rectangular (Figure 89). The nuclei, which may be irregularly shaped, are generally oriented parall e l to the posterior i r i s surface. The sphincter is a large, relatively tightly packed mass of small cells bounded anteriorly by a small amount of stroma and posteriorly by low epithelium (Figure 90).  The anterior surface of the sphincter is  scalloped in appearance and is separated from the stroma by an intercellular space.  The developing muscle cells are small with small, variously  irregularly shaped nuclei. Groups of muscle cells seem to associate together in groups and are separated from other muscle groups by distinctly  visible intercellular spaces (Figure 90).  However, the individual muscle  cells may be interconnected with each other.  The muscle cells generally  stain darker than the anterior epithelial cells.  The cytoplasm is uniform'  darkly staining for any one cell but staining intensities vary for the individual cells.  In the midst of this mass of differentiating muscle  cells, an occasional mitotic figure is encountered. The i r i s stroma is not extensive.  It is thin at the i r i s root  (Figures 87, 89) and increases only slightly in width towards the pupillary margin.  Blood vessels, nerves, stromal cells and connective tissue  are present.  In the f i r s t few days after birth, the stroma s t i l l over-  hangs the pupillary edge of the sphincter.  However, by the tenth day,  either the stroma has regressed or the neuroectodermal elements of the iris have stretched centrally.  Some blood vessels of the stroma curve  around the pupillary edge of the sphincter. Also, there are s t i l l some vessels which continue past the pupillary border of the i r i s into the pupil i t s e l f .  These small capillaries are usually strung out in a line  with connective tissue strands interconnecting the individual capillaries. It could be that even at this late stage of development (10 days postnatal), remnants of the pupillary membrane are s t i l l apparent.  Whether  the vessels are s t i l l functional cannot be determined from our present studies.  229.  Figure 87  5-10 Days Post-natal  (LM)  The i r i s (I) and c i l i a r y body (CB) are acquiring characteristics of the adult. Distinct c i l i a r y processes are seen although they are s t i l l not long. The c i l i a r y epithelium (ce) varies in height in different locations on the c i l i a r y process, being higher at the tip. At the tip of the ci-liary process, the cytoplasm of the cells is vesiculated and invaginated giving the cells a highly irregular outline (arrow). The c i l i a r y pigment epithelium (cpe) follows the c i l i a r y epithelium closely. Both the epithelia fold to form a hair-pin loop with the narrow intervening space being f i l l e d with stromal elements. The i r i s is thin. thelium are low.  Figure 88  The posterior (pe) and anterior (ae) epix 220  5-10 Days Post-natal  (LM)  The posterior epithelium of the i r i s (pe) is made up of squamous or cuboidal-type cells. There is slight wavy appearance to the posterior surface of the i r i s created by the bulging out of the cells. The anterior epithelium (ae) is relatively high and is very closely associated with the i r i s stroma (s). x 220  o CD  231.  Figure 89  5-10 Days Post-natal (LM) The anterior (ae) and posterior (pe) epithelium of the i r i s are of about the same height. The posterior epithelium consists primarily of squamous cells with flattened nuclei, whereas the anterior epithelium consists of cuboidal or rectangular cells. The two layers are closely apposed. The cells of the stroma (s) are a l l arranged parallel to the length of the i r i s and they abut onto the anterior surface of the anterior epithelium. x 220  Figure 90  5-10 Days Post-natal (LM) The sphincter (sph) is a cluster of small cells which appear to be divided into large muscle groups by intercellular spaces (arrows). Spaces are also found in between the individual muscle cells. x 220  232.  233.  5.  From 11 Days Post-natal On (Figures 91-95)  At about two weeks after birth, the rat eyes open.  It would seem  then that by the 14th or 15th day post-natal the i r i s would be fully functional in order to cope with the environment.  Structurally, the i r i s and  the c i l i a r y body would have attained the adult form.  This is found to be  so. By the 12th day after birth, there are numerous long, thin and finger-like c i l i a r y processes, much as in the adult rat eye (Figure 91). Each c i l i a r y process is essentially a vascular structure.  The c i l i a r y epi-  thelium and the pigment epithelium of the c i l i a r y processes are closely adherent to each other.  They make a long thin hair-pin loop, the bend of  the loop being the tip of the c i l i a r y process.  The pigment epithelium of  both arms of the loop are quite close together and are only separated by the stroma of the c i l i a r y body.  There are few stromal cells.  Normally a  large blood vessel completely occupies the cleft between the folds of the pigment epithelium such that the endothelium is closely associated with the c i l i a r y pigment epithelium. present.  Occasionally there may be a few stromal cells  Sometimes, the incoming blood vessel expands and becomes sac-  like at the tip of the c i l i a r y process.  Thus the tip of the c i l i a r y process  appears knob-like externally (Figure 91). The i r i s is long and thin and assumes the configuration of the adult i r i s in pupillary constriction. creases slightly  The antero-posterior width of the i r i s in-  from the periphery towards the pupil.  This increase is  due to the increase in.the width of the stroma up to the peripheral edge of the sphincter. The posterior epithelium of the i r i s consists of a single row of thick or brick-like cells (Figure 92). length of the cells.  The nuclei are elongated along the  The posterior surface of the i r i s is s t i l l relatively  smooth because of the shape and arrangement of the posterior epithelial cells.  The cell boundaries are not distinctly visible.  By the. 15th day  after birth, parts of the posterior surface of the i r i s becomes finely irregular (Figure 93).  Some of the nuclei of the squamous cells may bulge  out a l i t t l e posteriorly, thus contributing to the wavy appearance of the posterior i r i s surface.  In some specimens at 15 days post-natal, the  characteristic features of the posterior epithelium are as follows. epithelial cells often appear hemispherical in shape (Figure 94).  The They are  attached in a row to the anterior epithelium, with the curved portions of the hemispheres directed posteriorly into the posterior chamber.  The sides  of the hemispherical cells do not touch each other so that each individual epithelial c e l l is well demarcated from its neighbor.  The arrangement of  the hemispherical epithelial cells gives to the posterior i r i s surface a regularly scalloped appearance.  The plasma membrane of the individual  epithelial cells is not smooth but shows irregularities as well. dark nuclei are situated in the center of the cells.  The small  Some of the posterior  epithelial cells are s t i l l dividing two weeks after birth. The anterior epithelium is about the same width as the posterior epithelium by the 15th day (Figures 92-94).  It might even at times be a  l i t t l e narrower than the posterior epithelium.  The nuclei are generally  elongated parallel to the i r i s posterior surface. spaced out along this layer. ing.  They are not regularly  The cytoplasm is relatively uniformly stain-  At the anterior stromal surface of the anterior epithelium, there is  a smooth, dark, dense line which clearly sets off the anterior epithelium from the stroma. thelium.  This is the dilator muscle layer of the anterior epi-  The anterior epithelium is a myoepithelial layer.  The nucleus  and cell organelles are in the apical portion of the cell while the contractile elements are in the basal portion of the c e l l .  In general, the  boundary zone between the anterior epithelium and the stroma is a smooth straight line.  At times, individual epithelial cells, or groups of cells,  may bulge out into the stroma. The sphincter is s t i l l a large mass of small tightly packed cells which are darkly staining (Figure 95). place within the sphincter region.  Cell division is s t i l l taking  Besides the muscle cells, capillaries  and nerves are also detected within the sphincter. In the stroma, there is an increasing number of capillaries. Larger blood vessels have their lumens parallel to the posterior surface of the i r i s (Figures 93, 94).  Some of the large blood vessels consist of  only an endothelial lining, whereas others may have, in addition, a pericyte layer external to the endothelium.  In young eyes, the stromal cells  and blood vessel endothelium is always very closely associated with the anterior epithelium. together.  In fact, at times, the two layers seem to be fused  However, in development, the stromal elements slowly move away  from the anterior surface of the anterior epithelium.  It is very rare to  see a stromal cell abutting closely onto the anterior epithelium.  The  nuclei of the stromal cells are kept at a distance from the anterior epithelium.  Cytoplasmic processes of the stromal cells occupy the region  between the anterior epithelium and the stromal c e l l nuclei.  The nuclei  are large structures and would probably interfere mechanically with the mobility of the i r i s i f they are placed too close to the anterior epithelium.  The anterior surface of the i r i s stroma is relatively smooth although  at times i t appears that there are gaps in the lining endothelium.  236.  Figure 91 From 11 Days Post-natal On (LM) The c i l i a r y processes are vascular structures. The c i l i a r y epithelium (ce) and c i l i a r y pigment epithelium (cpe) make hair-pin loops enclosing capillaries and stromal cells. At the tip of the c i l i a r y process, the blood vessel (bv) may expand to give a knob-like appearance to the tip of the c i l i a r y process. The c i l i a r y epithelial cells possess a highly irregular outline. The c i l i a r y pigment epithelial cells are cuboidal or very low columnar. x 220  Figure 92 From 11 Days Post-natal On (LM) The posterior (pe) and anterior (ae) epithelium consists of two rows of brick-shaped cells. The posterior, epithelial cells are arranged end to end giving the posterior surface of the i r i s a smooth contour. The cytoplasm of the anterior epithelial cells stains relatively dark so that the boundary between the stroma (s) and the anterior epithelium is discernible. The stromal cells and blood vessels (bv) are a l l arranged parallel to the posterior surface of the i r i s . x 220  Figure 93 From 11 Days Post-natal On (LM) The posterior surface of the i r i s is finely irregular (compare with Figure 92). The individual cells of the posterior epithelium (pe) may bulge out a l i t t l e . The anterior epithelium (ae) or dilator is a dense staining layer distinctly separated from the stroma (s). The stromal elements do not come right next to the anterior surface of the anterior epithelium, as in younger eyes. x 220  237.  238.  Figure 94  From 11 Days Post-natal On (LM) The posterior surface of the i r i s has a regularly scalloped appearance. The posterior epithelial cells (pe) are hemispherical in shape. The cell cytoplasm is vesiculated and c e l l processes are beginning to be seen (arrows). The anterior epithelium or dilator (ae) is a distinct layer. The cells in the stroma are kept away from the dilator. x 220  Figure 95  From 11 Days Post-natal On (LM) The sphincter (sph) muscle cells are cut in cross-section. There is not such a clear well-defined space between the sphincter and the stroma (s) as is regularly seen in younger eyes. Capillaries are present in the loose i r i s stroma. A small part of the i r i s stroma s t i l l overhangs the pupillary edge of the sphincter and a blood vessel (bv) appears to be headed into the pupillary aperture. x 220  239.  240.  F.  A Scanning Electron Microscopic Study of the Posterior Surface of the Developing Rat Iris  1.  17 Days Fetal - Term (Figures 96-105)  The fetal i r i s in a scanning electron micrograph is a narrow rim of tissue surrounding a very large pupil. The posterior surface of the i r i s may be covered by an amorphous, sheet-like substance (Figure 96).  It  is sometimes pitted and pock-marked and appears capable of being flaked off (Figure 96).  At times, this amorphous layer is partially removed from the  posterior surface of the i r i s during the processing of the tissues (Figure 97).  It is then seen as a thin sheet adherent to the i r i s .  Near the  pupil, its surface appears relatively smooth except for bits of f i b r i l l a r material.  Superficial to but closely associated with this amorphous layer  are numerous blood vessels containing red blood cells (Figures 96, 97). The lining layer of the blood vessels is normally torn off when the surrounding tissues, such as the lens, are removed (Figure 96).  More often than  not, both the superficial blood vessels and the amorphous layer are removed during the preparation of the tissues to expose  the basement mem-  brane covering the posterior epithelium of the developing  i r i s (Figure 98).  The posterior surface of the i r i s near the pupillary margin is relatively smooth.  There may be a few dents and grooves, which presumably, are made  by the superficial blood vessels.  The more peripheral portion of the  posterior surface of the i r i s has a very slightly more rugged contour (Figures 98, 99).  A fine network of f i b r i l l a r material in varying amounts  (Figures 97, 99, 100) covers a l l of the posterior surface of the i r i s . From meridional sections of the developing rat i r i s , i t is observed that the posterior surface of the i r i s is lined by a layer of epithelial cells. However, the boundaries of the posterior epithelial cells are not visible  in these scanning electron micrographs.  The overlying basement membrane  of the posterior epithelial cells and the adherent f i b r i l l a r material would obscure any cell boundaries that may be present.  At this early stage in  the development of the rat i r i s , there is no indication at a l l from scanning electron microscopy that the i r i s posterior epithelium is in actuality made up of a layer of discrete cells.  The nuclei of the posterior epithelial  cells do not bulge out to indicate that this is so. There are some capillary systems associated with the fetal i r i s (Figure 101); a large annular vessel of the hyaloid system located at the region of the developing c i l i a r y body (Figure 102), capillaries arising from the annular vessel and traversing the posterior surface of the i r i s (Figures 101, 102) and capillaries of the pupillary membrane (Figures 101, 103). The annular vessel is relatively large in comparison to the feeder capillaries (Figure 102).  It is usually torn off during specimen prepara-  tion, but when left intact, i t is seen overlying the developing c i l i a r y body.  A group of interconnecting capillaries of the retinal portion of the  hyaloid vascular system meet the annular vessel (Figure 102).  These capil-  laries have relatively thick walls although the red blood cells within the lumens can be perceived  (Figure 104).  The highly branched capillaries are  held together and possibly to the surface of the retina by a dense meshwork of f i b r i l s  (Figures 102, 104). The f i b r i l s are of varying diameters (Fig-  ure 104).  Capillaries also radiate out from the annular vessel over the  posterior surface of the i r i s (Figures 101, 102). They are usually more delicate looking than the retinal hyaloid vessels.  They form a complicated  pattern on the posterior surface of the i r i s and are seen to reach the pupillary margin (Figure 101). The pupillary membrane (Figures 97, 98, 101, 103) extends centrally  from the anterior surface of the i r i s .  The pupillary membrane has not been  observed to cover a l l of the pupillary aperture but extends from the pupillary margin to varying degrees (Figures 98, 101, 103). The capillaries of the pupillary membrane (Figure 105) have thinner walls than those of the retinal hyaloid system (Figure 104).  They are almost transparent so that  the red blood cells within the capillary lumens impart to the capillaries a bead-like appearance.  The capillaries pursue a tortuous course and are  complexly interconnected with each other (Figures 101, 103, 105). The pupillary membrane blood vessels are held in the pupillary aperture by a scaffolding network of connective tissue fibers (Figures 103, 105). Rarely, some cells are seen within the connective tissue network. Some of the capillaries from the anterior portion of the i r i s do not extend centrally to form the pupillary membrane.  Instead, at the  pupillary margin, they turn around and traverse the posterior surface of the i r i s (Figures 97, 103).  243.  Figure 96  17 Days Fetal - Term (SEM) The posterior surface of the i r i s is covered by an amorphous, sheet-like substance. It may be pitted. Parts of the walls of the blood vessels on the posterior surface of the i r i s have been removed to expose the contents, the erythrocytes. x 820  Figure 97  17 Days Fetal - Term (SEM) Most of the superficial flaky amorphous substance is removed to reveal the underlying basement membrane covering the posterior surface of the i r i s . Parts of the walls of two blood vessels remain. These continue past the pupillary margin and are continuous with the blood vessels from the anterior surface of the i r i s . x 1,530  245.  Figure 98  17 Days Fetal - Term (SEM) The narrow zone around the pupil is relatively smooth with a few shallow grooves and low bulges. Peripheral to that, the posterior surface is covered with a finely f i b r i l l a r material. At the pupillary margin, the lumens of many blood vessels, suspended in a dense connective tissue framework, are seen. They have a l l come from the anterior surface of the i r i s . One blood vessel appears to be changing in direction at the pupillary margin. x 700  Figure 99  17 Days Fetal - Term (SEM) A high magnification scanning electron micrograph of the smooth and f i b r i l l a r parts of the posterior surface of the iris. x 3,800  246.  247.  Figure 100  17 Days Fetal - Term (SEM) Occasionally, there is a large amount of f i b r i l l a r material on the posterior surface of the i r i s . x 4,400  Figure 101  17 Days Fetal - Term (SEM) Three networks of blood vessels are seen in close association with each other, the blood vessels extending from the pupillary margin, the blood vessels on the posterior surface of the i r i s and the blood vessels on the inner surface of the retina. Most of the blood vessels on the posterior surface of the i r i s have been removed during specimen preparation. The developing i r i s is a narrow strip of tissue with a relatively smooth surface. x 150  248.  249.  Figure 102  17 Days Fetal - Term (SEM) At a high magnification, a large annular vessel is seen at the region of the developing c i l i a r y body. Blood vessels on the inner surface of the retina are held together by a dense connective tissue network. These capillaries link up with the annular vessel. At the opposite pole of the annular vessel, another series of capillaries (on the posterior surface of the iris) are connected into i t . Most of these have been broken off. x 380  Figure 103  17 Days Fetal - Term (SEM) The posterior surface of the i r i s is smooth except for a few pieces of amorphous material. The pupillary membrane extends for varying distances into the pupil from the anterior surface of the i r i s . The pupillary membrane consists of a network of thin-walled capillaries suspended in a f i b r i l l a r connective tissue framework. x 380  250.  251.  Figure 104  17 Days Fetal - Term (SEM) The capillaries on the inner surface of the retina have relatively thick walls. An occasional blood cell may be perceived. The capillaries branch and are quite extensively interconnected. The capillaries are held together by a very dense f i b r i l l a r meshwork. The f i b r i l s are of varying diameters. x 1,530  Figure 105  17 Days Fetal - Term (SEM) The capillaries of the pupillary membrane are highly branched, They have thin, almost transparent walls so that the red blood cells readily show through. A framework of mainly fine connective tissue fibers hold the capillaries in their relative positions. x 1,530  \  252.  2. 1-4 Days Post-natal (Figures 106-115) During the f i r s t four days after birth, certain changes are observed in the posterior surface of the i r i s .  Other features, present in  the fetal i r i s , are observed in more explicit detail, especially pertaining to the blood vessels of the pupillary membrane and the blood vessels on the posterior surface of the i r i s . In a low magnification scanning electron micrograph, i t is seen that the posterior surface of the i r i s can be divided into two regions, a central or pupillary and a peripheral region (Figure 106).  The pupillary  region makes up about one third of the length of the i r i s .  It is covered  by a layer of amorphous material, the basement membrane.  It is oftentimes  extensively and deeply grooved so that this region has a corrugated appearance (Figures 106, 107). In certain eyes, this characteristic is not as clearly observable and the two regions are not as distinctly delineated (Figure 108).  The grooves are radially disposed (Figures 106, 107). The  grooves are almost a l l of the same width and depth.  They may follow a  straight course peripherally, or they may branch as they do so (Figure 107). The peripheral two thirds of the posterior surface of the i r i s is beginning to show some contouring (Figure 106) as compared to the fetal i r i s . It seems to be made up of a myriad l i t t l e elevations (Figure 106), which at a higher magnification, are revealed to be the nuclei of the posterior epithelial cells. impressions.  Superimposed on the posterior surface of the i r i s are They are generally radially oriented, they may branch and  they are of about the same width as, but shallower than, the grooves on the pupillary zone of the posterior surface of the i r i s . At a higher magnification, the contours of the posterior surface of the i r i s are seen in a l i t t l e greater detail.  In the pupillary region,  relatively large, more or less oval elevations are present (Figures 108,  109).  The basement membrane covering the pupillary portion of the iris  posterior epithelial cells is quite smooth save for a few fine grooves and ridges. Tiny holes may be present (Figure 109).  Over the peripheral  portion of the posterior surface of the i r i s there is a mosaic of small round to oval forms, separated by shallow depressions, which bulge ever so slightly towards the posterior chamber (Figures 109-111).  These bulges,  most probably representing the nuclei of the posterior epithelial cells, are not oriented in any particular fashion either in their relationships with each other or with respect to the i r i s as a whole. Occasionally, crater-like formations (Figure 111) are seen within this mosaic arrangement of epithelial cells.  The basement membrane covers a l l of the c e l l surfaces  Particulate material, probably proteinaceous, is present on the basement membrane (Figure 111). The pupillary membrane is s t i l l as extensive four days after birth as i t is in the fetal eye (Figure 112).  Here, i t is observed that the  capillaries arising from the annular vessel and traversing the posterior surface of the i r i s , and the capillaries of the pupillary membrane are apparently closely inter-related (Figures 112-114).  The capillaries from  the annular vessel are morphologically similar to those of the pupillary membrane. They have thin walls so that their content of red blood cells shows through.  The capillaries tend to have a radial course with respect  to the i r i s , and they are linked to each other by sometimes smaller interconnecting vessels (Figure 114).  They are also supported by a dense net-  work of connective tissue fibers which appear to intermingle with the fibers of the pupillary membrane (Figures 112-115).  These capillaries  sometimes appear to link up with those of the pupillary membrane (Figure 114), although the image obtained in our scanning electron micrograph may actually be due to the superimposit ion of one set of capillaries on another  The pupillary membrane.blood vessels, as in fetal eyes, radiate from the pupillary margin held in its connective tissue framework. branch, join, form arcades and loops.  They '  The width of the pupillary membrane  as a whole differs for each part of the i r i s , being quite extensive in parts and practically non-existent  in others.  hugging the pupillary margin are broken off.  Most times, the capillaries However, they are usually  broken off in such a way as to suggest that they are turning around at the pupillary margin and apparently going for the posterior surface of the i r i s (Figures 113, 114).  Or, a part of a blood vessel may actually be seen  making this turn and becoming adherent to the posterior surface of the i r i s (Figure 113).  Occasionally, a capillary from the anterior i r i s stroma  encircles the pupillary margin and appears to become  continuous with or  as a capillary arising from the annular vessel of the hyaloid system (Figure 113).  256.  Figure 106 1-4 Days Post-natal (SEM) The posterior surface of the i r i s is distinctly divided into a central pupillary region which is deeply radially grooved, and a peripheral region which is speckled and appears to be made up of a mosaic of tiny elevations. Superimposed on the peripheral region are some radially oriented impressions, x 150  Figure 107  1-4 Days Post-natal (SEM) At a higher magnification, the radial grooves in the pupillary region are deep and wide. They a l l are of about the same widths. Sometimes they may bifurcate. x 760  257.  258.  Figure 108 1-4 Days Post-natal (SEM) The pupillary membrane extends past the pupillary margin. The pupillary region is relatively smooth with hints of a few elevations. The peripheral region consists of smaller polygonal elevations. x 790  Figure 109  1-4 Days Post-natal (SEM) The basement membrane covering the large, oval, low elevations of the pupillary region is relatively smooth. In the peripheral region, the basement membrane shows wrinkles. A mosaic of polygonal forms are barely perceptible. Some tiny holes are present. x 1,530  259.  260.  Figure 110  1-4 Days Post-natal (SEM) Most of the posterior surface of the i r i s is a mosaic of polygonal forms distinctly separated from each other by shallow depressions. These forms bulge out ever so slightly. They are not arranged in any particular relationship with each other. x 760  Figure 111  1-4 Days Post-natal (SEM) A high magnification of Figure 110. A crater-like formation is also seen. x 1,530  261.  262.  Figure 112  1-4 Days Post-natal (SEM) The capillaries arising from the annular vessel appear to be intimately associated with the those of the pupillary membrane. These capillaries l i e on the posterior surface of the i r i s , being held together by a connective tissue network. These capillaries are very similar to those of the pupillary membrane. They have thin walls so that the red blood cells show through. x 180  Figure 113  1-4 Days Post-natal (SEM) Not a l l of the blood vessels from the anterior surface of the i r i s extend centrally to become part of the pupillary membrane. Many of these blood vessels appear to turn at the pupillary margin and be headed posteriorly. Rarely, a capillary from the anterior surface of the i r i s turns around at the pupillary margin and becomes continuous with one of the capillaries arising from the annular vessel. x 450  263.  264.  Figure 114 1-4 Days Post-natal (SEM) The capillaries on the posterior surface of the i r i s arising from the annular vessel extend past the pupillary margin to join the capillaries of the pupillary membrane arising from the anterior surface of the i r i s . Both capillary systems are held in a fine f i b r i l l a r connective tissue framework. x 450  Figure 115  1-4 Days Post-natal (SEM) The connective tissue fibers of the pupillary membrane and of the capillaries on the posterior surface of the i r i s appear to merge and intermingle. x 900  265.  266.  3.  5-10  Days Post-natal (Figures 116-127)  The rate of development of the rat i r i s varies from one l i t t e r of rats to another and also among the members of any one l i t t e r of animals. This is seen in our studies where the irises from a l i t t e r of rats aged 6 days post-natal have a more mature form than those from a l i t t e r of rats aged 8 days post-natal. From the f i f t h to tenth days after birth, the rat i r i s is slowly acquiring characteristics of the adult i r i s , as exemplified by the posterior surface.  Initially, the pupil is s t i l l large but the i r i s has grown.  Around the pupillary margin, as previously, there is a region which is different in appearance from the rest of the posterior surface of the i r i s . This pupillary zone makes up only about one f i f t h or less of the total i r i s length.  The pupillary region assumes different forms.  It may be covered  by a continuous layer of amorphous, smooth and sheet-like material, or parts of this material may have flaked off to reveal the underlying epithelium (Figures 116, 117). throughout its extent.  This layer is not of a uniform thickness  Right at the pupillary margin, i t appears to be  relatively thick but as one moves along i t peripherally, this amorphous layer seems to thin out and becomes continuous with the basement membrane covering the posterior surfaces of a l l of the epithelial cells (Figure 117). Occasionally, there may be a quite sharp transition between this thick amorphous layer and the basement membrane of the posterior epithelial cells (Figure 116).  Blood vessels are oftentimes embedded within this amorphous  layer (Figure 118).at the pupillary zone but they become free and l i e superficial to the posterior epithelium at the peripheral extent of this layer.  These blood vessels are s t i l l interconnected by a few wisps of  connective tissue fibers, reminescent of the connective tissue framework holding together a l l of the capillaries on the posterior surface of the  i r i s which have come from the annular vessel.  These capillaries may  run  a l l the way peripherally to the region of the c i l i a r y body, or they may stop short midway on the i r i s . This amorphous sheet is sometimes not only confined to the pupillary region of the i r i s but extends into the pupil to varying degrees (Figure 119).  Capillaries from the anterior portion of the i r i s are seen completely  embedded in this amorphous substance (Figure 119). The posterior epithelial cells are now quite clearly visible over the rest of the posterior surface of the i r i s (Figures 116-118, 120).  In  a low magnification scanning electron micrograph, the posterior surface of the iris has a raspberry-like appearance (Figure 116).  Numerous, generally  round elevations are disposed throughout the posterior surface of the i r i s . They are relatively equally spaced from each other by shallow  depressions.  This is seen more clearly at a higher magnification (Figure 120).  Each  rounded protuberance represents the nucleus of a posterior epithelial c e l l . The nuclei are round and they are a l l of the same size.  A basement mem-  brane covers the posterior surfaces of the epithelial cells so that there are no distinct cell boundaries although the extent of each c e l l is perceptible. A l i t t l e further along in development, some changes are seen.  The  amorphous plaque-like material is s t i l l present along the pupillary zone, thicker at the pupillary border and diminishing in thickness peripherally (Figure 121).  Radially oriented blood vessels are s t i l l enmeshed within  this substance.  If these capillaries are removed in the preparation of the  specimens, deep grooves are present a l l along the pupillary region to suggest where these capillaries had been (Figure 122).  When the capillaries  are removed, part of the amorphous layer usually goes with them. However, a thin layer s t i l l remains.  Sometimes, an interesting configuration of  the amorphous layer in the pupillary region is observed (Figure 123).  The  peripheral extent of the layer is quite regularly scalloped in its outline so that a series of V-shaped structures are seen.  Each V is occupied by  a blood vessel or a groove suggesting that a blood vessel had been in position before.  In between the blood vessels, the amorphous layer has  apparently retracted towards the pupillary margin.  As has been previously  noted, there is most often no really sharp transition between the amorphous layer and the basement membrane of the posterior epithelial cells.  As the  amorphous layer retracts, i t exposes the underlying posterior epithelial cells with their basement membrane to varying degrees (Figure 123). The posterior epithelial cells are very distinctly visualised (Figures 123-126).  Each c e l l with its centrally located nucleus bulges  quite prominently into the posterior chamber. delineate the confines of each c e l l .  Relatively deeper depressions  However, unlike in the adult where  the posterior epithelial cells are arranged in ridges depending on the degree of pupillary dilation or constriction, here in the early post-natal i r i s , the posterior epithelial cells just cover a l l of the posterior surface of the i r i s without being arranged in any particular way. Occasionally, though, deeper grooves are present which separate cut groups of epithelial cells (Figure 124). ridges and grooves.  This may be the beginnings of the epithelial  Scattered along the posterior surface of the i r i s are  some protruding structures (Figures 122, 125, 126). They are crater-like in appearance and are elevated to different heights above the posterior surface of the i r i s .  They are many times larger than the surrounding  posterior epithelial cells.  They are in some ways similar to the bulbous  structures on the posterior surface of the adult rat Iris in extreme pupillary constriction. A basement membrane covers a l l of the posterior surfaces of the  epithelial cells and bulbous structures.  Unlike the relatively smooth  basement membrane in younger eyes, the basement membrane now shows wrinkles and crinkles (Figures 125, 126) as i t follows the contours of the posterior epithelial cells.  Over the crater-like structures, the basement membrane  appears to be a l i t t l e smoother than i t is over the rest of the posterior surface of the i r i s (Figure 126). The pupillary membrane, which is s t i l l prominent in the f i r s t few days after birth, has in large measure disappeared.  A few capillaries  supported by its connective tissue f i b r i l l a r network are seen extending centrally from isolated parts of the pupillary border (Figure 127). capillaries are s t i l l f i l l e d with red blood c e l l s .  The  In most instances, the  capillaries protrude just slightly from the pupillary margin.  270.  Figure 116  5-10 Days Post-natal (SEM) The pupillary region is covered by an amorphous material which has flaked off in parts. There is a sharp transition line between the pupillary and peripheral regions. The peripheral region consists of a mosaic of round to oval elevations separated by shallow depressions. x 380  Figure 117  5-10 Days Post-natal (SEM) The amorphous material is thicker at the pupillary region. It seems to thin out peripherally and becomes continuous with the basement membrane covering the rest of the posterior surface of the i r i s . Where the amorphous material has been removed, the underlying cells are seen. x 610  271.  272.  Figure 118 5-10 Days Post-natal (SEM) Some blood vessels are embedded in the amorphous material at the pupillary margin, but they become free peripherally. Wisps of connective tissue f i b r i l s interconnect these blood vessels. Some of these blood vessels extend a l l the way to the c i l i a r y region. x 760  Figure 119 5-10 Days Post-natal (SEM) Blood vessels from the anterior surface of the i r i s are embedded in amorphous material. Remnants of the walls of the blood vessels suggest that these anterior stromal blood vessels make a turn at the pupillary margin to traverse the posterior surface of the i r i s . x 1,220  273  274.  Figure 120  5-10 Days Post-natal (SEM) The posterior epithelial cells, covered with a basement membrane, possess round nuclei. The cells are equally spaced from each other by shallow depressions. Cell boundaries are not evident. x 1,530  Figure 121  5-10 Days Post-natal (SEM) Radially oriented blood vessels are embedded in the amorphous material at the pupillary margin but they become free more peripherally. These blood vessels occasionally branch. The amorphous material is thick at the pupillary region but thins out and merges with the basement membrane over the rest of the i r i s surface. x 390  275  276.  Figure 122 5-10 Days Post-natal (SEM) The blood vessels on the posterior surface of the i r i s have been removed but deep impressions made by the blood vessels in situ remain to mark their course in vivo. A large craterlike structure is seen in the midst of the posterior epithelial cells. x 410  Figure 123 5-10 Days Post-natal (SEM) The amorphous material appears to be continuous with the basement membrane in some parts and to be completely separate in other parts. It appears to be retracting towards the pupillary margin. A large blood vessel is adherent to the posterior surface of the i r i s only at the pupillary region. The posterior epithelial cells are distinctly seen. x 760  277.  278.  Figure 124 5-10 Days Post-natal (SEM) The posterior epithelial cells are relatively closely packed together being separated only by shallow depressions. Occasionally, deeper grooves are seen separating out groups of rows of epithelial cells. x 820  Figure 125 5-10 Days Post-natal (SEM) The posterior epithelial cells are covered by a basement membrane, which shows many crinkles, which are less prominent over the bulging nuclear region. A crater-like structure is present. It is elevated above the posterior surface . of the i r i s . The basement membrane covering i t is continuous with the basement membrane covering the other posterior epithelial cells. The basement membrane also shows some crinkles. x 1,630  279.  280.  Figure 126  5-10 Days Post-natal (SEM) This crater-like structure appears high above the plane of the other epithelial cells. It is generally bloated. It is collapsed in the middle. The covering basement membrane layer is relatively smooth. x 1,630  Figure 127 5-10 Days Post-natal (SEM) The pupillary membrane is s t i l l present in parts of the i r i s , but i t is not as extensive as in younger eyes. Other blood vessels on the posterior surface of the i r i s appear to be continuous with those of the anterior surface. x 390  281.  4.  From 11 Days Post-natal On (Figures 128-137)  By about two weeks after birth, the rat i r i s has attained the adult form in terms of the morphology of the posterior surface.  There are a few  remnants of the fetal and early post-natal vascular system s t i l l present and these w i l l be alluded to.  They are lost or present in only a limited  extent in the adult rat i r i s . The i r i s is usually observed in some degree of pupillary constriction (Figures 128, 129).  The posterior surface of the iris can be divided  into a pupillary and a peripheral region.  In a low magnification scanning  electron micrograph, the pupillary margin is not smooth.  In the pupillary  region there are numerous blood vessels embedded in an amorphous material (Figures 128, 129).  Some of the blood vessels remain only in the pupillary  region while others stretch out towards the c i l i a r y body.  The number of  these long blood vessels varies from one eye to the next (Figures 128, 129).  The rest of the posterior surface of the iris appears relatively  smooth at a low magnification. (Figure 128).  Faint circumferential lines may be seen  Along the periphery of the i r i s at the region of the c i l i a r y  body, there are some barely visible radial ridges (Figure 128). short and peter out almost immediately.  These are  Occasionally, wisps of f i b r i l l a r  material are seen stretching across the pupillary aperture.  These are  probably remnants of the connective tissue network of the pupillary membrane which has at this point virtually disappeared  (Figure 129).  At a slightly higher magnification, the differences between the pupillary and the peripheral regions are clearly seen (Figure 130).  Large,  rounded, relatively thin-walled capillaries stretch from the pupillary margin to the periphery. functional.  They are f i l l e d with red blood cells and appear  When the blood vessel is slightly displaced from its position,  it is observed that the vessel makes a radially oriented impression on the  circumferentially arranged rows of posterior epithelial cells.  Many of  the blood vessels, though, seem to end at the pupillary region.  It is  d i f f i c u l t to say whether this is artifactual, that is, the capillaries have been removed in the handling of the tissues, or whether this is a true indication of the regression of the capillaries taking place. A closer examination of the pupillary region reveals that most of the capillaries turn around and hug the pupillary margin i t s e l f (Figure 131).  A scanning electron micrograph of the pupillary margin viewed edge•  on (Figure 132) shows that these blood vessels come from the i r i s stroma and make a complete turn at the pupillary margin.  Most of the capillaries  l i e superficial to the underlying amorphous layer although a few are s t i l l embedded in i t (Figure 131).  Most of the capillaries that are confined to  the pupillary region, are no longer round tubes containing red blood cells. Instead, many of them show bulges on the sides (Figure 133).  It appears  as though the endothelial c e l l walls are thinned out so that any red blood cells in the lumens can push outwards. a state of atrophy (Figure 134).  Many of the capillaries appear in  The cells present do not look normal.  Highly coiled f i b r i l l a r material and cellular debris are common (Figures 133, 134). Beneath the atrophied capillaries, the posterior surface of the i r i s is smooth, except for a few striations.  This is very similar to  what is boserved in the adult sphincter region (Figure 134). The posterior epithelial cells are arranged in ridges which are in general circumferentially oriented (Figures 135, 136). It is d i f f i c u l t to trace the entirety of a row of epithelial cells as the epithelial ridges merge and branch in a complicated way (Figures 135, 136). The epithelial ridges are separated by grooves of varying depths.  The individual poster-  ior epithelial cells can be recognised as their nuclei bulge a l i t t l e more than the rest of the c e l l cytoplasm.  The basement membrane covering the  posterior epithelial cells is highly wrinkled but to a lesser extent over the nuclei.  The crinkles over the cytoplasmic portions of the cells  probably suggest that there are cytoplasmic cell processes underlying the basement membrane. Near the c i l i a r y body, the beginnings of the adult c i l i a r y - i r i s processes are observed (Figure 137).  These radial ridges are not as promi-  nent as those in the adult but they show the general characteristics of the adult c i l i a r y - i r i s processes.  It is a peripherally located, radially-  oriented ridge, being higher at the peripheral end and going deep into the i r i s tissue at the pupillary end (Figure 137).  There is a break in the  continuity of the posterior epithelial ridges over the highest point of the c i l i a r y - i r i s process. to some degree.  The other epithelial ridges tend to skirt around i t  Some epithelial ridges are seen going over the lower  portions of the c i l i a r y - i r i s process.  These ridges are not as high nor  are the grooves as deep as those of the rest of the posterior surface of the i r i s .  285.  Figure 128  From 11 Days Post-natal On (SEM) The pupil is in partial constriction. The posterior surface of the i r i s is relatively smooth. Some faint circumferential striations are barely perceptible. At the periphery of the iris in the region of the c i l i a r y body, a few small, short radial ridges are seen. The pupillary margin is slightly corrugated. Right around the pupillary margin, blood vessels of varying lengths are embedded in an amorphous layer. Some of the blood vessels terminate at the pupillary region while a few traverse the posterior surface of the i r i s to varying degrees. x 40  Figure 129  From 11 Days Post-natal On (SEM)  .  The pupil is much constricted. The pupil is small and wisps of connective tissue are seen across the pupillary aperture. The posterior surface of the i r i s is smooth except for a small zone around the pupil. An enormous number of blood vessels are embedded in the amorphous material around the pupil, but they are free peripherally. Many of these blood vessels traverse the entire length of the i r i s to the c i l i a r y region. x 40  286.  287.  Figure 130  From 11 Days Post-natal On  (SEM)  Circumferential rows of posterior epithelial cells are present over most of the posterior surface of the i r i s . Most of the blood vessels are limited to the pupillary region. A few stretch between the pupillary edge and the c i l i a r y body. These blood vessels have thin walls so that the red blood cells within the lumens give them a beaded appearance. When a blood vessel has been displaced from its original position, an impression of its path is observed running across the circumferential epithelial ridges. x 160  Figure 131  From 11 Days Post-natal On  (SEM)  A l l of the blood vessels on the posterior surface of the i r i s turn around and hug the pupillary margin itself. A small amount of amorphous material and some connective tissue fibers are s t i l l present in the pupillary region. The posterior epithelial cells are arranged in circumferential rows separated by narrow grooves. The individual nuclei of the epithelial cells can be seen bulging outwards. x 410  288.  289.  Figure 132 From 11 Days Post-natal On (SEM) An edge on view of the pupillary margin shows that the blood vessels from the anterior i r i s stroma turn around at the pupillary margin and apparently go posteriorly and peripherally. x 1,630  Figure 133 From 11 Days Post-natal On (SEM) Atrophying blood vessels are seen in the pupillary region together with some connective tissue remnants. The blood vessel walls bulge out in many places. x 820  290.  291.  Figure 134  From 11 Days Post-natal On (SEM) Only a few atrophied blood vessels and some connective tissue fibers are present. A few fine striations are seen in the pupillary region. x 700  Figure 135  From 11 Days Post-natal On (SEM) The circumferential rows of posterior epithelial cells are clearly visible. The individual cells in each epithelial ridge is demarcated by the bulging nuclei. Deep narrow grooves separate the epithelial ridges. x 820  292.  293.  Figure 136 From 11 Days Post-natal On (SEM) A higher magnification of Figure 135. The epithelial ridges bifurcate, taper down or blend with other epithelial ridges. The basement membrane covering the epithelial cells is wrinkled, but slightly less so over the bulging nuclear regions. x 1,630  Figure 137 From 11 Days Post-natal On (SEM) The beginning of the c i l i a r y - i r i s process is shown here. It is a low radial ridge. The circumferential epithelial ridges either skirt around the c i l i a r y - i r i s process, or there is a break in the continuity of the epithelial ridges. If the epithelial ridges go over the radial ridge, they seem to be flattened or stretched out. x 1,000  294.  295.  G.  Horse-radish Peroxidase (HRP)  Studies of the Iris in Fetal, Post-natal  and Adult Rats The glutaraldehyde-fixed  i r i s tissue is incubated according to the  method set out by Karnovsky (1967).  The reaction product, a brownish to  black precipitate, indicating the location of the HRP, the light microscope.  is observed with  In these studies, i t is found that neither the type  of anesthesia used nor the route of the intravascular infusion of the affected the localisation of the reaction product.  HRP  The eyes were removed  at different time intervals, from 1 to 45 minutes, following the injection of the HRP.  It is observed that there is no qualitative difference in the  localisation of the reaction product dependent on time.  Occasionally,  there are quantitative differences in the intensity of the reaction between the eyes that are removed soon after the injection of the HRP and those that are removed 45 minutes later.  Smith (1971) in his studies on the  mouse, observed that there is a gradual increase in the amount of tracer present with time.  However, in our investigation, this is not consistently  observed. In fetal eyes, (Figures 138,  139) the tracer HRP  is seen in the  developing c i l i a r y body and i r i s as early as 1 minute following the infusion of the HRP. varies.  The overall intensity of the reaction in the tissues  Only occasionally, but not consistently, does this variation in  reaction intensity appear to be time dependent, that is, there is an increase in the intensity of the reaction with time. When the reaction is light, the reaction product is present both in the c i l i a r y body and in the i r i s .  In the c i l i a r y body, the reaction pre-  cipitate is found along the stromal (basal) surfaces of the c i l i a r y pigment epithelium.  In the developing i r i s , the reaction product lines the stromal  surfaces of the anterior epithelium (Figure 138). When the reaction is intense (Figure 139) the reaction precipitate is found along the basal surfaces of the c i l i a r y pigment epithelium, in between the individual pigment epithelial cells in varying degrees, and at times in the space between the c i l i a r y and pigment epithelia.  The stromal  surfaces of the pigment epithelial cells are often coated with the precipitate, especially i f they are abutting directly against a blood vessel in the stroma.  The precipitate may be seen only between the basal halves of  the pigment epithelial cells, or i t may be found a l l along the lateral surfaces of the cells.  Some of the stromal cells in the c i l i a r y stroma are  also covered with precipitate.  In the developing fetal i r i s , the reaction  product is seen along the anterior surfaces of the anterior epithelium and occasionally in between some of the anterior epithelial cells.  As in the  c i l i a r y body, the reaction is always more intense i f a blood vessel is immediately adjacent to the anterior epithelium.  Some of the i r i s stromal  cells overlying the anterior epithelium also show reaction product along their c e l l surfaces.  Some precipitate is seen along the anterior and  pupillary borders of the developing sphincter muscles.  Sometimes, reaction  precipitate may outline the individual cells of the developing sphincter. Some precipitate is occasionally seen along the posterior surfaces of the i r i s posterior epithelium and of the c i l i a r y epithelium facing the posterior chamber of the eye. In young post-natal rat eyes, up to 12 days after birth (Figures 140-149), the localisation of the HRP reaction product is essentially similar to that observed in the fetal eyes.  In the c i l i a r y body, the reac-  tion precipitate is found along the stromal surfaces of the pigment epithelial cells, in between the individual pigment epithelial cells and also in between the c i l i a r y and pigment epithelia (Figures 141, 143, 145, 147,  149).  During this period of time, the c i l i a r y body is developing morpho-  logically.  Ciliary processes are beginning to form.  It is observed that  whenever c i l i a r y processes are present, the reaction precipitate is either more intense or is only localised at the tip of the developing c i l i a r y processes (Figure 149).  Here, the c i l i a r y blood vessel is usually very  intimately associated with the pigment epithelium without any intervening stromal cells.  The reaction precipitate may surround a l l of the surfaces  of the pigment epithelial cells, or the precipitate is found only between the pigment epithelial cells and between the c i l i a r y and pigment epithelia. Occasionally,  reaction product is found within the pigment cells them-  selves, as has been noted in the mouse (Smith, 1971).  This reaction pre-  cipitate is usually concentrated in the anterior basal poles of the cells. As one moves away from the tip of the developing c i l i a r y process, the reaction either diminishes or disappears.  The precipitate is then usually  located between the c i l i a r y and pigment epithelia (Figures 147, 149). In the i r i s , the reaction precipitate is always found along the stromal surfaces of the anterior epithelium,  thus demarcating the boundary  zone between the anterior epithelium and the stroma (Figures 140-144, 146148).  Some precipitate is also found in between the individual anterior  epithelial cells and in between the i r i s anterior and posterior epithelia (Figures 140,  141, 143, 144, 146-148).  This is more prominently evident  in the peripheral portion of the i r i s towards the root.  Usually some pre-  cipitate surrounds the pupillary and anterior borders of the i r i s (Figures 140, 144, 146).  sphincter  Reaction precipitate may be found in between a l l  of the muscle cells of the developing sphincter.  Most often, the reaction  precipitate is found amongst the cells in the posterior half of the sphincter (Figures 140, 142, 144, 146).  Some precipitate is occasionally  seen in the blood vessel overhanging the sphincter (Figure 140) and among  some of the stromal cells (Figures 147, 148). With further development and growth of the rat, some changes are seen in terms of the localisation of the HRP reaction product.  From 15 to  22 days after birth, the sites of the HRP precipitate in the c i l i a r y body are similar to those observed previously and also to those observed in the adult.  In the c i l i a r y body, the reaction precipitate is more definitively  localised at the tips of the c i l i a r y processes (Figure 150). the i r i s some changes occur.  However, in  At 15 days after birth, there is some, but  very l i t t l e , reaction precipitate seen in the i r i s tissue i t s e l f .  It is  usually localised at the very root of the i r i s near to the c i l i a r y body. By 22 days after birth, the reaction in the i r i s disappears.  As in the  adult rat eye, reaction precipitate for HRP is found only in the c i l i a r y processes but not in the i r i s .  299.  Figure 138 20-21 Days Fetal (HRP) The overall reaction for HRP is light. The precipitate is seen lining the stromal surfaces of the c i l i a r y pigment epithelium (cpe) and of the anterior epithelium (ae) of the i r i s . No reaction precipitate is seen in the c i l i a r y epithelium (ce), posterior epithelium of the i r i s (pe) or in the sphincter (sph). There is a small amount of precipitate in the c i l i a r y stroma (cs) but not in the i r i s stroma (s). x 250  Figure 139 20-21 Days Fetal (HRP) The reaction for HRP is strong. In the developing c i l i a r y body, reaction precipitate is found a l l along the basal (stromal) surface of the c i l i a r y pigment epithelium (cpe), outlining the individual cells of the c i l i a r y pigment epithelium and in between the c i l i a r y epithelium (ce) and the c i l i a r y pigment epithelium. In the developing i r i s , the reaction is intense a l l along the stromal surface of the anterior epithelium (ae) and this continues on to line the anterior and pupillary borders of the developing sphincter (sph). Some precipitate permeates in between some of the sphincter muscle cells. At the root of the developing i r i s , some precipitate is seen delineating the boundaries of the anterior epithelial cells. There is some precipitate in between some of the cells at the c i l i a r y - i r i s junction (arrow). Some precipitate is present both in the c i l i a r y (cs) and i r i s (s) stroma. x 220  300.  301.  Figure 140  1 Day Post-natal (HRP) In the developing sphincter (sph), the reaction precipitate is found in between almost a l l of the muscle cells. Some precipitate is present along the pupillary margin of the sphincter. There is a relatively intense reaction in the stroma (s) overhanging the pupillary margin of the sphincter and along the stromal surface of the anterior epithelium (ae). Reaction precipitate is seen in between the cells of the anterior epithelium and along the boundary between the anterior epithelium and the posterior epithelium (pe). x 350  Figure 141 1 Day Post-natal (HRP) In the developing c i l i a r y body, the reaction is most intense along the stromal surface of the c i l i a r y pigment epithelium (cpe), especially where i t is right next to a blood vessel (bv) of the c i l i a r y stroma (cs). The reaction precipitate lines a l l of the surfaces of the c i l i a r y pigment epithelial cells. At the bases of some of the c i l i a r y pigment epithelial cells, some precipitate appears to be within the c e l l cytoplasm. . ". . x 350  303.  Figure 142 4 Days Post-natal (HRP) There is much reaction precipitate a l l along the stromal surface of the anterior epithelium (ae). There is some precipitate in the stroma (s) at the peripheral part of the developing sphincter (sph). A very faint reaction is seen' in between some of the developing sphincter muscle cells. x 220  Figure 143 4 Days Post-natal (HRP) In the developing i r i s , reaction precipitate coats the stromal surface of the anterior epithelial cells (ae). Some precipitate is also found between the individual anterior epithelial cells, between the anterior and posterior epithelium (pe), .especially towards the root of the i r i s . The c i l i a r y body is beginning to form c i l i a r y processes. The reaction precipitate is found primarily in between the c i l i a r y epithelium (ce) and the c i l i a r y pigment epithelium (cpe) and in between the individual cells of the c i l i a r y pigment epithelium. Some precipitate is also found along the stromal surface of the c i l i a r y pigment epithelial cells towards the tip of the developing c i l i a r y process. x 220  304.  305 „  Figure 144 5 Days Post-natal (HRP) Reaction precipitate for HRP is seen a l l along the pupillary margin of the sphincter (sph), in between the muscle cells, along the stromal surface of the anterior epithelium (ae), in between the anterior epithelial cells and in between the anterior and the posterior epithelium (pe). In the i r i s stroma (s) immediately adjacent to the anterior epithelium and in the stroma overlying the tip of the sphincter, there is also some reaction. x 220  Figure 145 5 Days Post-natal (HRP) The c i l i a r y body consists of one large c i l i a r y process which is beginning to branch. The reaction appears more pronounced towards the tip of the c i l i a r y process, where the precipitate lines a l l of the surfaces of the c i l i a r y pigment epithelial cells (cpe). Some precipitate may be within the basal poles of the cells. Towards the base of the c i l i a r y process, the precipitate is found mainly between the c i l i a r y epithelium (ce) and the c i l i a r y pigment epithelium. There is also some precipitate in between a few of the cells of the c i l i a r y epithelium. x 220  307.  Figure 146 7 Days Post-natal (HELP) In the developing sphincter (sph) there is reaction precipitate only among the muscle cells in the posterior half of the sphincter. Some precipitate is seen coating the anterior, pupillary and posterior borders of the sphincter. Reaction is abundantly present along the stromal surface of the anterior epithelium (ae) and in the immediately adjacent stroma (s). There is a hint of some precipitate along the posterior surface of the iris. x 220  Figure 147  7 Days Post-natal (HRP) In the developing i r i s , the reaction precipitate clearly demarcates the extent of each anterior epithelial c e l l (ae). However, the reaction peters out toxvards the root of the i r i s . In the developing c i l i a r y process, the reaction is more intense towards the tip of the c i l i a r y process and gradually diminishes away from the tip. At the tip of the c i l i a r y process, the precipitate lines a l l of the surfaces of the c i l i a r y pigment epithelial cells (cpe). At the base of the c i l i a r y process, the precipitate is mainly seen in between the c i l i a r y pigment epithelium and the c i l i a r y epithelium (ce). x 220  308.  309.  Figure 148  12 Days Post-natal (HRP) The reaction precipitate is observed mainly in the i r i s stroma (s) and along the stromal surface of the anterior epithelium (ae). There is a faint reaction in between the anterior epithelial cells and in between the anterior and posterior epithelium (pe). x 220  Figure 149  12 Days Post-natal (HRP) Long c i l i a r y processes are observed. The HRP reaction precipitate is more intense at the tips of the c i l i a r y processes, especially where the blood vessels (bv) expand. At the tips of the c i l i a r y processes, the reaction is very intense along the stromal surface of the c i l i a r y pigment epithelial cells (cpe) and less so in between the individual pigment epithelial cells and in between the c i l i a r y pigment epithelium and the c i l i a r y epithelium (ce). Some precipitate is also found within the stromal poles of the c i l i a r y pigment epithelial cells. As one moves towards the root of the c i l i a r y process, the precipitate along the stromal surface of the c i l i a r y pigment epithelium disappears leaving some precipitate in between the individual c i l i a r y pigment epithelial cells and in between the c i l i a r y pigment epithelium and the c i l i a r y epithelium. x 220  310.  311.  Figure 150  Adult  (HRP)  The localisation of the reaction precipitate for HRP in the c i l i a r y process is similar to that in Figure 149. HRP reaction precipitate is only found in the c i l i a r y process and not in the i r i s (I). x 220  313.  IV.  DISCUSSION AND SUMMARY OF THE RESULTS  A. A Light and Transmission Electron Microscopic Study of the Rat Iris in Pupillary Dilation and Constriction  1. General The rat pupil is capable of extensive excursions in response to changing light conditions.  This can be quite easily observed by either  shining a bright light onto the eyes, at which point the pupils constrict to oftentimes a pinhole, or observing the eyes i n a slightly shaded condition, when the pupils dilate.  In normal laboratory conditions, the rat  pupils are in some degree of pupillary constriction.  In order to study the  structural changes of the i r i s that must necessarily occur as the pupil constricts or dilates, we must be able to maintain the i r i s in a fixed state of dilation or constriction. aid  of chemical mediators.  In our investigations this is done with the A mixture of phenylephrine hydrochloride and  cyclopentolate and echothiophate iodide are regularly used to dilate and constrict the pupils, respectively.  Phenylephrine hydrochloride (Goodman and  Gilman, 1967) with the chemical structure  /  V-CH —CH—NH  \J  i ii  / OH H CH3 OH mydriatic in ophthalmology. Dilation of the pupil occurs without any increase in intraocular pressure. Phenylephrine hydrochloride is used tois a sympathomimetic drug..  It is used as a  gether with cyclopentolate, (Goodman and Gilman, 1967) an antimuscarinic agent. Cyclopentolate, COOCH CH_N(CH ) | CH 2  blocks the action of acetylcholine on the i r i s sphincter muscle, thus resulting in mydriasis.  a*  OH  3  2  314.  Echothiophate iodide (Goodman and  0  Gilman, 1967) is a potent derivative  SCH CH N(CH3)3  of choline.  +  C2H5O  2  It is a powerful anti-cholinesterase agent.  to the eye, i t causes miosis within a few minutes.  I  2  When i t is applied  Concomitant with miosis  there is usually a f a l l in the intraocular pressure and thus i t is also used in the treatment of glaucoma. The mixture of phenylephrine hydrochloride and cyclopentolate, and echothiophate iodide are applied locally and the pupils reach maximal dilation or constriction after about 20 minutes.  Usually both dilation and  constriction is observed from the same animal as one eye is dilated while the other is constricted. enucleation occurs.  The.rat is placed under anesthesia while ;  The irises are fixed both by immersion and by perfusion in various fixatives.  When the irises are fixed by immersion, a few d i f f i c u l t i e s are  sometimes encountered.  As soon as enucleation occurs, dilation of the  pupil tends to take place unless i t is transferred almost immediately into the fixative and chemical fixation is almost instantaneous.  Any delay would  result in the pupil being in partial constriction rather than in extreme miosis.  If the pupil is already dilated, then there is no tendency for  constriction to occur.  This phenomenon of "death dilation" has been  commonly observed but not understood (Alphen, 1963; Kelly and Arnold, 1972). To prevent pupillary changes from taking place after the eyes are removed from the animal, Alphen (1963) froze the monkey eyes in situ, while Kelly and Arnold (1972) perfused the rats with f i r s t a salt solution which is then followed by solutions of osmium tetroxide or glutaraldehyde. Both methods are equally effective for maintaining the pupils in extreme miosis or mydriasis. In our investigation, i t is found that with speed and care the irises  315.  can be maintained in pupillary dilation or constriction using the method of immersion fixation. For light microscopic studies, the iris tissue appears relatively adequately fixed.  There are some vesicles in the posterior and  anterior epithelial layers but at the magnification and resolution obtainable with the light microscope i t cannot be determined i f these vesicles are artifactual or real.  However, the requirements for the preservation of  ultrastructural details of the tissue are much more stringent for transmission electron microscopy than for light microscopy.  Immersion fixation  does preserve the overall configurations and structural characteristics of the cellular components of the i r i s but there is some intracellular tissue disruption.  The mitochondria are primarily affected.  so that many vesicles with cellular debris are found.  The cristae break up This would contribute  in part to the vesiculated nature of some of the posterior and anterior epithelial cells as seen light microscopically. fusion fixation is used.  To circumvent this, per-  As the rat is being perfused with the fixative,  the pupils remain in the same degree of miosis or mydriasis suggesting that chemical fixation has happened almost instantaneously.  The presence of  both thick and thin filaments in the sphincter and dilator muscles is one criterion that fixation is adequate (Kelly and Arnold, 1972). observed in our perfused specimens.  These are  However, contrary to Kelly and Arnold's  findings, perfusion with a salt solution prior to the administration of the fixative is not essential for preserving the thick and thin filaments. Perfusion with the fixative alone is sufficient.  Even when the i r i s as a  whole appears well-fixed, some tissue disruption is s t i l l present in isolated parts.  This is especially noticeable in the posterior  epithelium.  The posterior epithelial cells appear to be particularly sensitive to any external changes.  Perhaps, the posterior epithelial cells are held in such  a fine balance with the surrounding aqueous humor that even the slightest  change in the milieu, like the presence of a chemical fixative, immediately evokes disruptive changes within the c e l l cytoplasm. Perhaps i t w i l l be questioned whether the histological and ultrastructural changes of the iris tissues that are seen in extreme pupillary dilation or constriction as induced by the local administration of drugs are truly indicative of the changes that normally occur in response to changing light conditions without the intervention of an external agent. From present studies this question remains unresolved.  Further studies  will have to be carried out to compare the histological characteristics of the i r i s in pupillary dilation and constriction during its normal responses to changing light conditions and also during its responses to different types of drugs.  2.  The Histological and Ultrastructural Features of the Iris in Pupillary Dilation and Constriction  The most striking differences between the histology of the i r i s when i t is in pupillary dilation from when i t is in pupillary constriction are primarily noted in the posterior neuroectodermally derived layers. Thus our attention and interest is focussed here.  Observations made on  the light microscope w i l l be correlated with observations .made on the transmission electron microscope. When the pupil is dilated, the i r i s is short and plump while when the pupil is constricted,  the i r i s is thin, delicate and tenuous.  There is  an apparent drastic change in the total exposed surface area of the i r i s . This is very clearly shown by scanning electron microscopy.  The shapes of  the posterior epithelial and dilator cells, and their relationships to each other alter in response to changes in pupillary size.  We are interested in  317.  probing into what these changes are and what are the characteristics of these layers which allow them to so very efficiently accommodate themselves to changes in pupillary diameter. The posterior epithelial cells form a layer covering the posterior surface of the i r i s .  In pupillary dilation, the posterior surface of the  iris is most often deeply convoluted as the result of the rows of peg-like epithelial cells which are discretely separated from each other.  Occasion-  ally, the posterior epithelial cells are very close together without any intervening gaps, as seen light microscopically.  The posterior surface of  the rat i r i s in pupillary dilation is not arranged in arcades as in the monkey iris where each arch is made up of eight to ten cells (Alphen, 1963). Rather, the posterior epithelial cells are arranged individually in rows. With the transmission electron microscope, the arrangement of the posterior epithelial cells with respect to each other, as seen with the light microscope, is verified.  In addition, the transmission electron microscope re-  veals that a typical basement membrane is associated with the c e l l membranes of the posterior epithelial cells.  There is always a clear zone between  the cell membranes and the electron-dense basement membrane. Perhaps, this space allows some freedom of movement to the epithelial cells. adherent membrane might be a mechanical disadvantage.  A closely  Light microscopically,  the posterior epithelial cells show large c e l l processes which are devoid of nuclei.  Electron microscopically, these processes consist essentially  of a mass of c e l l infoldings with some organelles.  In fact, c e l l infoldings  are extensively found on a l l the surfaces of the posterior epithelial cells leaving only a small rim of cytoplasm around the nucleus.  The c e l l in-  foldings are highly complex and they interdigitate with each other in a three dimensional meshwork. The basement membrane does not follow the outlines of the cell infoldings. Rather, i t covers the posterior surface  313.  of the i r i s like a loose sheet. In pupillary constriction, the configuration of the posterior epithelial cells is changed markedly.  From being high columnar cells in  pupillary dilation, the epithelial cells are flattened out so that they appear as a thin layer. longer apparent.  The gaps in between the individual cells are no  The posterior surface of the i r i s is relatively flat in  extreme miosis with only an occasional bulge. closely associated with its neighbour.  Each epithelial c e l l appears  In the rat i r i s , there are no  observable tight junctions between adjacent posterior epithelial cells, whereas tight junctions are present in the human i r i s (Hogan, Alvarado and Weddell, 1971).  This would probably account for the observation that the  rat i r i s can be more extensively dilated or constricted than the human iris (Newsome and Loewenfeld, 1971).  The c e l l infoldings are s t i l l present. It  is impossible to say whether some of the cell infoldings have opened out, as i t were, to provide more c e l l membrane to cover the posterior surface of the i r i s .  They s t i l l interdigitate complexly with each other.  The thick-  ness of the basement membrane is not measured in pupillary dilation. perhaps also thin out when the pupil is extremely constricted.  It may  In some of  our scanning electron micrographs of pupillary constriction, the basement membrane appears to have been thinned out to reveal the underlying c e l l interdigitations.  The basement membrane may have been a r t i f i c i a l l y removed  in the specimen preparation rather than being thinned out as a result of pupillary constriction. Within the substance of the posterior epithelial cells, certain changes are also seen, especially with respect to the shape and orientation of the nuclei and the disposition of the intracellular filaments. In pupillary dilation, the nucleus of the posterior epithelial cell has a very irregular outline.  The length of the nucleus lies along the long axis of  319. the cells.  In electron micrographs, the nuclei show deep indentations of  the nuclear membrane.  These indentations are occupied by tongues of uni-  formly staining cytoplasm devoid of cell organelles.  There is much dense  heterochromatin associated with the nuclear envelope and within the nuclear substance itself.  It seems that there is always a clear space between the  outer and inner nuclear membranes. This is seen in well-fixed material so that this space is unlikely to be artifactual.  From pupillary dilation to  pupillary constriction, the nuclei of the posterior epithelial cells change in shape and in orientation. The nuclei become oval or elongated in shape and are oriented parallel to the posterior surface of the i r i s , as are the epithelial cells.  The nuclear outline is relatively smooth.  very rarely any nuclear indentations.  There are  The nucleus of the posterior epi-  thelial cells, though large in relation to the cell volume, is highly pliable and readily alters its shape from pupillary dilation to pupillary constriction.  Perhaps, the looseness of the enveloping nuclear membranes  facilitates, or at least, does not hinder, the alteration of the nuclear configuration. The usual cell organelles are found in the cytoplasm. there are bundles of intracellular filaments.  In addition,  Such filaments have not been  described by other investigators. It is not known whether they are present only i n the posterior epithelial cells of the rat i r i s .  The other cell  organelles are not oriented in any particular way within the c e l l cytoplasm. These intracellular filaments, however, change in their orientation within the c e l l in miosis and mydriasis. close to the nucleus.  The filaments are usually found quite  This may not be of any significance since there is  only a thin rim of cytoplasm around the nucleus and the rest of the c e l l volume is made up of cell infoldings. In pupillary dilation, bundles of filaments seem to cascade around the nucleus, primarily along the lateral  320.  and posterior regions. cytoplasm.  There are occasionally some filaments in the anterior  These bundles of filaments may be quite large as is fortuitously  seen in a cross-section.  On the whole, the filaments appear to form a  loose 'hammock' around the nucleus.  In pupillary constriction, the f i l a -  ments are mainly found in the posterior half of the epithelial cells.  The  filaments run in bundles along the long axes of the cells and parallel to the posterior surface of the i r i s . they may  form a discrete bundle.  to attach  They form an intermeshing network, or Occasionally, a bundle of filaments appear  to dense areas of the cell membranes. We can only speculate as  to the possible nature and function of these filaments. only a structural component of the epithelial cells. elasticity to them.  Perhaps, they are  They may have a certain  But any elasticity would seem to be functional only i f  the filaments are anchored in position at certain points.  We cannot t e l l  whether this is so from our results. There is only a hint that some of the filaments are attached to the cell membranes. The posterior epithelium and dilator are intimately related to each other.  In pupillary dilation, the dilator muscle layer is readily seen  both with the light and transmission electron microscopes. thick as the epithelial cells are high.  It may be as  In mydriasis, the boundary between  the posterior epithelium and the dilator is not easily traced.  The poster-  ior epithelial cells interdigitate with the dilator cells in different planes so that the extent of each c e l l is not well-delineated.  In miosis,  however, the boundary between the two layers is a relatively smooth, undulating line.  Microvillous processes from both layers interdigitate loosely  with large gaps in between the interdigitations. together by cell junctions.  The two layers are bound  Tight junctions are the most common, although  some junctions occasionally show some semblance to desmosomes, but these are rare.  In the excursions of the i r i s , these cell junctions would play a  321.  role in maintaining the integrity of the two layers in their relationships to each other. The individual dilator cells also interdigitate extensively with each other.  This accounts for the light microscopic observation that the  boundaries of the dilator cells are not apparent.  In pupillary dilation,  the dilator muscle cells interdigitate with each other in a more three dimensional manner whereas in pupillary constriction, there appear to be fewer interdigitations which relate with each other in a somewhat two dimensional fashion. The nuclei of the dilator muscle cells behave similarly to those of the posterior epithelial cells.  In pupillary dilation, the nuclear outlines  of the dilator muscle cells are more highly indented than those of the posterior epithelial cells.  Again, cytoplasmic protrusions occupy the  depths of the indentations. In pupillary constriction, the nuclear outlines smooth out, generally to give a long cigar-shaped nucleus.  Occasionally, a  deep indentation is seen but this would be oriented parallel to the posterior surface of the i r i s . The most striking changes are seen along the boundary zone between the dilator muscle fibers and the stroma.  In pupillary dilation, numerous  arborescent processes are observed a l l along the boundary zone. very darkly with toluidine blue and are also very electron-dense.  They stain The  sarcoplasm is highly concentrated here so that the myofilaments are not readily visible in electron micrographs.  These dilator processes may be  simple protrusions of the dilator into the stroma, or they may arise from a hillock of dilator material, in which case, they branch and spread out in a fan-like manner. Occasionally not only dilator processes but groups of dilator cells encroach on the stroma as muscle spurs. commonly seen as the dilator processes.  This is not as  Since the dilator layer is attached  322.  to the posterior epithelium, which is i t s e l f a bulky structure, in extreme pupillary dilation, there is not enough room to accommodate a l l of the dilator cells.  Parts of the cells have to be squeezed outwards and this is  only possible anteriorly into the relatively loose stroma.  It would be  interesting to speculate that there are certain well-defined regions a l l along the stromal surfaces of the dilator muscle cells where dilator processes can protrude outwards. structured components.  The dilator processes do appear to be well  They do not seem to have been formed haphazardly.  Perhaps, the dilator cells are arranged in contractile units. the sphincter,  At least in  there is some indication that the muscle cells are arranged  in functional groups (Hogan, Alvarado and Weddell, 1971). case with the dilator as well. unit of cells may  The force of contraction  be concentrated at certain points.  This may  be the  for each functional  Here, the dilator  material would bulge out as a hillock from which would arise a series of profusely branched dilator processes. In pupillary constriction, the dilator layer is barely visible light microscopically.  The nuclei are large and occupy most of the c e l l volume.  The contractile portion is confined to a small strip along the boundary zone with the stroma, which appears as a dense line with the light microscope.  With the electron microscope, the myofilaments are quite readily  discernible as the sarcoplasm is not as dense as in pupillary dilation. stromal surface of the dilator is usually smooth.  The  The dilator processes, so  numerous and striking in pupillary dilation, are missing in pupillary constriction.  Very rarely, there may  into the stroma. flattened.  be a few short dilator processes jutting  The whole anterior surface of the dilator layer has been  In pupillary constriction, a l l of the dilator cells can be  accommodated within one layer and there is no necessity the dilator to occur.  for protrusions of  323.  A basement membrane lines the anterior surface of the dilator cells. As with the basement membrane of the posterior epithelial cells, the basement membrane of the dilator drapes loosely the dilator and its processes in pupillary dilation.  The basement membrane has to be relatively malleable so  that the dilator processes can protrude into the stroma without any d i f f i culty.  In pupillary constriction, the basement membrane appears to be  qualitatively thinner, almost as i f i t had been stretched. The various types of cells in the stroma are not identified as the main concern here is in the overall arrangement of the cells and connective tissue framework in the stroma.  In mydriasis, the stromal cells and their  processes appear to be disposed in vertical columns extending from the anterior surface of the i r i s to the boundary zone with the dilator.  The  spaces in between the cells accentuate this impression of vertical linearity. Only the processes of the stromal cells, but not the nuclei, come relatively close to the dilator processes, so as not to impede the conformational changes of the dilator.  The basement membrane of the dilator cells and the  surrounding collagen fibers around the dilator processes impart a 'halo' to them which is free of cells.  In miosis, the stromal cells and the assoc-  iated intercellular spaces are no longer perpendicular but parallel to the posterior surface of the i r i s .  With a special stain for collagen, Mallory's  Trichrome Stain, i t is seen that the collagen network within the i r i s stroma follows closely the changes in orientation of the stromal cells.  The colla-  gen fibers are parallel to the posterior surface of the i r i s in pupillary constriction but they are perpendicular to the posterior surface of the iris in pupillary dilation.  Thus between the two extremes of pupillary size, the  stromal components apparently go through a shift in position.  In some  studies (Tousimis and Fine, 1959b) the collagen appears to surround the stromal cells and to delineate out various intrastromal tissue channels  324.  which are quite different and separate from the readily identifiable blood vessels (Gregersen, 1958a, 1958b, 1959b; Francois, Neetens and Collette, 1960). Perhaps the whole collagen network in the stroma is interconnected  and i t  serves to maintain the stromal cells in their positions relative to each other, as well as to keep open the tissue spaces or channels.  Thus the func-  tional integrity of the stroma is maintained through the extreme changes in structural orientation that occur in pupillary dilation and constriction.  B.  A Scanning Electron Microscopic  Study of the Rat Iris in-Pupillary  Dilation and Constriction  1.  Comments on the Methodology  The scanning electron microscope is used in our present investigation to study the ultrastructure of the anterior and posterior surfaces of the rat i r i s .  In light microscopic studies of meridionally sectioned rat  i r i s , i t is observed that the posterior surface of the i r i s is covered by a layer of posterior epithelial cells while the anterior surface of the i r i s is covered by a layer of squamous endothelial cells.  Our main interest is  focussed on the configurations of these surface cells and their relationships to each other.  In addition, we know that the i r i s is not a static  structure but i t is constantly changing its total surface area, thus regulating the size of the pupil, in response to external changing light conditions.  In our light microscopic studies, the shapes of the posterior epi-  thelial cells likewise change to accommodate to these excursions of the i r i s tissue in pupillary dilation and constriction.  It is not possible, however,  to t e l l whether the shapes of the anterior endothelial cells alter with pupillary dilation and constriction although i t is observed that the overa l l contour of the anterior i r i d i a l surface is different when the i r i s is  in pupillary dilation than when i t is in pupillary constriction. was  Thus it  deemed useful to employ the scanning electron microscope as a tool to  compare the ultrastructural features of the anterior and posterior surfaces of the rat i r i s in pupillary dilation and constriction, as such a study has not been carried out before. The pupil constricts or dilates not only in response to light conditions but also in response to certain drugs, the miotics and mydriatics.  In  our studies a mixture of phenylephrine hydrochloride and cyclopentolate,  and  echothiophate iodide are regularly used to respectively dilate and constrict the pupils.  The eyes are removed immediately and fixed by immersion.  Hansson (1970), in his investigations of the ultras trueture of the surface of the rat i r i s , mentions that by using fixation by immersion, the tissues become twisted and curled. studies.  However, this problem is not encountered in our  The i r i s tissues do occasionally curl a l i t t l e , but this is the  result of later steps in the preparative procedures, namely, during the drying of the tissues.  Such slight twisting or curling of the i r i s tissue  does not affect the morphological characteristics as examined with the scanning electron microscope. The i r i s tissues are fixed in glutaraldehyde, or in glutaraldehyde followed by post-fixation in osmium tetroxide.  It was  thought that perhaps  the incorporation of a heavy metal into the tissue itself would give better resolution, or allow the tissues to stand up better under the electron beam. In our investigation, there appears to be no difference between the tissues that are fixed in glutaraldehyde alone and those which had a double fixation. Freeize-drying  is commonly used to prepare biological specimens for  examination with the scanning electron microscope. method used by Hansson (1970).  Freeze-drying is the  However, freeze-drying  has certain dis-  326.  advantages.  During the i n i t i a l freezing process, ice-crystals inevitably  form and disrupt the tissue components.  Ice-crystal artifacts can be re-  duced, but not eliminated entirely, by numerous methods (Boyde and Wood, 1969)..  To circumvent these problems, two other methods have been intro-  duced for drying the tissues, the camphene method (Watters and Buck, 1971) and the c r i t i c a l point drying method (Boyde and Wood, 1969; Smith and Finke, 1972).  These two methods are utilised in our studies to prepare the tissues  for scanning electron microscopy.  Both are as effective in giving a con-  sistent image of the posterior and anterior surfaces of the rat i r i s in pupillary dilation and constriction. Following the camphene method (Watters ,and Buck, 1971), the fixed tissues are f i r s t dehydrated, in a graded alcohol series and then transferred into propylene oxide. The tissue is infiltrated with liquid camphene at o 45 C. When brought to room temperature, the camphene solidifies.  It is  sublimed from the tissue in a vacuum evaporator leaving a dried specimen. The advantage of this method is that a fluid-air interphase is eliminated and with i t any damages or distortion which can occur as a result of surface tension forces. The c r i t i c a l point drying method (Boyde and Wood, 1969; Smith and Finke, 1972; Hollenberg and Erikson, 1973) of dehydration of tissues gives consistently good results with the scanning electron microscope.  The water  content in the tissues is replaced in a series of steps by alcohol, amyl acetate and liquid carbon dioxide.  The tissues, immersed in liquid carbon  dioxide, is enclosed in a pressurized 'bomb' which is then heated to above the c r i t i c a l point of the carbon dioxide.  At this c r i t i c a l point, the  liquid and gaseous phases of the carbon dioxide are in equilibrium, so that there is no surface tension on the tissues as the carbon dioxide is removed from the specimens.  Distortions that occur as a result of phase boundaries  327.  are eliminated. Certain differences are perceived between the results of Hansson's studies (1970) and our studies. parts of the discussion.  These w i l l be alluded to in the appropriate  The most likely explanation lies in the differ-  ences in methodology for drying of the i r i s tissues. The differences in morphology of the posterior and anterior surfaces of the rat i r i s in pupillary dilation and constriction is of special interest to us.  Observations made with the scanning electron microscope are corre-  lated with observations made with the light microscope on plastic sections of the rat i r i s which have been cut meridionally. The changes in the contours of both the posterior and anterior surfaces of the i r i s from pupillary dilation to pupillary constriction are quite dramatic.  A hint of these surface changes is given by studying meri-  dional sections of the rat i r i s with the light microscope.  However, the  total view of the surfaces is not possible, due partly to the limited extent of the sampling of the tissues in sections and also partly to the fact that the posterior and anterior surfaces are only seen in two dimensions. The scanning electron microscope has these two advantages over the light microscope.  A large area of the tissue surface can be viewed concurrently and  what is perceived is a three dimensional rather than a two dimensional image. In addition, new information on the surface structures of the i r i s is also obtained.  2.  The Posterior Surface of the Iris in Pupillary Dilation and Constriction  In pupillary dilation, the posterior surface of the rat i r i s , as viewed light microscopically, is lined by a row of large, peg-like, columnar epithelial cells from the root of the i r i s to the peripheral extent of the  328.  sphincter.  The heights of the epithelial cells vary.  The grooves in between  the individual posterior epithelial cells also vary in width and depth depending on the degree of pupillary dilation.  In general, the posterior  surface has a highly convoluted appearance up to the periphery of the sphincter.  The posterior surface of the sphincter is lined by a squamous  epithelium and this presents a smooth outline.  With the light microscope,  i t is not possible to visualise the basement membrane which covers the posterior surfaces of a l l the epithelial cells but with the transmission electron microscope, the basement membrane is readily apparent. A scanning electron -micrograph of the posterior surface of the i r i s in pupillary dilation shows that i t is deeply grooved.  Circumferentially  oriented ridges are separated by grooves over most of the i r i s posterior surface.  These ridges represent rows of posterior epithelial cells. The  height of these ridges vary corresponding to the differing heights of the epithelial cells in varying degrees of pupillary dilation.  The whole post-  erior surface is covered by an amorphous basement membrane so that the outlines between individual cells in a single epithelial ridge are not discernible.  The posterior epithelial cells are arranged in circumferential but  not concentric rows.  Neighboring rows of cells may come together, or a row  of cells may bifurcate to form two rows which then blend with other rows of cells.  This aspect of the arrangement of the posterior epithelial cells in  the whole i r i s is not evident in light microscopic  studies.  According to  Fine and Yanoff (1972), in the human i r i s , the posterior epithelial cells within any one ridge are arranged in a staggered fashion.  This  observation  is also most probably applicable to the rat posterior epithelium, although we are not able to make this observation ourselves owing to the superficial layer of basement membrane covering the posterior i r i d i a l surface.  This  staggered arrangement of the epithelial cells in each row would explain what  329.  is seen in a meridional section of the rat i r i s .  The widths of the epi-  thelial cells are not always similar and the nuclei are not regularly observed within each and every one of the cells from the root of the i r i s to the periphery of the sphincter. Light microscopic studies show that the individual posterior epithelial cells have a highly irregular outline as a result of variously sized cytoplasmic cell processes.  With the scanning electron microscope, i t is  observed that the basement membrane follows the overall contours of the cells but i t does not extend into the cells to cover a l l of the smaller cytoplasmic cell processes. scopic picture.  This is verified by the scanning electron micro-  The basement membrane is uneven over the ridge of epithel-  i a l cells and shows bumps and corrugations.  The basement membrane extends  deeply into the grooves between the epithelial ridges but i t does not extend into the row of cells, that is, into each c e l l . At the pupillary margin, the posterior surface of the i r i s in pupillary dilation is relatively smooth as viewed with the scanning electron microscope.  It corresponds to the flattened epithelium which lines the  posterior surface of the sphincter. Quite distinct differences are seen between the posterior surface of the i r i s in pupillary dilation and the posterior surface of the i r i s in pupillary constriction.  Unlike the extensively ridged i r i d i a l  posterior  surface seen in pupillary dilation, in pupillary constriction the posterior surface is relatively smooth.  The appearance of the i r i d i a l posterior sur-  face varies slightly depending on whether the pupil is partially or extremely constricted. In less extreme states of pupillary constriction, referred to here as simply pupillary constriction (refer to the results), the posterior epithelial cells are low cuboidal, as seen with the light microscope.  They may  330.  bulge a l i t t l e , thus giving the posterior surface a very slightly scalloped appearance in meridional sections.  In a scanning electron micrograph, the  i r i s posterior surface in pupillary constriction exhibits slight and bulges.  corrugations  Each bulge presumably represents a single posterior epithelial  c e l l which is polygonal or slightly spindle-shaped, as has been previously observed (Hansson, 1970).  The circumferential arrangement of the epithelial  cells in rows in pupillary dilation is lost over most of the posterior surface of the i r i s .  Occasionally, the epithelial cells may appear to be dis-  posed in just perceptible circumferential rows but the deep grooves and high ridges have been flattened out as the i r i s goes from pupillary dilation to pupillary constriction. The basement membrane s t i l l covers the posterior c e l l surface and obscures the outlines of the individual cells.  However,  the limits of each cell can be vaguely made out by assuming that each bulge represents the nucleus of a posterior epithelial c e l l .  A line drawn midway  between the nuclei would give us an idea of the size of the posterior epithelial cells at a particular stage of pupillary constriction. Such measurements of c e l l size are not made in our investigation.  Occasionally the  basement membrane is thinned out or removed so that what appears to be cytoplasmic processes of the epithelial cells are seen. In extreme pupillary constriction, the posterior epithelial cells are flattened, in light microscopic as a smooth line.  studies.  The posterior surface appears  The sphincter consists of a bundle of closely packed  muscle cells located at the pupillary margin.  In scanning electron micro-  scopy, the posterior i r i d i a l surface is smooth and quite non-descript most of the i r i s , except for a small zone around the pupil.  over  This difference  is not observed in plastic sections probably because of the sampling size. The posterior epithelial cells are completely flattened out.  Only slight  variations in the contours of the posterior surface suggest that the  331.  posterior surface is actually made up of numerous epithelial cells. Around the pupillary edge, the sphincter muscle is gathered very tightly together as a series of overlapping humps.  These are covered by a  smooth surface layer, the basement membrane, which overlies the flat cells lining the posterior surface of the sphincter.  Peripheral to the sphincter  region, the posterior epithelial cells are arranged in rows.  Unlike in  pupillary dilation, these rows are oriented radially rather than circumferentially.  Thus, the posterior epithelial cells are capable of being  lined up either circumferentially (in pupillary dilation), or radially (in pupillary constriction).  There are also  ridges of epithelial cells sepa-  rated by grooves of varying depths and widths.  The epithelial ridges also  bifurcate, taper down and blend with adjacent ridges.  Nearer the pupil,  the epithelial cells are packed closer together but as one moves peripherally the grooves and ridges become shallower and lower, respectively, until the posterior surface is seen as a smooth sheet.  Within this zone of epi-  thelial ridges and grooves, large bulbous structures are seen. be puffed up or partially collapsed.  They may  They are of differing sizes and  shapes and are covered with a basement membrane similar to that covering the rest of the i r i s surface.  It is suggested that these bulbous structures  represent posterior epithelial cells which have been crowded out of their normal position because of the extreme pupillary constriction.  The equiva-  lent in meridional sections has not been observed. In addition to what has been presented,  two new features of the  posterior surface of the rat i r i s , which have only been made visible by scanning electron microscopy, are described here. At the periphery of the rat i r i s near the c i l i a r y body there are numerous, large, radial processes connecting the c i l i a r y body to the i r i s . Thus, they are named here as c i l i a r y - i r i s processes.  The number of these  332.  c i l i a r y - i r i s processes varies^from one iris to the next. slightly differing lengths in an individual i r i s .  They are of  They act, as i t were, to  anchor the i r i s to the ciliary body and thus stabilising the root of the i r i s during the extensive excursions of the iris in pupillary dilation and constriction.  In pupillary constriction, the posterior epithelium and  basement membrane covering the c i l i a r y - i r i s process is similar to what is observed over the rest of the i r i d i a l posterior surface. However, in pupillary dilation, the posterior epithelial cells over the c i l i a r y - i r i s process do not form ridges or grooves, which are very prominently seen over the rest of the i r i s posterior surface.  Instead, there is a break in the  continuity of the epithelial ridges over the body of the c i l i a r y - i r i s process.  At the pupillary end of the c i l i a r y - i r i s process where i t dives  deeply into the i r i s tissue itself, the other epithelial ridges skirt around i t , as though the c i l i a r y - i r i s process were a relatively solid obstruction. Superficial to the posterior surface of the i r i s are capillaries which radiate from the pupillary margin to the region of the c i l i a r y body. These capillaries vary in number and size in different irises.  Unfortunately,  the beginning and termination of these blood vessels are not well defined. At the pupillary margin, the blood vessel appears to have come from the i r i s stroma.  It then turns around at the pupillary border and traverses  the posterior surface of the i r i s .  We speculate that these are connected to  the i r i s stromal capillaries and that they are functional in the adult, otherwise they would have atrophied.  Possibly, they are remnants of the  fetal circulatory system.  3.  The Anterior Surface of the Iris in Pupillary Dilation and Constriction  The anterior surface of the adult i r i s is covered by an anterior  333.  endothelium with intervening crypts and pores (Vrabec, 1952; 1958b, 1959,  1961;  Gregersen, 1958a,  Coulombre, 1961; Klika and Kloucek, 1962; Purtscher,  Newsome and Loewenfeld, 1971).  1962;  However, the nature of these large-sized  crypts and smaller-sized pores can only be adequately studied by scanning electron microscopy. In meridional sections, i t is seen that the contour of the anterior i r i d i a l surface in pupillary dilation is highly scallopeddue to large i r i s stromal blood vessels which bulge out anteriorly. The larger blood vessels are present in most of the i r i s stroma whereas capillary-sized blood vessels are predominant over the sphincter region.  Using the scanning electron  microscope on the rat i r i s in pupillary dilation, i t is seen that the anterior i r i d i a l surface is indeed covered by a mass of. blood vessels of varying sizes which are closely packed together in a generally circumferent i a l direction. The blood vessels show different configurations. bifurcate or branch, they may join, they may deep into the i r i s stroma.  They may  travel superficially or dive  In the region around the pupillary margin, the  blood vessels are smaller and do not bulge as much anteriorly. These represent the capillary system over the sphincter.  In extreme pupillary dila-  tion, this capillary region becomes telescoped into the more peripheral region so that i t is no longer apparent. The morphology of the anterior surface of the rat i r i s and the course of the blood vessels is best studied when the pupil is constricted, and with scanning electron microscopy.  In the constricted pupillary condi-  tion, the blood vessels are spread out and the anterior endothelial cells are well exposed and the morphology of the i r i s crypts and pores are displayed to their best advantage.  Some crypts, though, are visible in the  pupillary region in the dilated pupil. The i r i s blood vessels are believed to pursue a slight corkscrew  334.  course in the i r i s (Hogan, Alvarado and Weddell, 1972).  In our investiga-  tion, i t appears that the larger blood vessels are arranged in a more planar fashion, much like the pleats of an accordion. periphery of the i r i s to the pupillary margin.  They zig-zag from the  Branches are given off at  each external bend of the parent blood vessel and these change direction to go deep into the i r i s stroma.  At the pupillary margin, the capillaries  are more tortuous and inter-relate with each other in a complicated  pattern.  At times, these capillaries form arcades which hug the pupillary margin itself.  Some blood vessels seem to go over the pupillary edge. Perhaps,  such blood vessels do indeed turn posteriorly to traverse the posterior surface of the i r i s to the c i l i a r y region. The detailed anterior surface structure of the rat i r i s is seen in high magnification scanning electron micrographs.  In our investigation,  the morphology of the anterior endothelial cells and of the i r i d i c crypts and pores is much more well-defined and seen in finer detail than that presented by Hansson (1970).  The structural differences observed are  probably a result of the difference in methodology employed in preparing the i r i s tissues for scanning electron microscopy, especially with regards to the drying process.  Hansson (1970) used fixed or unfixed tissues which  are frozen in isopentan-propane and then freeze-dried.  The tissues prepared  by this method are very susceptible to ice crystal and phase-boundary artifacts.  Distortions then generally occur.  In our case, the tissues are  dried by the camphene (Watters and Buck, 1971) and c r i t i c a l point drying (Boyde and Wood, 1969; Smith and Finke, 1972) methods, which have been proven to be superior to the freeze-drying method for preserving the ultrastructural details of tissues for examination with the scanning electron microscope. The anterior surface of the rat i r i s is covered incompletely by a  335.  layer of endothelial cells, substantiating what has been previously observed (Vrabec, 1952). shape.  The anterior endothelial cells are variously polygonal in  The oval nucleus is located approximately in the center of each  anterior endothelial c e l l and bulges slightly into the anterior chamber. The rat anterior endothelial cells do not show microvilli in our study, unlike what is described by Hansson (1970). cell surface is quite smooth.  In fact, the anterior endothelial  There are occasionally some blebs on the cell  surface which in no way resemble microvilli.  Intercellular gaps occur in  between the anterior endothelial cells but these gaps are bridged by numerous cytoplasmic processes of the surrounding endothelial cells.  The  resulting defects are probably equivalent to the i r i s pores of light microscopy.  In addition, there are larger holes or crypts, in contrast to the  smaller pores.  The crypts are of varying sizes and are found throughout the  anterior surface of the i r i s .  A number of crypts may be grouped together at  a specific location, oftentimes around a blood vessel. iously round or oval in shape with well-defined an open hole.  borders.  The crypts are varThe crypt is not  The openings are spanned by cytoplasmic processes of the  anterior endothelial cells and of the underlying stromal cells, and by f i b r i l l a r material, presumably, belonging to the collagen connective tissue network in the i r i s stroma.  C.  A Light Microscopic  Study of the Development of the Rat Iris Using  Toluidine Blue Stained Plastic  Sections  The developing rat i r i s , like that of the adult, is very susceptible to the effects of chemical fixation. posterior epithelium.  This is particularly evident in the  Different fixatives combined with different buffers  were tried but a l l apparently s t i l l produce some degree of tissue disruption. In this study, the development of both the i r i s and the c i l i a r y  body are observed as they are so closely associated. The bulky immature ciliary body, through a process of mitotic division, differentiation and reorganisation of the cells, is transformed into a series of delicate, finger-like c i l i a r y processes. The anterior neuroectodermal  The i r i s grows in length and in complexity.  layer develops into the sphincter and dilator  muscles by a gradual process.  A l l this takes place in the latter pre-natal  days and during the f i r s t two weeks after birth, so that by the end of the second week the adult form is attained. The rate of growth and differentiation of the c i l i a r y body and i r i s varies from one rat to the next and between different l i t t e r s of rats. Mitotic figures are often encountered in a l l parts of the developing rat i r i s and c i l i a r y body.  There do not appear to be foci of c e l l division.  Cell division is s t i l l taking place in the posterior epithelium of the i r i s and in the developing sphincter as late as two weeks after birth.  Accord-  ing to Imaizumi and Kuwabara (1971) the neuroepithelium ceases to divide actively by the 18th pre-natal day.  This is not upheld by our observations.  At 19 days fetal, the i r i s and c i l i a r y body are i n i t i a l l y coextensive with each other and with the retina.  The c i l i a r y body is seen  only as a mere bulge interposed between the retina and the i r i s . cytologically the  However,  c i l i a r y epithelium can be demarcated from the retina by  virtue of its staining characteristics with toluidine blue.  At 19 days  fetal, the c i l i a r y epithelium consists of a thick, stratified mass of cells, contributing to the bulkiness of the c i l i a r y body. At term, the c i l i a r y body as a whole becomes more well delineated. In the first few days after birth, the beginnings of the division of the c i l i a r y body into c i l i a r y processes are observed. arrangement and form.  -The c i l i a r y epithelial cells alter both in their Instead of a stratified mass of cells, there is now  usually a single layer of cells which are elongated and closely packed to-  gether.  As c i l i a r y processes become distinguishable, through the folding of  both the c i l i a r y epithelium and the c i l i a r y pigment epithelium, the c i l i a r y epithelial cells at the tips of the developing c i l i a r y processes are higher than those at the bases of the processes.  From the 5th to the 10th days  after birth the c i l i a r y processes grow in length.  The c i l i a r y epithelial  cells differ in their appearance according to their locations along the c i l i a r y processes.  At the tips of the developing c i l i a r y processes, the  cells are high columnar but they are lower along the sides of the processes. Concomitant with the changes seen in the c i l i a r y epithelium are the changes in the c i l i a r y pigment epithelium.  In the pre-natal eye, the c i l i a r y  pigment epithelium is a light staining layer of high columnar cells.  Its  posterior surface closely follows the contours of the c i l i a r y epithelium whereas its anterior surface is molded by the blood vessels and cells of the c i l i a r y stroma. creased.  In development, the height of the epithelium is de-  As well, extensive folding of the pigment epithelium occurs as  c i l i a r y processes are being formed.  In fact, the foldings of the c i l i a r y  pigment epithelium always precedes and are more extensive than the foldings of the c i l i a r y epithelium.  The troughs in between the folds of the pigment  epithelium are f i l l e d with blood vessels and some stromal cells. By the 14th or 15th post-natal day, the c i l i a r y processes are long and finger-like.  Each c i l i a r y process consists of two closely adherent  layers, the c i l i a r y epithelium and pigment epithelium making a hair-pin loop enclosing many capillaries and a few stromal cells within the loop. It is essentially a vascular structure. The blood vessels at the tip of the c i l i a r y process may expand to give a knob-like appearance to the tip of the c i l i a r y process. The i r i s is relatively undeveloped at birth.  It is short and stubby.  In our observations, most of the growth in length of the i r i s occurs prim-  arily after the 5th post-natal day.  This growth is probably the result of  both mitotic division and the postulated stretching of the epithelial layers (Imaizumi and Kuwabara, 1971).  The ratio of the bulk of the devel-  oping sphincter to the total length of the i r i s is a good indication of the rate of growth.  The bulk of the sphincter remains relatively constant  although the number of cells in the sphincter increases with growth. Initially, the sphincter makes up half to one third of the i r i s length at 19 days fetal, but in the f i r s t few days after birth, i t makes up only one quarter or one f i f t h of the i r i s length. The posterior and anterior epithelia are neuroectodermal in origin. The posterior epithelium retains its epithelial character while the anterior epithelium differentiates into smooth muscle, namely, the sphincter, and into myoepithelium, namely, the dilator. The posterior epithelium in the pre-natal i r i s is not of uniform height, being columnar near to the c i l i a r y junction and gradually decreasing in height to the peripheral extent of the developing sphincter.  Gradually,  with development, the posterior surface of the i r i s becomes lined by a layer of brick-like cells.  The posterior surface of the i r i s is smooth  until relatively late in development.  This is more clearly visualised in  scanning electron micrgoraphs of the posterior surface of the developing iris.  The posterior epithelial cells are formed into rows separated from  each other so as to impart a regularly scalloped appearance to the posterior surface of the i r i s . The development of the anterior epithelium has been an area of much interest.  It has been debated whether the sphincter muscle, in particular,  is really of neuroectodermal rather than of mesodermal origin.  Our observa-  tions corroborate those of other investigators that both the sphincter and dilator muscles arise through differentiation of the anterior epithelium  (Imaizumi and Kuwabara, 1971;  Lai, 1972a, 1972b; Tamura and Smelser, 1973).  By 19 days fetal, the developing sphincter is seen as a distinct knob-like' mass of elongated, centripetally directed cells at the pupillary tip of the anterior epithelium. together.  The cells are relatively large and closely packed  The developing sphincter is distinctly separated from the i r i s  stroma by an intercellular space.  In terms of its stainability with tolui-  dine blue, these cells do not stain differently from those of the rest of the anterior epithelium. increased.  By 4 days post-natal,  the number of cells have  Small intercellular spaces appear in between the developing  muscle cells giving the sphincter a crackled glass effect.  The developing  muscle cells show different staining intensities between individual cells, but the cytoplasm is uniformly dense within any one c e l l . indication, light microscopically,  This is the  first  that these cells are cytologically differ  ent from those of the rest of the anterior epithelium.  Presumably, the  bundles of myofilaments stain more intensely with toluidine blue.  With the  transmission electron microscope, myofilaments are observed in the developing muscle cells as early as 19 days fetal (Lai, 1972a), or at the latest by term (Imaizumi and Kuwabara, 1971).  However, the toluidine blue stain  for light microscopy is probably not able to differentiate out the myofilaments t i l l they are present in large amounts. Within the developing sphincter, the cells are in different stages of differentiation.  Some cells  are s t i l l dividing while others have acquired a f u l l complement of myofilaments.  This mosaic characteristic of the developing sphincter is not as  readily apparent in electron microscopic studies.  With development, there  is a decrease in the diameter of the individual cells, as has been previousl observed (Lai, 1972a), so that by the second week after birth the sphincter consists of a mass of small cells.  They are usually quite closely packed  together although small intercellular spaces are found in between the cells.  340.  In addition, groups of muscle cells seem to associate together and be separated from other muscle groups by intercellular spaces.  This is probably  the beginning of the division of the sphincter into functional units.  In  the human sphincter, the muscle cells are linked in groups which function as a unit (Hogan, Alvarado and Weddell, 1971).  Capillaries and nerves  insinuate themselves in between the muscle cells during development. The anterior epithelium is usually a uniformly high columnar epithelium throughout its extent.  Up to the f i r s t few days after birth, the  anterior epithelium is two to three times the height of the posterior epithelium.  But by 10 days post-natal, the anterior epithelium appears to have  been stretched out, as i t were, so that i t is about the same height of the posterior epithelium.  The development of the muscular portion of the  anterior epithelium, the dilator, is more tardy than that of the sphincter. In transmission electron microscopic studies, there is a discrepancy as to the f i r s t appearance of myofilaments in the developing dilator cells of rats.  According' to Lai (1972b) myofilaments appear as early as 20 days  fetal, whereas, according to Imaizumi and Kuwabara (1971) myofilaments are seen two days after birth.  In light microscopic studies, patches of densely  staining material, presumably the myofilaments, are seen in the basal cytoplasm of the anterior epithelium only by 3 or 4 days post-natal. dilator has begun to develop.  The  By three weeks after birth, the dilator ap-  pears asa dense line at the stromal boundary, much as in the adult. The stroma of the c i l i a r y body and of the i r i s are considered together as they are in continuity. Stromal cells and blood vessels make up the stroma. Large blood vessels are often seen extending from the developing c i l i a r y stroma to the i r i s . to the length of the i r i s .  Their lumens are always patent and parallel  In the c i l i a r y body, some of the blood vessels  and cells change direction to tumble into the clefts of the developing  ciliary processes.  In both the iris and the c i l i a r y body, the endothelium  of the blood vessels is characteristically very intimately associated with the anterior surface of the dilator or pigment epithelium, respectively. There is no perceivable gap as seen with the light microscope, between the dilator cells or the c i l i a r y pigment epithelium and the endothelium of the stromal blood vessels. Rarely are there cells interposed between the blood vessels and the dilator or c i l i a r y pigment epithelium.  In the adult i r i s ,  blood vessels are never seen in such close apposition with the dilator.. Thus with development, there must be a slight reorientation of the stromal elements with respect to the posterior epithelial layers.  In the adult  eyes, the proximity of blood vessels or stromal cells to the dilator would be mechanically disadvantageous  in the movements of the iris tissue and  the conformational changes that occur at the anterior surface of the dilator.  In the very young developing eyes, this would seem to be of no  consequence.  Although tests were not made to determine when the sphincter  and dilator become functional, i t would seem logical to postulate that this does not happen t i l l quite late in development.  Structurally, the sphincter  and dilator muscles are relatively undeveloped at birth.  Besides, the rat  eyes are not opened or exposed to varying light conditions t i l l about two weeks after birth, thus not necessitating an early functional i r i s . The blood vessels and cells of the iris stroma do not end at the pupillary margin, as has been reported (Imaizumi and Kuwabara, 1971).  In  fact, in fetal and young post-natal eyes, the i r i s stroma always overhangs the pupillary tip of the i r i s .  With development, • the relative position of  the stroma with respect to the iris shifts so that in the adult, the iris stroma does not extend beyond the pupillary margin.  Capillaries of the iris  stroma are often seen streaming out past the pupillary margin to form the pupillary membrane. These capillaries are strung together by connective  342.  tissue strands.  Some i r i s stromal capillaries also turn around at the  pupillary margin and appear to be going posteriorly. often seen along the posterior surface of the i r i s .  Capillaries are quite A l l these features of  the fetal vasculature are better observed and appreciated in scanning electron micrographs and have been described.  The possible inter-relationships  of these blood vessels are speculated upon in scanning electron microscopic studies of the developing rat i r i s .  D. A Scanning Electron Microscopic Study of the Posterior Surface of the Developing Rat Iris  1.  General  The scanning electron microscopic studies of the posterior surface of the fetal and young post-natal rat i r i s are carried out to complement the light microscopic studies of the development of the rat i r i s as observed on toluidine blue stained plastic sections which have been meridionally cut. Owing to the limited sampling size and the two dimensional nature of tissue sections, very l i t t l e information can be gleaned from a study of tissue sections pertaining to the development of the surface configurations of the posterior epithelial cells, the development of the c i l i a r y - i r i s processes, the topography of the pupillary membrane, and the relationships of other associated capillary networks to the pupillary membrane.  A wealth of infor-  mation is obtained from our scanning electron microscopic studies. Much as i t would have been desired to carry out a concomitant  study  on the anterior surface of the i r i s in fetal and young post-natal rats, to elucidate the development of the i r i d i c crypts and pores, this is not done at the present time due to technical d i f f i c u l t i e s . short and fragile.  The developing i r i s is  In order to study the anterior surface of the i r i s , the  cornea would have to be dissected away. This was tried but without the support of the cornea, the i r i s becomes highly curled and distorted duringspecimen preparation and is not easily visualised by scanning electron microscopy.  2.  The Posterior Surface of the Developing Rat Iris  In the fetal i r i s , the posterior surface is often covered by an amorphous sheet-like layer which is capable of being flaked off during the preparative procedures for scanning electron microscopy. often embedded in this material.  Blood vessels are  This amorphous layer may be removed com-  pletely to expose the basement membrane covering the posterior epithelial cells.  In the f i r s t few days after birth, this layer is s t i l l present but  is usually confined to the pupillary region. the pupil i t s e l f .  It may extend somewhat into  This amorphous layer is usually associated with the blood  vessels found a l l along the pupillary region. nature of this amorphous layer.  We can only speculate on the  It is perhaps a modification of the base-  ment membrane, which acts as an adhesive to hold the capillaries of the pupillary margin onto the posterior surface of the i r i s .  Peripheral to  this layer, the capillaries become free on the surface of the i r i s and can be dislocated from their positions during the handling of the tissue.  Their  original paths are suggested by impressions left on the posterior surface of the i r i s .  In addition, this amorphous layer merges into the basement  membrane covering the posterior epithelial cells.  Very often, there is no  sharp transition between this amorphous layer and the basement membrane. It almost seems as i f the amorphous layer thins out and becomes the basement membrane of the posterior epithelial cells.  By two weeks after birth, when  most of the blood vessels at the pupillary margin are atrophying, much of this amorphous layer is no longer present.  In parts, only the basement  344.  membrane covering the squamous epithelial cells of the sphincter region is visible. The basement membrane always covers the posterior surface of the epithelial cells.  It is so very closely related with the epithelial cells  that i t is rarely removed during specimen preparation.  The surface charac-  teristics of the basement membrane change with the development of the posterior epithelial cells. i r i s is smooth.  In fetal eyes, the posterior surface of the  From scanning electron microscopy, i t is not possible to  say that the posterior surface of the i r i s is lined by a layer of epithelial cells, as is readily seen in light microscopic studies.  The pupillary region  is usually smoother than the peripheral region where there might be a few irregularities on the posterior surface of the i r i s . is also smooth.  The basement membrane  Occasionally, there is a mass of fine filamentous material  on the i r i s surface.  The source and nature of these filaments is not known.  In the f i r s t four days after birth, some contouring of the posterior surface of the i r i s is seen, primarily in the peripheral two thirds of the iris.  These are the f i r s t superficial signs that the posterior surface of  the i r i s is indeed made up of myriads of c e l l s .  In a low magnification  scanning electron micrograph, the posterior surface of the i r i s has a speckled appearance.  At a higher magnification, i t is observed that the  posterior surface of the i r i s consists of a mosaic of round to oval slight elevations separated by barely discernible depressions.  The elevations  represent the nuclei of the posterior epithelial cells.  The basement mem-  brane covering the posterior epithelial cells is s t i l l relatively smooth except for a few irregularities.  The posterior surface of the pupillary  region does not show these elevations and depressions but is smooth. The basement membrane may show a few fine striations.  This is the epithelium  345.  which underlies the developing sphincter muscle. Up to 10 days post-natal, the posterior surface of the i r i s appears smooth in light microscopic show some waviness.  still  studies, although occasionally i t may  With scanning electron microscopy, certain changes in  the contours of the posterior surface of the i r i s are observed.  The indivi-  dual posterior epithelial cells slowly become more well-defined.  Initially,  the posterior surface of the i r i s has a raspberry-like appearance.  The  posterior epithelial cells, covered by a smooth basement membrane, bulge a l i t t l e more into the posterior chamber.  With further development, the  posterior epithelial cells become almost similar to those of the adult. They bulge out quite prominently. However, they are s t i l l not arranged in any particular way.  Sometimes, a few grooves are seen parcelling out groups  of posterior epithelial cells.  This would be the beginnings of the epi-  thelial ridges and grooves of the adult posterior surface of the i r i s .  The  basement membrane is no longer smooth but shows many crinkles. Some bulbous structures are seen scattered throughout the posterior surface of the i r i s . They remind us of the bulbous structures seen on the posterior surface of the adult rat i r i s when i t is in extreme pupillary constriction. In light microscopic  studies of the developing rat i r i s , i t is observed that mitotic  figures are often found in the posterior epithelium of young post-natal rats.  Perhaps, these bulbous structures, as seen by scanning  microscopy, represent the i r i s .  electron  foci of c e l l division in the posterior epithelium of  These newly formed cells have not as yet been fitted, as i t were,  into the posterior epithelium as a whole.  Thus, they bulge outwards much  as the posterior epithelial cells of the adult iris are crowded out of position during extreme pupillary constriction. By two weeks after birth, the posterior epithelium has practically attained the adult form as seen by both light and scanning electron micro-  346.  scopy.  The individual posterior epithelial cells are more readily apparent  than in the adult i r i s .  The epithelial cells are arranged in circumferential  rows or ridges separated by grooves of varying widths and depths. The branching and merging of the epithelial ridges appears to be a l i t t l e more profuse than in the adult.  The basement membrane covers the posterior epi-  thelial cells and dips deep into the grooves between the epithelial ridges. The basement membrane is highly crinkled as in the adult, suggesting the numerous cytoplasmic processes underneath, as revealed by light microscopy. At this time too, the beginnings of the c i l i a r y - i r i s processes are observed.  As opposed to the circumferential posterior epithelial ridges,  the c i l i a r y - i r i s processes are much larger radial ridges located only towards the periphery of the i r i s .  The developing c i l i a r y - i r i s process interferes  with the continuity of the posterior epithelial ridges, much as in the adult, but to a lesser extent.  3.  The Peri-natal Vascular System of the Rat Iris  In our investigation of the developing rat i r i s , and in the investigations of Matsuo and Smelser (1971) of the rabbit, the pupillary membrane is present not only in the fetal animals but persists in young post-natal animals.  Unlike in humans (Mann, 1964) where the pupillary membrane is a  transient fetal structure, in the rabbit and in the rat, i t is more correctly thought of as being a transient peri-natal structure.  The pupillary membrane  is presumably s t i l l functional after birth, as suggested by the large number of red blood cells that are always present in the lumens of the thin-walled capillaries. In the rat, the pupillary membrane is detected as early as 17 days fetal when the i r i s is s t i l l relatively undeveloped.  It may be present  even earlier, but from our results we are not able to say when the pupillary  membrane begins to form. after birth.  The pupillary membrane is s t i l l seen up to 10 days  At this time, i t is usually not as extensive as in younger  eyes and is seen only in some specimens.  By two weeks after birth, the  pupillary membrane is virtually non-existent. connective tissue fibrils a cobweb.  Sometimes strands of fine  are left and they span the pupillary aperture like  In our investigation, the pupillary membrane has not been observed  to extend completely across the pupil, as has been observed with the s l i t lamp in the rabbit (Matsuo and Smelser, 1971).  The pupillary membrane is a  very fragile structure. The most likely explanation is technical.  When the  lens is removed, part of the pupillary membrane is most probably removed with i t .  Thus the topography of the rat pupillary membrane is not examined  in its totality. The rat pupillary membrane consists mainly of a series of thinwalled capillaries which issue from the i r i s stroma. connect with each other by branches. lary arcades and loops.  The capillaries inter-  They form a complex pattern of capil-  A l l of the capillaries are held together by a  scaffolding of dense connective tissue fibers.  The fibers are of varying  diameters and they form a complicated meshwork around and in between the capillaries.  These fibers are probably collagen fibers.  They serve perhaps  to hold the capillaries of the pupillary membrane in a.relatively stable configuration with respect to each other and with respect to the lens so as to ensure that blood flow through the capillaries is not impeded.  Since the  lens has been removed, i t is impossible to say whether these fibers also anchor the pupillary membrane capillaries to the capsule of the lens. In light microscopic studies of the developing rat i r i s , i t is observed that the i r i s stromal blood vessels extend centrally into the pupil, being strung together by connective tissue strands. ing electron microscopy.  This is well substantiated by scann-  It is very rare to find cells associated with the  348.  capillaries, or enmeshed in the connective tissue skeleton of the pupillary membrane.  In transmission electron microscopic studies of the rabbit  pupillary membrane (Matsuo and Smelser, 1971), fibroblasts and macrophages are also present.  This may be a species difference.  The i r i s stromal capillaries, besides extending forward as the pupillary membrane, also turn around at the pupillary margin, change direction and travel towards the periphery of the i r i s .  In light microscopic  studies of the developing rat i r i s , an occasional i r i s stromal blood vessel is observed to encircle the pupillary border.  These capillaries are  closely applied to the posterior surface of the i r i s , especially in the pupillary region.  In the pupillary region of fetal and young post-natal  irises, these capillaries are embedded to a certain extent in an amorphous layer.  More often than not, these capillaries are removed during specimen  preparation.  However, they leave quite readily visible impressions on the  posterior surface of the i r i s which are especially concentrated pupillary region.  The capillaries are radially oriented.  in the  They may branch  and link up with each other. At the region of the c i l i a r y body, there is a large annular vessel. This is connected to the hyaloid system (Hollenberg and Dickson, 1971). From the optic nerve head, the hyaloid vessels separate into two groups. One group of vessels runs directly to the inner surface of the lens while the other group radiates out onto the inner surface of the retina. hyaloid vessels on the retina are seen in some of our specimens.  The Much like  the pupillary membrane, the hyaloid vessels on the retina form a complicated pattern.  The capillaries are also held together by a connective tissue  framework.  These capillaries are connected to the annular vessel located  in the region of the developing c i l i a r y body. Another system of capillaries arises from the other side of the  annular vessel.  These capillaries have a l l the characteristics of the capi  laries in the pupillary membrane.  They are thin-walled so that the red  blood cells show through the almost transparent walls. form an interconnecting vascular network. f i b r i l l a r connective tissue framework.  The capillaries  The capillaries are enmeshed in  Oftentimes, these capillaries from  the annular vessel are seen extending into the region of the pupillary membrane and may even appear to be incorporated into some of the i r i d i a l vessels of the pupillary membrane. Rarelyj a capillary from the annular vessel runs along the posterior surface of the i r i s and becomes continuous with a capillary which has come from the i r i s stroma and has made a turn at the pupillary margin. From our rather limited scanning electron microscopic data on the peri-natal vasculature of the rat i r i s , we cannot say with absolute certainty that the hyaloid vessels are connected to the i r i s stromal vessels to any considerable extent.  But the data we have at the present moment  suggest that this is a possibility. The rat pupillary membrane consists mainly of capillaries from the i r i s stroma.  However, i t may receive a contribution from the branches of  the annular vessel. In the developing rat i r i s , there are numerous capillaries traversing  its posterior surface.  Many of the i r i s stromal vessels turn at the  pupillary margin to l i e on the posterior surface of the i r i s .  There are  also branches of the annular vessel which l i e on the posterior surface of 'the iris and seemingly extends into the pupillary membrane.  The question  here is whether the two systems of capillaries, from the i r i s stroma and from the annular vessel, are interconnected.  There is a hint that at least  some of the i r i s stromal capillaries are continuous with the capillaries of the annular vessel, thus linking the hyaloid vessels to the i r i s  stromal  350.  blood vessels.  The capillaries from the annular vessel atrophy and dis-  appear during development, much like the capillaries of the rest of the hyaloid system.  The iris stromal capillaries on the posterior surface of  the iris atrophy to varying degrees, so that even in the adult i r i s ,  there  are a few such capillaries remaining on the posterior surface of the i r i s . It is problematic as to the termination of these capillaries, which is unfortunately not observable in our present investigation. The pupillary membrane and the capillaries on the posterior surface of the i r i s are part of the transitory vascular system of the eye, which also includes the hyaloid vessels.  The underlying mechanisms of the atrophy  of these blood vessels is obscure.  In the regressing pupillary membrane of  the rabbit, Matsuo and Smelser (1971) hypothesise that cessation of blood flow through the pupillary membrane capillaries, due perhaps to mechanical and/or physiological factors, precedes the actual atrophy of the capillaries.  This is followed by an accelerated synthetic activity of an in-  creased number of fibroblasts in the pupillary membrane. The fibroblasts and the collagen f i b r i l s degenerate, perhaps releasing some enzymes which have a destructive effect on the junctions of the endothelial cells. atrophied capillaries are then slowly removed by phagocytic  E.  The  activity.  Horse-radish Peroxidase (HRP) Studies of the Iris in Fetal, Post-natal and Adult Rats The i r i s is bathed in aqueous humor of the anterior and posterior  chambers of the eye.  The aqueous humor is not a static component but i t is  constantly being produced and removed from the eye.  Its composition is  presumably modified in its passage from the posterior to the anterior chamber by the surrounding tissues, namely, the iris and the lens (Kinsey and Palm, 1955).  Morphologically,  the i r i s tissue is intimately associated  351.  with the surrounding aqueous humor. There is a system of tissue spaces within the i r i s stroma which communicates with the aqueous humor by means of the i r i d i c crypts and pores (Vrabec, 1952; Gregersen, 1958a, 1958b, 1959, 1961; Klika and Kloucek, 1962; Purtscher, 1962; Coulombre, 1961; Newsome and Loewenfeld, 1971).  Substances injected into the anterior chamber of the  eye make their way into the i r i s stroma via these i r i d i c crypts and pores (Gregersen, 1958a, 1958b, 1961).  Conversely, i t is conceivable that sub-  stances from the i r i s tissues also enter the anterior chamber in a similar manner. The i r i s may, to a certain extent, modify the composition of the aqueous humor. The permeability of the i r i s blood vessels to small molecules would give some indication of the role the i r i s might play in the modification of the aqueous humor in the eye. Horse-radish Peroxidase (HRP) is a protein with a low molecular o weight of about 40,000 and a molecular diameter of 44-47A (Vegge, 1971b). It has a lower molecular weight than many serum proteins.  The HRP tracer  technique was originally developed by Karnovsky (1967) for studying the ultrastructural basis of capillary permeability. Since then, HRP has also been used as a tracer substance in other diverse studies (Webber and Blackbourn, 1971).  The reaction product is easily visualised both with the  light and electron microscopes as a brownish-black or electron-dense precipitate, respectively.  The sites of the reaction precipitate indicate  the location of the HRP.  Thus the pathways of the HRP can be followed.  In the present investigation, the permeability of the c i l i a r y and i r i s capillaries to HRP is studied in fetal, post-natal and adult the light microscopic level. vascular injection of HRP,  rats at  At different time intervals after an intra-  the eyes are removed and fixed in glutaraldehyde.  The eyes are then appropriately incubated to demonstrate the sites of deposition of the colored reaction precipitate.  352.  In the adult rat, the c i l i a r y capillaries are readily permeable to the HRP, as has also been observed in the mouse (Smith, 1971) and in the Vervet monkey (Vegge, 1971a).  In the c i l i a r y processes, the capillaries at  the tip appear to be more permeable to the HRP than those towards the base of the c i l i a r y processes, and in the main mass of the c i l i a r y body. Once the HRP leaves the c i l i a r y capillaries, i t makes its way outwards towards the posterior chamber.  The HRP reaction product is seen a l l along the  stromal surfaces of the pigment epithelial cells.  The precipitate also  permeates in between the individual pigment cells and accumulates a l l along the boundary zone between the c i l i a r y and the pigment epithelia.  This  region is sometimes known as the apical extracellular space (Smith, 1971). There is no precipitate in the intercellular spaces of the c i l i a r y epithelial cells.  There is thus an apparent barrier to the outward movement of the  HRP from the apical extracellular space between the c i l i a r y and pigment epithelia to the posterior chamber of the eye. the apices of the c i l i a r y epithelial cells.  This barrier is located at  Electron microscopic studies,  using HRP as an ultrastructural cytochemical tracer, have been performed on the mouse (Smith, 1971) and on the Vervet monkey (Vegge, 1971a). findings are consistent with our observations.  Their  At the electron microscopi-  cal level, i t is seen that there is an epithelial blood-aqueous barrier to HRP in the c i l i a r y processes (Vegge, 1971a).  A tight junction is always  found at the apex of the c i l i a r y epithelial cells.  This would structurally  exclude the HRP reaction precipitate in the apical extracellular space from entering the intercellular spaces of the c i l i a r y epithelial cells.  In the  formation of aqueous humor in the c i l i a r y processes, a f i l t r a t e of blood leaves the ciliary capillaries to enter the c i l i a r y stroma. However, the composition of the fluid in the c i l i a r y stroma (the potential aqueous humor) and that in the posterior chamber is somewhat different, especially with  353.  regards to the amount of proteins present.  The aqueous humor has a low  protein content whereas quite a considerable amount of proteins leave the c i l i a r y capillaries (Vegge, 1971a).  In our system, the c i l i a r y capillaries  are readily permeable to HRP, a low molecular weight protein.  But not a l l  of the proteins that leave the c i l i a r y capillaries enter the posterior chamber and contribute to the make-up of the aqueous humor. There is a structural blood-aqueous barrier to proteins (in this case, HRP), which has been demonstrated to be located at the apices of the c i l i a r y epithelial cells (Smith, 1971; Vegge, 1971a).  Perhaps, not only proteins but other  anions and non-electrolytes are also partially held back from entering the posterior chamber.  It is suggested that the apical extracellular space may  be the site where.there is selective absorption of the proteins and other substances which readily f i l t e r out of the c i l i a r y capillaries (Smith, 1971). Coupled with this, there may be some selective secretion as well. In comparison with the c i l i a r y capillaries, the i r i s capillaries of the adult rat, mouse (Smith, 1971) and Vervet monkey (Vegge, 1971b) are highly impermeable to HRP.  In our studies, no HRP reaction precipitate is  found within the i r i s tissue of adult rats.  The HRP reaction product is  frequently observable only within the lumens of the i r i s capillaries.  In  the adult rat, proteins, as exemplified by HRP, do not leave the i r i s capillar ies . to. enter the i r i s stroma.  In the i r i s , the blood-aqueous barrier to  proteins lies in the walls of the i r i s blood vessels, whereas in the c i l i a r y processes the blood-aqueous barrier lies not in the c i l i a r y capillary walls but in the c i l i a r y epithelium.  In the adult rat, mouse and monkey, i t  appears that the i r i s capillaries do not at any rate contribute to the protein composition of the aqueous humor whereas the c i l i a r y capillaries do, although to a very limited extent.  In the mouse, Smith (1971) found that  some of the HRP does reach the posterior chamber.  But the HRP enters the  354.  posterior chamber probably not by an extracellular route because of the" tight junctions at the apices of the c i l i a r y epithelial cells, but by an . intracellular route, that is, by traversing the cytoplasm of the c i l i a r y epithelial cells.  These observations made with protein tracers do not,  however, totally exclude the possibility that the i r i s capillaries might contribute other substances, which are not demonstrable histologically with our present day techniques, to the aqueous humor of the anterior chamber of the eye.  This suggestion is elicited by the observation that there are  two kinds of aqueous humor in the eye (Kinsey and Palm, 1955).  The aqueous  humor in the posterior chamber of the eye is different in composition from that in the anterior chamber.  Since the fluid in the i r i s stroma is in  open communication with the aqueous humor of the anterior chamber, i t seems logical to postulate that there is an interchange of materials between the i r i s and the aqueous humor. One highly possible pathway where such an interchange of substances can take place is across the i r i s capillary walls. Kinsey and Palm (1955) found that thiocyanate and sodium enter the anterior chamber of the eye partly by flow from the posterior chamber and partly by diffusion through the i r i s vessels. HRP tracer studies have not, as far as is known, been carried out in fetal and young post-natal rats.  This is done in our present investiga-  tion to compare the permeability of the c i l i a r y and i r i s capillaries to HRP in the adult and in the developing eye.  As in the adults, in the fetal and  post-natal rats, the HRP is introduced intravascularly, via the umbilical veins in fetal rats, and via the retro-orbital, jugular or saphenous veins in the post-natal rats.  The eyes are removed, fixed and incubated for HRP  in exactly the same manner as for the adult eyes. In fetal eyes, the c i l i a r y body is perceptible only as a bulge interposed between the retina and the developing i r i s .  Even at this early  355.  stage, the c i l i a r y capillaries are very similar to those in the adult rat with respect to their permeability to HRP.  The HRP leaves the c i l i a r y  capillaries and the reaction precipitate is localised on the stromal surfaces of the pigment epithelial cells, in between the individual pigment epithelial cells and along the space between the c i l i a r y epithelium and the pigment epithelium.  Although the c i l i a r y body in the fetal rat is morpho-  logically quite different from the fine, long, finger-like processes which make up the adult c i l i a r y body, the locations of the reaction precipitate of HRP are qualitatively similar. As in the adult, the fetal c i l i a r y capillaries are permeable to proteins, in this instance, HRP.  The exit  barrier to proteins into the posterior chamber is epithelial and lies at the apices of the c i l i a r y epithelial cells.  Very rarely, some HRP reaction  product lines the posterior surface of the c i l i a r y body.  It is not known  i f this HRP reaction product has reached the posterior chamber following an extracellular or intracellular route across the c i l i a r y epithelium.  It  could possibly be that in the fetal c i l i a r y body, the epithelial bloodaqueous barrier is not as well established so that some HRP occasionally leaks through extracellularly. In the ensuing days after birth, the c i l i a r y body gradually develops and acquires the characteristics of the adult form, that is, the single bulky c i l i a r y body is divided up into numerous long thin c i l i a r y processes. Initially, the reaction precipitate is present in the locations  mentioned  in a l l of the c i l i a r y body but as the c i l i a r y processes develop, the reaction precipitate is concentrated at the tips of the c i l i a r y processes, as in the adult. The fetal rat i r i s is short and quite undeveloped when compared with the adult rat i r i s .  However, very early in development, the component parts  of the mature i r i s are easily perceptible.  The fetal rat i r i s consists of a  356.  posterior and anterior epithelium, a highly vascular stroma and a developing sphincter muscle bundle at the pupillary end.  In the f i r s t two weeks after  birth, the i r i s grows in length and histological changes occur within the i r i s tissue i t s e l f .  The adult form is attained after this time.  capillaries in the fetal and young post-natal rats, up to 14-15  The  iris  days after  birth, are quite different from the i r i s capillaries in the adult rat in terms of their permeability to intravascularly injected HRP.  Unlike the  adult i r i s capillaries, these capillaries are readily permeable to HRP.  In  younger eyes, the i r i s blood vessels are always closely associated with the anterior epithelium, much as the c i l i a r y capillaries are associated with the pigment epithelium.  HRP reaction product is usually found along the  stromal surfaces of the anterior epithelium, in between the anterior epithelial cells and between the anterior and posterior epithelia. with the observations on the c i l i a r y body is unmistakeable.  The analogy  There is^also  some reaction precipitate in the stroma immediately adjacent to the anterior epithelium as well as some precipitate in amongst the cells of the developing sphincter.  Thus in young eyes, both sets of capillaries, those of the  c i l i a r y body and those of the i r i s , are permeable to HRP.  In the c i l i a r y  body, a blood-aqueous barrier exists at the c i l i a r y epithelium, whereas in the young i r i s the blood-aqueous barrier exists at the posterior epithelium. However, i t must be borne in mind that any HRP  that permeates out of the .  i r i s capillaries into the i r i s tissue can conceivably enter the aqueous humor by an anterior route, that is, through the anterior surface of the iris.  But i t is not known in the rat whether the i r i d i c crypts and pores  are formed at this stage, so that an anterior endothelial barrier may exist. Thus, in young eyes, proteins readily leave the i r i s capillaries but to what extent these proteins contribute towards the composition of the aqueous humor is not known. Since the i r i s capillaries are capable of leaking out  357.  proteins, other substances might leak out as well.  The developing i r i s  may  be a partial source of aqueous humor in the eye. The i r i s capillaries are only permeable to HRP up to about two weeks after birth.  Then the capillaries become like those in the adult and are  impermeable to HRP.  There is a change in the permeability of the i r i s capil-  laries with developmental age. Cotran and Karnovsky (1967) have cautioned us that HRP may  artifi-  cially increase the permeability of small blood vessels and induce vascular leakage so that the observations with HRP tracer studies do not give a true indication of the actual permeability of the blood vessels under study. This increase in permeability is apparently due to the release of histamine. However, in our investigation, a r t i f i c i a l l y increased permeability of the capillaries can be considered an unlikely possibility for the following reasons.  In the adult rat, the c i l i a r y capillaries are permeable to HRP  whereas the i r i s capillaries are not.  If histamine is released into the  blood stream and capillary permeability is increased a r t i f i c i a l l y , i t seems highly unlikely that i t should only be limited in its effect and be confined to the c i l i a r y capillaries, and even then, only to those capillaries at the tip of the c i l i a r y processes, and not affect the i r i s capillaries at a l l . In young eyes, both the c i l i a r y and i r i s capillaries are permeable to HRP. But the i r i s capillary permeability is lost quite abruptly at about two weeks after birth whereas the c i l i a r y capillary permeability is retained throughout l i f e .  The only variable here is the developmental age of the eyes.  Again i t does not seem likely that an a r t i f i c i a l l y HRP-induced vascular leakage is age dependent for the i r i s and not for the c i l i a r y body.  For  these reasons, we contend that in our present investigation, the permeability or impermeability of the capillaries to HRP natural condition.  is a true reflection of its  358.  V.  CONCLUDING REMARKS  The present studies (I) have given us new information on the structure of the adult and developing rat i r i s , and (2) have shown us the dynamic, rapidly altering nature of the histology of the adult rat i r i s in pupillary dilation and constriction. These aspects of the i r i s w i l l be mentioned in brief here as they have been adequately described in the appropriate sections of the discussion and summary of the results. 1.  The new information on the structure of the i r i s is as follows:  (a) In the adult rat, varying numbers of blood vessels are present on the posterior surface of the i r i s , as observed on scanning electron micrographs.  These come from the i r i s stroma, turn around at the pupillary  margin and traverse the whole extent of the posterior surface of the i r i s to the region of the c i l i a r y body. the posterior epithelium.  These blood vessels l i e superficial to  These blood vessels are remnants of the fetal  and early post-natal circulatory system associated with the development of the i r i s .  In the developing  i r i s , most of the blood vessels from the i r i s  stroma extend centrally as the pupillary membrane. Other blood vessels turn around at the pupillary margin to l i e on the posterior surface of the iris.  At times, these blood vessels on the posterior surface of the i r i s  appear to be connected to the hyaloid system via the large annular vessel. With development many of these blood vessels on the posterior surface of the iris regress, so that in the adult rat i r i s only a few such blood vessels are observed.  A number of questions are immediately raised as to:  i . the function of these blood vessels on the posterior surface of the i r i s , i i . the continuity of these vessels in the region of the c i l i a r y body with the overall vascular system of the eye,  i i i . the direction of blood flow in the vessels, and iv. the extent of the presence of these blood vessels in other adult vertebrate eyes. In the developing rat eye, both the blood vessels on the posterior surface of the i r i s and the blood vessels of the tunica vasculosa lentis may be important for the nutrition of the lens.  However, in the adult, these  blood vessels would represent the closest source of nutrients and also a means of removing metabolic waste products from the lens.  The answers to  the other questions have as yet to be explored. (b) At the periphery of the adult i r i s in the region of the c i l i a r y body there is a series of c i l i a r y - i r i s processes.  These are relatively  large radial ridges, distinctly separate from but associated with the c i l i a r y processes.  They are high close to the c i l i a r y processes but they  then go deeply into the i r i s tissue more centrally.  The c i l i a r y - i r i s proces  ses are covered by the posterior epithelial cells and the overlying basement membrane much like the rest of the posterior surface of the i r i s .  However,  the posterior epithelial cells over the c i l i a r y - i r i s processes are lower than those lining the posterior surface of the i r i s . processes appear to be relatively solid structures.  These c i l i a r y - i r i s Possibly they serve to  stabilize the root of the i r i s during the extensive excursions of the pupil in dilation and constriction. In fetal and early post-natal eyes, the c i l i a r y - i r i s processes are absent.  At about two weeks after birth, the beginnings of the development  of the c i l i a r y - i r i s processes are observed.  At f i r s t , they are relatively  low radial ridges but these develop in height and bulk with age.  It is  significant that these c i l i a r y - i r i s processes are only beginning to develop at two weeks after birth, that is at the time when the rat eyes open and presumably when the irises start to function.  At present i t is not known  360.  whether such c i l i a r y - i r i s processes are found in a l l vertebrate irises. (c) Early light microscopic studies have shown that there are crypts and pores on the anterior surface of the i r i s allowing an intercommunication of the aqueous humor and the ground substance of the i r i s stroma (Vrabec, 1952; Gregersen, 1958a, 1958b, 1959, 1961; Coulombre, 1961; Klika and Kloucek, 1962; Purtscher, 1962; Newsome and Lowenfeld, 1971). However, the morphology of the iridic crypts and pores is not well established owing to the inherent limitations posed by the two dimensional nature of tissue sections.  An i n i t i a l attempt was made by Hansson (1970)  who applied scanning electron microscopy to the study of the anterior surface of the i r i s .  However, the tissues were inadequately preserved.  In  our studies we have been able to take advantage of the improvements in the preparative procedures for scanning electron microscopy. The i r i d i c crypts are oval or round, vary in size and have well defined borders.  Occasionally cytoplasmic processes of the anterior endo-  thelial cells making up the crypt border may span the opening of the crypt. A network of the cytoplasmic processes of the underlying stromal cells as well as a network of fine f i b r i l l a r material are seen occupying the crypt openings.  The smaller iridic pores are formed as a result of intercellular  gaps between the cells lining the anterior surface of the i r i s . (d) In transmission electron micrographs of the posterior epithelial cells of the adult rat i r i s , the usual organelles are present, as has been observed by other invesitgators.  In addition there are bundles of intra-  cellular filaments which may be relatively large which have not been previously noted.  The filaments are most often associated with the nucleus.  Occasionally, a bundle of intracellular filaments appears to insert into dense areas of the basal plasma membranes of the posterior epithelial cells. Unlike the other c e l l organelles, the intracellular filaments appear to  change in orientation during pupillary dilation and constriction.  In  pupillary dilation, the filaments cascade around and form a hammock for the nucleus.  In pupillary constriction, the filaments are oriented parallel to  the length of the cells.  From our present studies, i t is not possible to  say whether the filaments are contractile, or whether they may have some degree of elasticity, or whether they are just a structural component of the posterior epithelial cells. (e) Light microscopic  studies of the development of the rat i r i s  have given us an overall view of the changes that take place in different parts of the i r i s tissue.  A time sequence for the development of the i r i s  is established, which can thus serve as a reference for developmental histochemical and physiological studies.  The changes of the posterior and  anterior neuro-ectodermal layers into the posterior epithelium and the dilator and sphincter muscles and the relationships of these layers to the stroma have been described and w i l l not be reiterated. (f) From scanning electron microscopic  studies, i t is observed that  in the adult i r i s the posterior epithelial cells are arranged in rows. These may may  be circumferentially oriented as in pupillary dilation, or they  be radially oriented around the pupil as in pupillary constriction.  The shape of the individual posterior epithelial cells and the arrangement of the cells with respect to one another contribute to the surface configuration of the posterior surface of the i r i s .  The contour of the posterior  surface of the adult i r i s is constantly changing with changes in pupillary s ize. In the fetal i r i s , the individual posterior epithelial cells are not discernible in scanning electron micrographs.  With development, the pos-  terior epithelial cells f i r s t appear as a mosaic of l i t t l e elevations on the i r i s surface.  The cells are arranged in a haphazard fashion with re-  spect to each other.  Later, the bulging outwards of the posterior epi-  thelial cells becomes more prominent.  The overlying basement membrane,  i n i t i a l l y smooth in fetal and early post-natal irises, becomes wrinkled. Grooves begin to appear in between groups and rows of posterior epithelial cells.  By the end of the second week after birth, the posterior epithelial  cells are arranged in rows which bifurcate, taper down or blend with adjacent rows of cells, as on the posterior surface of the adult rat i r i s .  By  two weeks after birth, the posterior epithelial cells are structurally adapted to accommodate the changes in total surface area of the i r i s in dilation and constriction. (g) The architecture of the pupillary membrane is well illustrated in the scanning electron microscopic  studies of the developing rat i r i s .  Most of the blood vessels of the pupillary membrane come from the i r i s stroma, with some contribution, perhaps, from the hyaloid system. The blood vessels of the pupillary membrane form an interconnecting network supported by a fine f i b r i l l a r connective tissue meshwork. The pupillary membrane blood vessels have thin, almost transparent, walls.  In the rat,  the pupillary membrane is not only present in the fetal eye but also in the early post-natal eye. (h) Changes in the permeability of the i r i s capillaries to an intravenously injected tracer substance, Horse-radish Peroxidase (HRP) occur with development.  In fetal and young post-natal eyes, up to two week  after birth, the iris capillaries are permeable to HRP, an average sized protein molecule.  However, there is an epithelial barrier to the outflow  of the reaction product for HRP to the posterior chamber of the eye, as in the c i l i a r y processes (Vegge, 1971a).  It is not known whether there is an  outflow of the HRP reaction product directly to the anterior chamber through the anterior endothelial layer.  In the adult rat i r i s , the i r i s  capillaries are impermeable to HRP. 2.  These studies have given us an insight into the changes in the  morphology of the i r i s associated with its function of dilating and constricting the pupil.  These studies demonstrate clearly that the morphology  of the i r i s must always be considered in relation to the pupillary size. The following features are characteristic of the i r i s in pupillary dilation: (a) The posterior epithelial cells throughout the i r i s are arranged in circumferential!  rows or ridges with grooves of varying depths in be-  tween the epithelial ridges. (b) The individual posterior epithelial cells are discretely separated from the adjacent cells. envelope.  The nuclei show indentations of the nuclear  The intracellular filaments form a loop around the nucleus.  (c) The dilator muscle layer is thick.  Dilator hillocks and dilator  processes are the prominent structures associated with pupillary dilation. The nuclei of the dilator muscle cells also show indentations. (d) The stromal cells and the intercellular connective tissue network appear to be oriented in columns perpendicular to the posterior surface of the i r i s . The following features are characteristic of the i r i s in pupillary constriction: (a) Most of the posterior surface of the i r i s is smooth.  The cir-  cumferential epithelial ridges seen in pupillary dilation have been flattened out.  In extreme pupillary constriction, the posterior epithelial  cells around the pupil are disposed radially.  These decrease in height and  peter out peripherally. Large, bulbous structures are sometimes seen in amongst the radial epithelial ridges.  These may represent sites where  eversions of the posterior epithelium occur.  (b) The posterior epithelial cells are generally low. continuous layer over the posterior surface of the i r i s . nuclei have smooth nuclear outlines. the length of the cells.  They form a  The elongated  The length of the nucleus lies along  The intracellular filaments are seen as longitu-  dinal bundles parallel to the length of the cells and situated mainly in the posterior portions of the epithelial cells. (c) The dilator muscle layer is low.  Dilator hillocks are absent.  There may be an occasional simple dilator process breaking the smooth, straight contour of the stromal surface of the dilator muscle layer.  The  nuclei generally have smooth outlines and they are' oriented parallel to the posterior surface of the i r i s . (d) The stromal components are oriented parallel to the posterior surface of the i r i s . In our studies, chemical mediators were used to maintain the pupil in dilation or constriction.  Similar changes in the histology of the i r i s  in pupillary dilation and constriction are also observed without the inter vention of drugs.  It is uncertain however whether the very extreme form  of pupillary constriction is obtainable as a natural response to intense light conditions. 3.  A number of experiments may be carried out as an extension of  the present study. (a) Comparative studies:  Vertebrates possess not only round but  also s l i t and variously shaped pupils (see Introduction).  Comparative  scanning electron microscopic studies of the posterior surface of the i r i s would reveal the arrangement of the posterior epithelial cells during pupillary dilation and constriction, the presence or absence of blood vessels along the posterior surface of the i r i s , and the presence or absence  of the c i l i a r y - i r i s  processes.  The blood vessels on the posterior surface  of the i r i s come around the pupillary margin from the anterior i r i s stroma to traverse the posterior surface of the i r i s .  At the present time it is  not known how these blood vessels relate to the overall vascular system of the eye.  Further scanning electron microscopic  studies may reveal this. It  would also be interesting to see i f there is a correlation between the number of c i l i a r y - i r i s  processes present and the mobility of the i r i s tissue.  This would give some indication as to the function of the c i l i a r y - i r i s processes. (b) Developmental studies:  There is some indication that  iridic  crypts and pores are absent in young eyes and that the anterior surface of the i r i s is covered by a continuous layer of cells.  With development,  crypts and pores are formed (Vrabec, 1952). Scanning electron microscopic studies of the anterior surface of the developing i r i s would show how the formation of the crypts and pores take place. Rat irises from their earliest fetal stages to the f i r s t few days after birth could be specifically prepared for scanning electron microscopy to study the formation and loss of the pupillary membrane. (c) Functional studies: i r i s is immature histologically.  At birth the rat eyes are closed.  The  By two weeks after birth, the rat eyes  open and the i r i s has acquired the adult form.  It would be interesting to  find out when the i r i s can first respond to external chemical mediators and whether this response can be correlated with the histological structure of the i r i s . (d) Permeability studies:  With the light microscope i t has been  found that the iris capillaries in the developing HRP, whereas those in the adult are not. electron microscope would reveal  i r i s are permeable to  Examination with the transmission  i . the exit pathways of the HRP at the ultrastructural level, and i i . the ultrastructure of the endothelial layer of the i r i s blood vessels both in the adult and in the developing rat i r i s . In addition, other electron microscopic  tracers, for example, f e r r i t i n and  lanthanum, could also be used. (e) Miscellaneous studies:  In some vertebrates, for example, the  newts, i f the lens is removed, a new lens is formed from the posterior epithelial cells of the i r i s .  In other vertebrates, for example, man,  regeneration does not take place.  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