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Morphological changes in the photoreceptor cells and retinal epithelium of the albino wistar rat in vitamin.. 1975

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MORPHOLOGICAL CHANGES IN THE PHOTORECEPTOR CELLS AND . RETINAL EPITHELIUM OF THE. ALBINO WISTAR RAT IN , .- VITAMIN A DEFICIENCY by Wan Ching Yang B. Sc., Nangyang University, 1968 M. Sc., University of Waterloo, 1970 A thesis sumitted i n pa r t i a l fulfilment of the requirements for the degree of Doctor of Philosophy i n the Departn^nt of Anatomy We accept this thesis as conforming to the r ^ u i f e d standard THE UNIVERSITY OF BRITISH COLUMBIA April 1975 In presenting th i s thesis in p a r t i a l fu l f i lment of the requirements fo an advanced degree at the Univers i ty of B r i t i s h Columbia, I agree that the L ibrary shal l make it f ree ly ava i lab le for reference and study. I further agree that permission for extensive copying of th is thesis for scho lar ly purposes may be granted by the Head of my Department or by his representat ives . It is understood that copying or pub l i ca t ion of th is thesis for f inanc ia l gain sha l l not be allowed without my writ ten permission. Department of The Univers i ty of B r i t i s h Columbia Vancouver 8, Canada - i i - V ABSTRACT S t r u c t u r a l changes i n the o u t e r r e t i n a s o f r a t s k e p t on a vitamin. A f r e e d i e t supplemented with, v i t a m i n A a c i d were s t u d i e d by l i g h t and e l e c t r o n m i c r o s c o p i c techniques. R e t i n a s were sampled a t f r e q u e n t i n - t e r v a l s o v e r a p e r i o d o f 11 months o f v i t e m i n A d e f i c i e n c y and t h e i r morphology ccmpared w i t h t h a t o f r e t i n a s from r a t s o f t h e same /age, f e d : a normal d i e t . Other s t u d i e s done i n c o n j u n c t i o n w i t h the above i n c l u d e d , (1) measurements o f weight g a i n and plasma v i t a m i n A c o n t e n t i n control.. . - 1 and v i t a m i n A d e f i c i e n t r a t s (2) l o c a l i z a t i o n o f a c i d phosphatase a c t i v i t y i n the r e t i n a l e p i t h e l i u m i n v i t a m i n A d e f i c i e n c y (3) r a d i o a u t o g r a p h i c 3 ~ study 0 f H -:rrethicmne i n c o r p o r a t i o n i n t o r e t i n a s o f c o n t r o l and . v i t a m i n A d e f i c i e n t r a t s . The r e s u l t s showed t h a t v i t a m i n A d e f i c i e n t animals g a i n e d weight more s l o w l y than the c o n t r o l s and s u f f e r e d a r a p i d d e c l i n e i n plasma v i t a m i n A content a f t e r 3 weeks on t h e s p e c i a l d i e t . L i g h t m i c r o s c c p i c study r e v e a l e d t h a t t h e f i r s t p o r t i o n s o f t h e photo- r e c e p t o r c e l l t o s u f f e r degeneration i n v i t a m i n A d e f i c i e n c y were the o u t e r segments f o l l o w e d s u c c e s s i v e l y by t h e i n n e r segments, s y n a p t i c p r ocesses and photoreceptor n u c l e i . E l e c t r o n m i c r o s c o p i c study r e v e a l e d t h a t a f t e r 3-4 weeks on the s p e c i a l d i e t , c o r r e s p o n d i n g t o the f a l l i n plasma v i t a m i n A l e v e l s , the l a m e l l a r d i s c s o f the p h o t o r e c e p t o r o u t e r segments began t o break doT.vn i n t o v e s i c l e s and t u b u l e s . A f t e r 2.5 months o f v i t a m i n A d e f i c i e n c y , the photoreceptor i n n e r segments began t o s w e l l and s h o r t e n and d i s p l a y l o s s o f m i t o c h o n r i a and ribosomes i n t h e i r cytoplasm. A f t e r 4-5 months, many o u t e r segments had c o m p l e t e l y disappeared - i i i - and further shortening of the inner segments was evident. After 6 norths, very few of the "remaining outer segments v;ere intact although i t was s t i l l possible to identify small number of polysomes and initochondria within the proximal inner segment cytoplasm next to the photoreceptor nuclei. At this stage, the photoreceptor synaptic processes were also severely affected. The number of synaptic vesicles i n each process was greatly reduced and large gaps appeared i n the plasma membranes. The synaptic processes were considerably shortened and synaptic sites reduced i n number. After 9 months, the outer segments had completely disappeared except for a few rerriaining clusters of disordered saccules. Both the inner segments and the synaptic processes had retracted markedly towards the photoreceptor nuclei. For the f i r s t time, the photoreceptor nuclear envelope began to break down although the nuclear chromatin appeared unchanged. After 10-11 months, only a single irregular row of photoreceptor nuclei remained and each nucleus was surrounded by a narrow rim of cytoplasm containing only a few recognizable organelles. Each photoreceptor c e l l was surrounded by several layers of membranes probably from g l i a l c e l l processes. The c e l l junctions normally forming the outer limiting membrane were now absent i n many places. Due to the loss of the photoreceptors the inner neural retina lay very close to the pigment epithelium and the outer processes of the Miller c e l l s were deflected l a t e r a l l y , contributing to the c e l l membranes surrounding the photoreceptor remnants. Progressive changes were also noted i n the structure of the retinal epithelium of the vitamin A deficient animals which were not present i n control animals of the same age. By 4-5 months of vitamin A deficiency, large numbers of lysosomes had accumulated i n - i v - the retinal e p i t h e l i a l cytoplasm close to i t s inner border. This increase i n lysosomes persisted throughout the study as the degeneration of the photoreceptors continued. The lysoscmes and the Golgi complexes of the r e t i n a l epithelium were found to contain the enzyme, acid phos- phatase. The inner or apical processes of the re t i n a l epithelium also proliferated markedly and became very proniinent after 11 months of vitamin A deficiency. Radioautographic studies showed that H^-methionine was incorporated into photoreceptors of both, control and vitamin A deficient animals, thus indicating that protein synthesis continued i n photoreceptors i n vitamin A deficiency. CONTENTS Page Abstract .. i i L i s t of Figures . i x . Acknowledgements x v i i i I Introduction X II Historical Review 7 III Materials and Methods 1) Animals and diets 32 2) Determination of blood plasma viterain A level 35 3) Determination of feed vitamin A content .38 4) Light microscopy 39 5) Electron microscopy 40 6) Acid phosphatase histochemistry -̂0 7) Radioautography-light microscopy Jf2 IV Observations Introductory note ^ 3 Notes on observations hk A) Growth curves k5 B) Plasma vitamin A levels i n control and vitamin A deficient animals kb -VI- C) Light microscopy 1) Retinal epithelium and photoreceptors i n control rats 46 2) Retinal epithelium and photoreceptors i n vitamin A deficiency 48 D) Electron microscopy 1) Normal r e t i n a l morphology a) The retinal epithelium 51 b) The photoreceptor outer segments ....... 5 3 c) The photoreceptor inner segments 5^ d) The outer limiting membrane . 5 5 e) The photoreceptor synaptic processes ... .55 2) Retinal morphology i n vitamin A deficiency a) Retinal changes after 1 month of vil^min A deficiency 58 b) Retinal changes after 1.5 months of vitamin A deficiency 58 c) Retinal changes after 2 months of vii^min A deficiency 5 9 d) Retinal changes after 2.5 months of vitamin A deficiency 5 9 e) Retinal changes after 4-5 months of vitamin A deficiency 60 f) Retinal changes after 6 months of vitamin A deficiency 61 g) Retinal changes after 7-8 months of vitamin A deficiency 62 - v i l - li) Retinal changes after 9 months of vitamin A deficiency 6 3 i) Retinal changes after 1 0 months of vitamin A deficiency ... 6 5 j) Retinal changes after 1 1 months of vitamin A deficiency 6 6 E) Acid phosphatase localization i n the ret i n a l epithelium 6 8 3 . F) Methionane-H incorporation m the retina i n vitamin A deficiency gg V Discussion A) Resume of the most pertinent results 1 3 2 B) Storage and metabolism of vitamin A 1 3 3 C) The photoreceptors i n vitamin A deficiency 1 ) The outer segments 1 3 5 2) The inner segments, synaptic processes and photoreceptor nuclei 1 3 7 D) The retinal epithelium i n vitamin A deficiency .. 12+2 E) Muller c e l l s i n vitamin A deficiency Iif4 F) Light damage to photoreceptors 147 G) Normal loss of photoreceptors 1 4 9 H) Glycogen filled-mitochondria 1 4 9 VI Summary 1 5 1 Original Contributions Bibliography Vit a - I X - LIST OF FIGURES Page Notes on Figure Legends 71 Figure 1. Graph showing the growth rate of the control and vitamin A deficient animals 72' 2a. Graph showing how the maximum absorbance of vitamin A i s obtained ....73 2b. Graph showing the blood plasma vitamin A levels i n the control and vitamin A deficient animals y^" 3. Light micrograph from a 2 month old control animal showing the overall structure of the normal retina posterior to the equator of the eye 75 4. Light micrograph at higher magnification showing the posterior retina of a 2 month old control animal 75 5. Light micrograph showing the retina peripheral to the equator of the eye from a 7 month old control animal ..........75 6. Light micrograph showing the peripheral outer retina from the same specimen as Figure 5 75 7. Light micrograph showing the peripheral retina from a 12 month old control animal 76 8. Light micrograph at higher magnification showing the outer retina from the same specimen as Figure 7 76., Figure Page 9. Light micrograph of the posterior retina from an animal which was on a vitamin A-free diet for 3.5 months 76. 10. Light micrograph at higher magnification showing the outer retina from the same specimen as Figure 9 76 11. Light micrograph showing the posterior retina from a 6 month vitamin A deficient animal 77 12. Light micrograph at higher magnification showing the same specimen as Figure 11 • 77 13. Light micrograph showing the posterior retina from a 9 month vitamin A deficient animal 77 14. Light micrograph at higher magnification showing the same specimen as Figure 13 77 15. Light micrograph showing the posterior retina of a 10 month vitamin A deficient animal 78 16. Light micrograph at higher magnification showing the same specimen as Figure 15 78 17. Light micrograph showing the posterior retina from an 11 month vitamin A def icient"'animal 78 18. Light micrograph at higher magnification showing the same specimen as Figure 17 78 19. Electron micrograph showing a portion of the ret i n a l epithelium from a 1.5 month old control animal 79 - x i - Figure Page 20. Electron micrograph showing the inner r e t i n a l epithelium and photoreceptor outer segments from a 7 month old control animal 80 21. Electron micrograph showing the r e t i n a l epithelium and the photoreceptor outer segments from the same specimen as Figure 20 g2 22. Electron micrograph showing the fine structure of two adjacent r e t i n a l e p i t h e l i a l c e l l s from a 9 month old control animal 82 23. Electron micrograph showing photoreceptor inner and outer segments from a 1.5 month old control animal 83 24. Electron micrograph showing the photoreceptor outer segments, connecting cilium and inner segments from a 7 month old control animal 84 25. Electron micrograph showing photoreceptor outer and inner segments from the same specimen as Figure 24 84 26. Electron micrograph showing photoreceptor inner segments from a 9 month old control animal 85 27. Electron micrograph at higher magnification showing photoreceptor inner segments and the outer limiting membrane from the same specimen as Figure 26 86 28. Electron micrograph showing i n i t s center a rod synaptic process from a 1.5 month old control animal 87 - x i i - Figure Paga 29. Electron micrograph showing the outer plexiform layer from a 1.5 month old control animal 88 30. Electron micrograph showing a cone synaptic process from a 1.5 month old control animal , 89 31. Electron micrograph showing rod spherules from a 1.5 month old control animal 8g 32. Electron micrograph showing the outer plexiform layer from a 12 month old control animal •• 90 33. Electron micrograph showing the photoreceptor outer segments from a 1 month vitamin A deficient ̂ animal 91 34. Electron micrograph showing the ret i n a l epithelium and photoreceptor outer segments from a 1.5 month vitamin A deficient animal 92 35. Electron micrograph showing photoreceptor outer segments from the same specimen as Figure 34 93 36. Electron micrograph showing photoreceptor outer and inner segments from a 2 month vitamin A deficient animal 9^ 37. Electron micrograph showing the junction between the r e t i n a l epithelium and photoreceptor outer segments from a 2.5 month \dtamin A deficient animal 95 38. Electron micrograph showing disintegrating photoreceptor outer segments from the same specimen as Figure 37 96 - x i i i - F i g u r e P a g e 39. E l e c t r o n rnicrograph showing t h e p h o t o r e c e p t o r i n n e r segments o f t h e same specimen as F i g u r e 37 .97 40. E l e c t r o n micrograph showing p h o t o r e c e p t o r o u t e r segments from a 4 month v i t a m i n A d e f i c i e n t animal 9 8 41. E l e c t r o n micrograph showing pho t o r e c e p t o r o u t e r segments, i n n e r segments and the o u t e r l i m i t i n g membrane from t h e same specimen as F i g u r e 40 99 42. E l e c t r o n micrograph showing pho t o r e c e p t o r i n n e r segments ' _" from t h e same specimen as F i g u r e 40 100 43. E l e c t r o n micrograph showing two adj a c e n t r e t i n a l e p i t h e l i a l c e l l s f r c m a 5 month v i t a m i n A d e f i c i e n t a n i m a l 101 44. E l e c t r o n micrograph showing t h e r e t i n a l e p i t h e l i u m , photo- r e c e p t o r o u t e r segments and i n n e r segments from a 6 month v i t a m i n A d e f i c i e n t animal ........ 102 45. E l e c t r o n micrograph showing t he r e t i n a l e p i t h e l i u m and t h e p h o t o r e c e p t o r o u t e r segments from t h e same specimen as F i g u r e 44 103 •j 46. E l e c t r o n micrograph showing t h e photo r e c e p t o r o u t e r segments from t h e same specimen as F i g u r e 44 104 •47. E l e c t r o n micrograph showing m a i n l y p h o t o r e c e p t o r i n n e r segments from t n e same specimen as F i g u r e 44 105 48. E l e c t r o n micrograph showing t he o u t e r p l e x i f o r m l a y e r from t h e same specimen as F i g u r e 44 106 -XXV- Figure Page 49. Electron micrograph showing the photoreceptor synaptic processes from the same specimen as Figure 48 107 50. Electron micrograph showing the r e t i n a l epithelium and photoreceptor outer segments from a 7 month vitamin A deficient animal 108 51. Electron micrograph shewing the ret i n a l epithelium, photoreceptor outer segments and inner segments from a 8 month vitamin A deficient animal • 109 52. Electron micrograph showing inner segments, photoreceptor outer segments and part of the re t i n a l epithelium from the same specimen as Figure 51 n o 53. Electron micrograph showing photoreceptor inner segments from the same specimen as Figure 51 m 54. Electron micrograph showing the outer plexiform layer from the same specimen as Figure 51 112 55. Electron micrograph showing the outer plexiform layer from the same specimen as Figure 54 113 56. Electron micrograph showing the outer retina from a 9 month vitamin A deficient animal .. 114 57. Electron micrograph showing the ret i n a l epithelium, remnants of photoreceptor outer segments and portions of photoreceptor inner segments from a 9 month vitamin A deficient animal 115 -XV- Figure Page 58. Electron micrograph showing the outer retina from a 9 . month vitamin A deficient animal 116 59. Electron micrograph showing at higher magnification photoreceptor inner segments and the outer limiting membrane from the same specimen as Figure 58 117 60. Electron micrograph shewing degenerating photoreceptors from a 9 month vitamin A deficient animal 118 61. Electron micrograph showing the posterior outer retina from a 10 month vitamin A deficient animal 119 62. Electron micrograph showing the close association between the retinal epithelium and the neural retinal layer at the 10th month of vitamin A deficiency 120 63. Electron micrograph showing at higher magnification the close association between the apical processes of the retinal epithelium and the processes of the Muller c e l l s from the same specimen as Figure 61 121 64. Electron micrograph showing the outer retina from the same specimen as Figure 61 1 2 2 65. Electron micrograph shewing the outer retina from an 11 month vitamin A deficient animal 123 66. Electron micrograph showing the retinal epithelium and the outer retina from the same specimen as Figure 65 124 - x v i - Figure Page 67. Electron micrcgraph shewing the outer retina from an 11 month vitamin A deficient animal . .. 1 2 5 68. Electron micrograph showing the outer retina from an 11 month vitamin A deficient animal 1 2 6 69. Electron micrograph from the same specimen as Figure 68 showing prcminent apical processes of the ret i n a l epithelium and layers of membranes probably of a g l i a l nature surrounding each of the remaining photo- receptor c e l l s 1 2 7 70. Electron micrograph showing acid phosphatase localization i n the ret i n a l epithelium from the posterior retina of a 6 month vitamin A deficient animal 1 2 3 71. Electron micrograph showing acid phosphatase localization i n the ret i n a l epithelium from the same specimen as Figure 70 1 2 9 72. Light microscopic radioautograph showing the posterior outer retina from a 1 0 month old control animal, 4 hours 3 after i n t r a v i t r e a l injection of H -methionine 130 73. Light microscopic radioautograph showing the posterior outer retina from a 1 0 month old control animal, 24 hours 3 after labelling intravitreally with H -methionine 130 74. Light microscopic radioautograph showing the posterior outer retina from a 2.5 month vitamin A deficient animal, . 3 . 4 hours after intravitreal labelling with H -methionine..... 1 3 0 - x v i i - Figure Page 75. Light microscopic radioautograph from a 2.5 month vitamin A deficient animal showing the posterior outer retina, 24 hours after i n t r a v i t r e a l labelling with 3 H -methionine 1 3 0 76. Light microscopic radioautograph from an 8 month vitamin A deficient animal, showing the posterior outer retina, 3 4 hours after intravitreal labelling with H -methionine 131. 77. Light microscopic radioautograph from an 8 month vitamin A deficient animal, showing the posterior outer retina, . . 3 24 hours after i n t r a v i t r e a l labelling with H -methionine ..... 131 78. Light microscopic radioautograph from a 10 month vitamin A deficient animal showing the ret i n a l epithelium and the neural retina, 4 hours after intravitreal labelling with 3 H -methionine 131 79. Light microscopic radioautograph from a 10 month vitamin A deficient, animal showing the same structures as Figure 78, 3 24 hours after intravitreal labelling with H -methionine 131 - x v i i i - AGKN^vTLEDGEMEOTS I em much indebted to r r r y supervisor, Dr. M. J. Hollenberg for his assistance, guidance and encouragement throughout the course of this study. I am also grateful to members of my thesis advisory cormdttee, Drs. S. M. Brance, C. E. Slonecker, W. A. Webber for their c r i t i c a l review and suggestions during the preparation of the thesis. I would l i k e also to thank Dr. H. W. J. Regetli, Head of .Virus . Chemistry Division, Agricultural Canada Research Station, Vancouver, B.C. for the use of the Beckman DK-2A spectrophotometer i n his lab- oratory, the Microbiology Department of Macdonald College for the use of darkroom f a c i l i t i e s and Miss Lorraine Leroux for typing the f i n a l draft of the thesis. The fin a n c i a l support from the Medical Research Council i s gratefully acknowledged. 1 I KnMDDUCTION B l i n d n e s s due t o e i t h e r c o r n e a l , r e t i n a l o r o p t i c nerve damage i s t h e most important and s t r i k i n g f e a t u r e o f v i t a m i n A d e f i c i e n c y . D e s t r u c t i o n o f the cornea i n xerophthalmia and k e r a t o m a l a c i a i n man and most experimental animals i s due t o d e f i c i e n c y o f v i t a m i n A (Johnson 1939, 1943; D e a l i n g and Wald, 1958). The l o s s o f the p r o s t h e t i c group, r e t i n e n e , from the r o d v i s u a l pigment, rhodopsin, l e a d s t o n i g h t b l i n d - ness i n man and experimental animals ( F r i d e r i c i a and Holm, 1925; Tansley, 19 31;.". Wald, 1935a, 1936; Wald e t a l . , 1938; Haig e t a l 1938; Wald and Steven, 1939; Steven and Wald, 1940). Dowling and Wald (1960) have shown t h a t v i t a m i n A d e f i c i e n t r a t s kept a l i v e by ad T n i n i s t r a t i o n o f v i t a m i n A a c i d e v e n t u a l l y l o s e p h o t o p i c as w e l l as s c o t o p i c v i s i o n , presumably because r e t i n e n e i s the p r o s t h e t i c group f o r cone as w e l l as r o d v i s u a l pigment. I n c a t t l e and dogs, v i t a m i n A d e f i c i e n c y a l s o l e a d s t o f a u l t y growth o f bone and nervous t i s s u e which can cause c o n s t r i c t i o n o f t h e o p t i c nerve (Moore e t a l . , 1935; Hart and G u i l b e r t , 1937; Moore, 1939). Tansley (1933, 1936) f i r s t d e s c r i b e d the s t r u c t u r a l damage t o t h e r o d photoreceptors i n v i t a m i n A d e f i c i e n t r a t s and dogs. L a t e r Johnson' (1939, 1943) confirmed and extended Tansley's o b s e r v a t i o n s i n the r a t . He observed by l i g h t microscopy t h a t a f t e r 7-13 weeks o f v i t a m i n A d e p r i v a t i o n i n young r a t s , many photoreceptor o u t e r segments disappeared and those t h a t remained s t a i n e d abnormally. As the. d e f i c i e n c y p r o g r e s s e d , the r o d i n n e r segments a l s o degenerated, and then s u c c e s s i v e l y t h e e x t e r n a l 2 l i m i t i n g membrane, t h e o u t e r n u c l e a r l a y e r and the i n n e r n u c l e a r l a y e r . Tne o u t e r segments o f rods which had d e t e r i o r a t e d o n l y s l i g h t l y showed c o n s i d e r a b l e r e g e n e r a t i o n w i t h i n 24 hours o f r e a d t i t i r d s t r a t i o n o f v i i ^ m i n A. Even rods which had degenerated completely regenerated w i t h i n 10-18 weeks o f d i e t a r y v i t a m i n A supplementation. F i f t e e n years l a t e r , i n . a n attempt t o map t h e e n t i r e course of v i t a m i n A d e f i c i e n c y and i t s cure i n t he r a t , Dowling and Wald (1958) approached t h e problem from p h y s i o l - o g i c a l , b i o c h e m i c a l and h i s t o l o g i c a l aspects. They found t h a t , i n weanling r a t s a f t e r 3 weeks o f v i t a m i n A d e p r i v a t i o n , the l i v e r v i t a m i n A content had d e c l i n e d and a week l a t e r the b l o o d v i t a m i n A content a l s o f e l l . A t . t h i s p o i n t , t h e r e t i n a l rbodopsin content began t o d e c l i n e l i n e a r l y and r e g u l a r l y , marking the onset o f n i g h t b l i n d n e s s . By the beginning o f t h e seventh week, l e v e l s o f o p s i n too began t o d e c l i n e and h i s t o l o g i c a l d e t e r i o r a t i o n o f the r e t i n a was observed. I n these s t u d i e s , t h e experimental animals u s u a l l y d i e d w i t h i n 2-3 months o f vitamin. A d e p r i v a t i o n . I t was then d i s c o v e r e d t h a t v i t a m i n A a c i d , f i r s t prepared by Aran and Van Dorp (1946) , can m a i n t a i n growth i n t he r a t w i t h o u t a f f e c t i n g the d e l e t e r i o u s e f f e c t s o f v i t a m i n A d e f i - c i e n c y on the v i s u a l system. This made a more d e t a i l e d i n v e s t i g a t i o n o f the anatomical changes i n the r e t i n a p o s s i b l e s i n c e animals are unable t o reduce v i t a m i n A a c i d t o v i i ^ m i n A aldehyde and thus i n f l u e n c e the v i s u a l system (Moore, 1957). A s e r i e s o f s t u d i e s o f the e f f e c t s o f v i t a m i n A d e f i c i e n c y c n the r e t i n a i n animals supplemented w i t h v i t a m i n A a c i d were then undertaken by Dowling and h i s a s s o c i a t e s (Dowling and Wald, 1960; Dowling and Gibbons, 1961; Dowling, 1966). H i s t o l o g i c a l s t u d i e s on r e t i n a s o f a l b i n o r a t s were c a r r i e d out over a p e r i o d o f 10 . months. Dowling and Gibbons (1961) observed by l i g h t microscopy t h a t the degeneration of the photoreceptors followed the same pattern seen e a r l i e r by Johnson (1939, 1943) . Electron microscopic studies (Dowling and Gibbons, 1961) shewed that after 2 months on a vitamin A free d i e t , supplemented with vitamin A acid, the f i r s t sign o f degeneration of the photoreceptor outer segments was breakdown of the outer segment discs into vesicles and tubules. After a high proportion of the discs had degenerated, the outer segments began to lose j t h e i r normal elongated, c y l i n d r i c a l shape and became almost spherical. After 6 months of vitamin A deprivation, only fragments of photoreceptor outer segments remained. The photoreceptor n u c l e i and inner segments were greatly reduced i n number and the inner segments that remained were short and thicker than normal. The fine structure of the inner segments, however, "appeared normal. ' ::" Muller c e l l processes became highly conspicuous i n the spaces l e f t between the remaining segments. By 10 months of vitamin A deficiency, the neural r e t i n a and the r e t i n a l epithelium adhered t i g h t l y t o one another, the photoreceptor inner and outer segments had disappeared, and the photorecep- to r nuclei had been reduced to one irregular row which, however, appeared ' normal. The rest of the r e t i n a and the r e t i n a l epithelium also appeared.'" normal. The outer p i e x i f orm layer was i n d i r e c t contact with the r e t i n a l epithelium but was somewhat reduced i n thickness due to a loss of synaptic processes from the v i s u a l c e l l s . The photoreceptors were capable of r e - generation as long as the inner segments were present. Prom the above observations, Dowling and Gibbons (1961) concluded that loss of opsin, a .major component of the outer segments, (14% dry weight i n c a t t l e and 40% dry weight i n frogs) was probably the primary cause of the s t r u c t u r a l damage to the outer segments. More recently, Nee 11 £t a l . (1971) have shown-that the ef f e c t s 4 of vitamin A deficiency on the rat eye are dependent upon the levels of illumination to which the rats are exposed d a i l y . Animals kept in darkness r e t a i n t h e i r normal electroretincgraphic (ERG) function and r e t i n a l rhodopsin content much longer i n vitamin A de- ficiency than those exposed to weak c y c l i c l i g h t . Shear et al.,(1973) reported that the r e t i n a of albino rats which have been kept i n a c y c l i c environment (14 hours of low intensity illumination and 10 hours o f darkness) show degeneration when they are k i l l e d a f t e r a period of 6 to 12 hours illumination. However, the retinas from those animals that have spent several hours i n darkness before they are k i l l e d are normal. The structural degeneration was due to a separation of the adjacent pigment- e p i t h e l i a l c e l l s , retraction of e p i t h e l i a l apical processes and change of the lamellar discs of the apical 1/3 of a l l photoreceptor outer segments into tubules. Recently, Eerron and Riegel (1974a, 1974b) have shown by radioautographic study that production of photoreceptor outer segment protein i s decreased i n temin A deficient rats and suggest that vitamin A must be available f o r rod outer segment production as lack of i t slows - • the production rate. Despite the foregoing work, a detailed study o f the sequential break- down and f i n e structural changes i n a l l regions of the photoreceptor c e l l i n vitamin A deficiency i s s t i l l lacking. Although i t has been suggested that the r e t i n a l epithelium i s unaffected i n vitamin A deficiency (Johnson, 1939, 1943; Dowling and Vvald, 1958; Dowling and Gibbons, 1961) , there i s as yet l i t t l e evidence to support t h i s conclusion. Furthermore, i t seems i l l o g i c a l that the pig-rent epithelium would be unaffected during the photoreceptor brea>:down process since the r e t i n a l epithelium normally i s active i n the phagocytosis of rod outer segment fragments (Dowling and 5 Gibbons, 1962; B a i r a t i and O r z a l e s i , 1963; Ishikawa and Yamada, 1970; Young, 1967, 1971a Young and Bok, 1969; S p i t z n a s and Hogan, 1970). One vrould a n t i c i p a t e t h a t t h e r e would be a marked i n c r e a s e i n the a c t i v i t y o f the pigment e p i t h e l i u m i n t h i s r e g a r d as p h o t o r e c e p t o r s are destroyed due t o l a c k o f •vitamin A. A l s o i n t h e c l a s s i c a l s t u d i e s o f v i t a m i n A d e f i c i e n c y by Dowling and h i s a s s o c i a t e s (Dowling and Wald, 1960; Dowling and Gibbcns, 1961; Dowling, 1966) the p o s s i b l e e f f e c t o f l i g h t damage t o the r e t i n a was n o t taken i n t o account. There i s no mention o f the l e n g t h o f time each day the animals were exposed t o l i g h t and the l i g h t i n t e n s i t y . As mentioned above l i g h t i n g c o n d i t i o n s are now known t o markedly a f f e c t photoreceptor c e l l morphology ( N o e l l and A l b r e c h t , 1971; Shear e t s i . , 1973). The p r e s e n t study has been undertaken i n an e f f o r t t o overcome these d i f f i c u l t i e s and augment our knowledge o f the event, p a r t i c u l a r l y t h e morphological changes t a k i n g p l a c e d u r i n g v i t a m i n A d e f i c i e n c y . The study examines, by l i g h t and t r a n s m i s s i o n e l e c t r o n microscopy, t h e s t x u c t u r a l changes t a k i n g p l a c e i n the photoreceptors and r e t i n a l e p i t h e l i u m of the a l b i n o r a t maintained on a v i t a m i n A f r e e d i e t sup- plemented v / i t h v i t a m i n A a c i d . The animals v/ere k e p t under s t r i c t l y c o n t r o l l e d l i g h t i n g c o n d i t i o n s o f 12 hours o f low i n t e n s i t y l i g h t and 12 hours o f darkness per day. The f i n e s t r u c t u r e , o f the photoreceptors and r e t i n a l e p i t h e l i u m has been s t u d i e d i n d e t a i l i n v i t a m i n A d e f i c i e n t and c o n t r o l animals o f v a r y i n g ages. Growth r a t e s o f v i t a m i n A d e f i c i e n t and c o n t r o l animals have been compared. .Blood v i t a m i n A- l e v e l s . f r o m animals t h a t were on the v i t a m i n A f r e e d i e t have been analysed and com- pared w i t h c o n t r o l s . The d e t a i l s and sequence o f d e s t r u c t i o n o f the 6 v a r i o u s p o r t i o n s o f the photoreceptors i n v i t a m i n A d e f i c i e n c y has been examined a t frequ e n t i n t e r v a l s o v e r a p e r i o d o f 11 months and compared w i t h photoreceptor s t r u c t u r e i n c o n t r o l r a t s d u r i n g aging. M o r p h o l o g i c a l changes i n the r e t i n a l e p i t h e l i u m i n c o n t r o l and v i t a m i n A d e f i c i e n t r a t s a l s o have been examined s i m i l a r l y . A c i d phosphatase l o c a l i z a t i o n has been used t o t e s t f o r the presence o f lysosomes i n the r e t i n a l 3 e p i t h e l i u m o f the v i t a m i n A d e f i c i e n t animals. F i n a l l y , H -methionine was administered t o the v i t a m i n A d e f i c i e n t animals t o determine i f p r o t e i n s y n t h e s i s s t i l l o ccurs i n the d i s i n t e g r a t i n g photoreceptors. The o b j e c t s o f the present study a r e : 1). t o f u r t h e r o u r under- sta n d i n g o f the d e t a i l e d s e q u e n t i a l breakdown o f the e n t i r e photo- r e c e p t o r i n c l u d i n g i t s o u t e r segment, i n n e r segment, nucleus and s y n a p t i c process i n v i t a m i n A d e f i c i e n c y and 2). t o e l u c i d a t e the sub- sequent e f f e c t o f v i t a m i n A d e f i c i e n c y on the s t r u c t u r e and f u n c t i o n o f the r e t i n a l e p i t h e l i u m and g l i a l c e l l s . A complete knowledge o f the above events w i l l be b e n e f i c i a l i n understanding the c e n t r a l r o l e p l a y e d by v i t a m i n A i n v i s i o n . 7 I I HISTORICAL REVIEW The E a r l y H i s t o r y o f R e t i n a l I n v e s t i g a t i o n Although the v i s u a l organ was one o f the foremost s u b j e c t s o f i n t e r e s t among a n c i e n t s c i e n t i s t s , d e t a i l e d study o f t h e s t r u c t u r e and f u n c t i o n o f the r e t i n a has not taken p l a c e u n t i l r e c e n t time (Polyak, 1941). The Greeks considered the r e t i n a as a " n e t - l i k e t u n i c " (Polyak, 1941). K e p l e r (1604) was the f i r s t t o demonstrate the e s s e n t i a l r o l e o f the r e t i n a as a photoreceptor. Lack o f i n s t r u - ments and the p r i m i t i v e c o n d i t i o n s o f o p t i c a l techniques a t the time prevented a more advanced i n v e s t i g a t i o n o f i t s s t r u c t u r e . Development o f the f i r s t microscope l e d t o more p r o f i t a b l e i n v e s t i g a t i o n o f the v i s u a l organ. Antony-van Leeuwenhoek (1674) i n a l e t t e r t o the p u b l i s h e r o f the P h i l o s o p h i c a l T r a n s a c t i o n s o f the Royal S o c i e t y o f London r e p o r t e d what he saw i n the r e t i n a o f the cow i n the f o l l o w i n g s h o r t statement: "The t h i r d t u n i c l e was e x c e e d i n g l y t h i n and tender and having viewed i t , I found i t a l s o c o n s i s t s o f g l o b u l e s u n i t s " . Ten years l a t e r , i n a l e t t e r t o the s e c r e t a r y o f the Royal S o c i e t y o f London, Leeuwenhoek (1684) again mentioned the r e t i n a , t h i s time, o f the f r o g . He again mentioned the blood v e s s e l s and the g l o b u l e s . He was the f i r s t t o see the blood c a p i l l a r i e s , nerve c e l l s and a l s o the rods and cones. Un- f o r t u n a t e l y h i s o b s e r v a t i o n r e c e i v e d l i t t l e a t t e n t i o n . During the e a r l y p a r t of the e i g h t e e n t h century the f u n c t i o n o f 8 the retina was thought t o be as f o l l o w s : l i g h t rays caused v i b r a t o r y motion o f the o p t i c nerve f i b e r s , which i n co-operation w i t h the " s p i r i t s " mediated the r e c e p t i o n and t r a n s m i s s i o n o f the v i s u a l impres- s i o n s t o the b r a i n , where the o b j e c t s were recognized by the " s o u l " (Maitre-^Jan, 1725). Towards the end o f the e i g h t e e n t h c e n t u r y , i n v e s t i g a t i o n o f the r e t i n a was a i d e d by the i n t r o d u c t i o n o f a number o f chemicals as f i x - a t i v e s and by h i g h power microscopes. There were w i d e l y d i v e r g e n t views r e g a r d i n g the s t r u c t u r e o f t h e r e t i n a . Some b e l i e v e d i t t o be a pure web o f nerve f i b e r s . Others c o n s i d e r e d i t as an expansion o f the medullary p a r t o f the o p t i c nerve and s t i l l o t h e r s came t o the c o n c l u s i o n t h a t the r e t i n a was composed o f the mucous substance o f the b r a i n . Fontana (1782, 1795) was the f i r s t t o throw l i g h t upon the e x i s t - i n g chaos o f o b s e r v a t i o n s and hypotheses on the r e t i n a . He s t u d i e d t h e r e t i n a o f the r a b b i t and v i s u a l i z e d o p t i c nerve f i b e r s , g a n g l i o n and ot h e r nerve c e l l s suspended i n a sup p o r t i n g framework o f n e u r o g l i a and b l o o d c a p i l l a r i e s . U n f o r t u n a t e l y he overlooked t he rods and cones. T r e v i r a n u s , between 1835-1838, c a r r i e d out a s y s t e m a t i c study o f th e r e t i n a marking the b e g i n n i n g o f modern i n v e s t i g a t i o n o f r e t i n a l s t r u c t u r e and f u n c t i o n . H i s r e s e a r c h animals ranged from f i s h t o mammals. He d e s c r i b e d the r e t i n a as b e i n g e n t i r e l y composed o f t h i c k l y packed and extremely d e l i c a t e tubes whose- b l i n d ends resembled t i n y warts o r " p a p i l l a e " which he b e l i e v e d t o protrude onto the v i t r e a l face o f the r e t i n a (Treviranus, 1835). He b e l i e v e d t h e " p a p i l l a e " t o be photoreceptors (Treviranus, 1835) , but by mistake, he p l a c e d them on the v i t r e a l f a c e . F o l l o w i n g T r e v i r a n u s , i t was V a l e n t i n (1837) who demonstrated 9 t h a t t h e r e t i n a was oorrpcsed o f p a r a l l e l , r e g u l a r l y a r r a n g e d l a y e r s . He c o r r e c t l y r e c o g n i z e d t h e p o s i t i o n o f the b a c i l l a x y l a y e r ( l a y e r o f ro d s and cones) b u t by m i s t a k e r e v e r s e d t h e o r d e r o f t h e l a y e r s i n t h e r e s t o f t h e r e t i n a . The o u t e r p o s i t i o n of. t h e b a c i l l a r y l a y e r was l a t e r c o n f i r m e d b y B i d d e r (1839) who found t h a t t h e f r e e ends o f t h e r o d s were •always t u r n e d t o w a r d t h e c h o r o i d membrane. . The s t u d y o f r e t i n a l s t r u c t u r e was advanced when Hannover (1840) demonstrated how t o a p p l y chromic a c i d t o harden t h e r e t i n a . T h i s improvement made i t p o s s i b l e bo c u t t h i n s e c t i o n o f r e t i n a l t i s s u e . D u r i n g t h e decade f o l l o w i n g the i n t r o d u c t i o n o f chr o m i c a c i d ; i n t o l a b o r a t o r y t e c h n i q u e s , t h e s t r u c t u r e o f t h e human and o f o t h e r v e r t e - b r a t e r e t i n a s was e x t e n s i v e l y s t u d i e d by P a c i n i (1845) ,. Bowman (1849), V i n t s c h g a n (1853), K511iker (1854) and e s p e c i a l l y by M u l l e r (1852, 1853, 1854, 1856-1857). These s t u d i e s r e v e a l e d t h e s t r a t i f i c a t i o n o f t h e . r e t i n a . I n a d d i t i o n , M u l l e r (1851) d i s c o v e r e d f i b r o u s s t r u c t u r e s w h i c h passed through t h e e n t i r e t h i c k n e s s o f t h e r e t i n a . He named them " r a d i a l f i b e r s " and t h e y s t i l l b e a r h i s name. By means o f p h y s i o l o g i c a l o b s e r v a t i o n s and w i t h t h e h e l p o f p e r t i n e n t h i s t o l o g i c a l d a t a , 2 - i l l e r (1853,-1854, 1856-57) computed t h e l o c u s o f p h o t o r e c e p t i o n t o be i n t h e b a c i l l a r y l a y e r . The f i r s t corrprehensive d e s c r i p t i o n s o f r e t i n a l h i s t o l o g y and f u n c t i o n a l i n t e r p r e t a t i o n s o f r e t i n a l s t r u c t u r e were p u t f o r w a r d by •Muller (1853, 1854, 1855-57) and K o l l i k e r (1854). They b e l i e v e d t h a t t h e r e d s and cones were t r u e p h o t o r e c e p t o r s . Because o f the o u t e r p o s i t i o n o f the rods and cones, t h e s t i m u l a t i n g r a y s o f l i g h t have t o pass t h r o u g h a i i r o s t t h e e n t i r e t h i c k n e s s o f t h e r e t i n a . 10 However, t h i s o b s t a c l e was l a r g e l y reduced by the g r e a t transparency and homogeneity o f t h e r e t i n a l substance. They suggested t h a t e x c i t a t i o n i n the rods and t h e cones t r a v e l l e d , i n the d i r e c t i o n o p p o s i t e t o l i g h t , ' along t h e " r a d i a l f i b e r s " , through the v a r i o u s i n t e r c a l a t e d n u c l e a r and g a n g l i o n c e l l s , and along the o p t i c nerve f i b e r s u n t i l f i n a l l y e n t e r i n g the v i s u a l c e n t e r s o f the b r a i n . • • • H i s t o l o g i c a l technique was f u r t h e r improved when Schultze and Rudneff (1865) i n t r o d u c e d osmium as a f i x a t i v e . They s u b s t a n t i a t e d M i l l e r and K o l l i k e r ' s views r e g a r d i n g t h e f u n c t i o n a l i n t e p r e t a t i o n o f r e t i n a l s t r u c t u r e . S c h u l t z e (1872) s u b d i v i d e d the r e t i n a s t r e t c h - i n g from the c h o r o i d t o the v i t r e o u s i n t o the present 10 l a y e r s , namely, 1) pigment e p i t h e l i u m , 2) rods and cones, 3) o u t e r l i m i t i n g membrane 4) o u t e r n u c l e a r l a y e r , 5) o u t e r g r a n u l a r (plexiform) l a y e r , 6) i n n e r n u c l e a r l a y e r , 7) i n n e r g r a n u l a r (plexiform) l a y e r , 8) g a n g l i o n c e l l s , 9) o p t i c nerve f i b e r s and 10) i n n e r l i m i t i n g membrane. He (1873) f u r t h e r e l u c i d a t e d the' h i s t o l o g i c a l d e t a i l s o f the p h o t o r e c e p t o r c e l l s which he c a l l e d cones and rods. The two types o f photoreceptors were c l a s s i f i e d ' b n the b a s i s t h a t 1) oone n u c l e i occupied a more s c l e r a l . p o s i t i o n i n comparison w i t h those o f the r o d s , 2) r o d o u t e r segments were elongated, t h i n and c y l i n d r i c a l w h i l e those o f the cone were t h i c k and tapered and 3) the rod c e l l had a k n o t - l i k e ending and the' cone had a • f o o t - l i k e ending. The area o f the photoreceptor c e l l s c l e r a l t o the photoreceptor nucleus was d i v i d e d i n t o an i n n e r and o u t e r segment because o f d i f f e r e n c e s i n s t a i n i n g . I n a d d i t i o n t o h i s important c o n t r i b u t i o n d u r i n g t h i s e r a , S c h u l t z e (1872) introduced the important " d u p l i c i t y theory" o f v i s i o n which i n d i c a t e d t h a t the rod photoreceptors, were f o r dim l i g h t (scotopic) v i s i o n and cones were f o r c o l o u r and d i s c r i m i n a t i v e 11 bright l i g h t (photopic) vision. Towards the end of the nineteenth century, Camillo Golgi (1873, 1878) discovered that a solution of s i l v e r nitrate stained nerve c e l l s black. Tartuferi (1887) applied this method to the retina and found that the cone fibers s p l i t at their v i t r e a l ends into a number of delicate filaments which entered the outer plexiform layer. Here these cone filaments met the scleral expansions of the bipolar and other c e l l s whose bodies formed the inner nuclear layer and whose v i t r e a l processes descended into the inner plexiform layer. In the lat t e r , these descending processes seemed again to merge with each other and with the scleral expansions of the ganglion c e l l s , thus establishing a connection between the photoreceptors, on the one hand, and the optic nerve fibers, on the other. I t was Ramon Cajal (1892, 1911) who supplemented the duplicity theory by assuming a duplex conducting mechanism a l l along the visual pathway. According to Cajal the nerve currents e l i c i t e d i n the receptive elements by the photic stimuli had to pass through the following three sets of neurons before they reach the brain: 1) the rods and cones, 2) the bipolars and 3) the ganglion c e l l s . The rods and cones each had their own sets of bipolars and ganglion c e l l s . Cajal also confirmed the "Neurone Theory" which proposed that the'neurones comprising nerve tissues were each structural and functional entities. No longer was. nerve tissue to be considered a continuous reticulum. 12 F i n e S t r u c t u r e o f the Photoreceptor The o u t e r segment Photoreceptor f i n e s t r u c t u r e was f i r s t s t u d i e d by S j o s t r a n d (1949, 1953a). He d e s c r i b e d t h e outer segment o f the rod photoreceptors o f the guinea p i g and perch, and cone photoreceptor o f the perch as being composed o f a p i l e o f membranous d i s c s enclosed w i t h i n a c e l l membrane. I n the r o d photoreceptor o f the guinea p i g , each d i s c was found t o be formed by a double membrane which appeared t o be f r e e o f the c e l l plasma membrane. Each d i s c a l s o possessed a s i n g l e i n c i s u r e The i n c i s u r e s o f the d i s c s were found t o be a l i g n e d forming a l o n g i t u d i n a l groove. These f i n d i n g s were a l s o r e p o r t e d i n photoreceptors o f o t h e r rode n t s ; e.g. the mouse (Cohen, 1960) and the r a t (Dowling and Gibbons, 1961). S i m i l a r l y , i n amphibia, P o r t e r (1956), Yamada (1957) and Wald (1958a) showed t h a t the r o d o u t e r segment a l s o c o n s i s t s o f a p i l e o f membranous d i s c s . Instead o f a s i n g l e i n c i s u r e i n amphibia each d i s c has numerous i n c i s u r e s r e s u l t i n g i n a s c a l l o p e d o u t l i n e . P o r t e r (1956) p o i n t e d o ut t h a t the i n c i s u r e s i n c r e a s e d the s u r f a c e area o f the d i s c s . Subsequent s t u d i e s o f rod o u t e r segments i n pigeon (Cohen, 1963), and i n man and monkey (Cohen, 1965) have a l s o r e v e a l e d a s c a l l o p e d appearance of the d i s c s . The i n c i s u r e s appear t o be a l i g n e d w i t h each o t h e r (Fernandez-Moran, 1961; de R o b e r t i s and Lasansky, 1961). I n cone o u t e r segment, most o f the d i s c s , except f o r a few a t t h e outermost t i p , have t h e i r membranes continuous w i t h t h e s u r f a c e membrane o f the c e l l s uggesting t h a t the d i s c s are made up o f a c t u a l i n f o l d i n g s o f 13 the c e l l membrane (Sj o s t r a n d , 1959 [perch]; Lasansky and de R o b e r t i s , 1960 [toad]; Moody .and Robertson, 1960, Yamada, 1960 [ f r o g ] ; Cohen, 1961a, 1961b [monkey], 1964 [ s q u i r r e l ] 1968 [ f r o g ] ) . In r o d o u t e r segments, the m a j o r i t y o f the d i s c s are i s o l a t e d and separated, although a few are s a i d t o ocmminicate w i t h t h e s u r f a c e membrane a t the base o f an o u t e r segment (Robertson, 1965; Cohen, 1964, 1965, 1968). Young (1971a, 1971b) has shown by r a d i o a u t o g r a p h i c studies, t h a t t h e d i s c s o f the r o d o u t e r segment are c o n s t a n t l y shed and i n t e r m i t t e n t l y renewed w h i l e those o f the cone are l o n g l a s t i n g . The d i s c s o f the r o d o u t e r segment are made up o f two membranes. Each membrane c o n s i s t s o f two p a r a l l e l dense l a y e r s each 20 A t h i c k , separated by a l i g h t i n t e r s p a c e 35 A t h i c k (Moody and Robertson, 1960). The d i s c has been shown by the f r e e z e - f r a c t u r e technique t o possess two d i s s i m i l a r membrane s u r f a c e s . A "rough" s u r f a c e studded w i t h s p h e r i c a l and l i n e a r p r o t r u s i o n s f a c i n g the i n t r a d i s c space and a "smooth" s u r f a c e f a c i n g the i n t e r d i s c space (Clark and Branton, 1968; Leeson, 1970, 1971b). More r e c e n t l y , C a p a l d i (1974) has suggested a new model f o r the c e l l merrbrane which c o n s i s t s o f a l i p i d b i l a y e r 45 A t h i c k and g l o b u l a r p r o t e i n s forming i n t r i n s i c and e x t r i n s i c p a r t s o f the membrane. The i n t r i n s i c p r o t e i n forms the i n t e g r a l p a r t o f the membrane. The d i s c membrane has been suggested t o c o n t a i n o n l y i n t r i n s i c p r o t e i n which i s the photopigment, rhcdopsin. In darkness, rhodopsin molecules are sub- merged f o r about 1/3 o f t h e i r diameter i n the d i s c membrane's o u t e r surface. Vthen i l l u m i n a t e d , the rhodopsin molecules s i n k deeper i n t o the membrane u n t i l they are half-submerged. 14 The connecting c i l i u m The o u t e r segment i s connected t o the i n n e r segment by a co n n e c t i n g c i l i u m . The c i l i u m i s e c c e n t r i c a l l y p l a c e d and e n t e r s the o u t e r segment o f the r o d a t t h e base o f t h e groove formed by the s e r i e s o f d i s c i n c i s u r e s (de R o b e r t i s , 1956). P o r t e r (1956) and de R o b e r t i s (1956) both demonstrat- ed t h a t t h e membrane c o v e r i n g the o u t e r segment i s continuous w i t h t h e c e l l plasma membrane by way o f the connecting c i l i u m . They noted t h a t the c i l i u m contains 9 p a i r s o f p e r i p h e r a l t u b u l e s b u t l a c k s the c e n t r a l p a i r c h a r a c t e r i s t i c o f a m o t i l e c i l i u m . Such p a i r e d p e r i p h e r a l t u b u l e s have been d e s c r i b e d i n the guinea p i g r o d ( S j o s t r a n d , 1953a) , i n r a b b i t r o d s (de R o b e r t i s , 1956), and cones (de R o b e r t i s and Lasansky, 1958) and i n human rods ( M i s s o t t e n , 1955a; Yamada e t a l . , 1958b) and oones (Yamada e t a l . , 1958b). The p e r i p h e r a l t u b u l e s e n t e r t h e a p i c a l p o r t i o n o f t h e i n n e r segment and end i n a b a s a l body o r c e n t r i c l e (Cohen, 1961a, 1961b; de R o b e r t i s , 1956; de R o b e r t i s and Lasansky, 1958; S j o s t r a n d , 1953b; Yamada e t a l . , 1958b). A second c e n t r i o l e , o r i e n t a t e d a t r i g h t angle t o the b a s a l body, has been d e s c r i b e d i n human rods and oones by Yamada e t a l . , (1958b) and i n monkey rods and cones by Cohen (1961a, 1961b). The i n n e r segment The i n n e r segment o f the photoreceptor i s marked by a c o n c e n t r a t i o n cf m i t o c h o n d r i a a t i t s apex. T h i s accumulation corresponds t o t h e e l l i p s o i d seen by l i g h t microscopy (Walls, 1942). I n the. r o d , t h e m i t o c h o n d r i a are o r i e n t e d w i t h t h e i r long axes p a r a l l e l t o the a x i s cf the c e l l (de R o b e r t i s , 1956; Cohen, 1960; Yamada, 1957). However, t h e r e 15 appears t o be no such o r i e n t a t i o n i n the cone (de Robertis and Lasanksy, 1958). In the rod e l l i p s o i d , endoplasmic reticulum has been observed between the mitochondria, i n man (Missotten, 1965a) , i n the r a b b i t (de Robertis, 1956) and i n the frog (Yamada, 1957), but there appears to be l i t t l e endoplasmic reticulum between, the mitochondria i n the cone e l l i p s o i d (de Robertis and Lasanksy, 1958). From the basal body a r i s e the c i l i a r y r o o t l e t s which run the entire length of the inner segment between two systans of vacuoles (Cohen, 1961a, 1961b; Uga et a l . , 1970; B a i r a t i and Q r z a l e s i , 1963). Cohen (1960) suggested that the c i l i a r y r o o t l e t s may be concerned wi t h conduction of e x c i t a t i o n while Uga e t a l . (1970) indicated they may serve as a s k e l e t a l support fo r the receptor inner segments. Below the e l l i p s o i d , the cytoplasm contains ribosomes, rough endoplasmic reticulum and a Golgi complex situated j u s t above the external l i m i t i n g membrane (Sjostrand, 1953b; Cohen, 1961b, 1963). This region i s known as the myoid because i n some lower vertebrates, i t i s c o n t r a c t i l e and responds t o changes i n r e t i n a l i l l u m i n a t i o n (Young, 1969). Membrane bound o i l droplets are found i n the cone inner segments of some non mammalian species (Walls, 1942; Duke- Elder, 1958; Pedler and Tansley, 1963; Sjostrand and E l f v i n , 1957; Berger, 1965, 1966; Borwein and Hollenberg, 1973). The o i l droplets are believed to be formed by nrLtochondrialfusion i n a v i t r e a l to s c l e r a l gradient (Berger, 1964; Ishikawa and Yamada, 1969; Borwein and Hollenberg, 1973). Vertebrate photoreceptor n u c l e i are t y p i c a l l y oval or spherical i n shape (Nilsson, 1964; Hollenberg and Berstein, 1966; Morris and Shorey, 1967; Dowling and Werblin, 1969) but i n the newt, they are elongated, c y l i n d r i c a l structures (Dickson and Hollenberg, 1971). Rod n u c l e i are l b more electron dense than the cones (Nilsson, 1964; Dickson and Hollenberg 1971). In the frog, the cone nuclei are located closer to the outer plexiform layer than the rods while the opposite i s the case i n the newt retina. The nuclei are surrounded by narrow rims of cytoplasm. Cohen (1960) noted that the rod nuclei are often packed together suggesting the p o s s i b i l i t y of interaction between rods at the nuclear le v e l . Where the nuclei are separated, processes of Muller's fibers l i e between them (Cohen, 1961b). The rod spherule The basic morphology of the photoreceptor synaptic terminals i s - remarkedly uniform i n a l l classes of vertebrates (de Robertis, 1958;- Cohen, 1969; Dartnall and Tansley, 1963; Evans, 1966; S t e l l , 1967, Dowling, 1968, 1970; Kolb, 1970). The rod synaptic terminal often enlarges to form a spherule. A single mitochondrion has been found i n the rod spherule i n the rat (Ladman, 1958) and the mouse (Cohen, 1960). Mitochondria have also been reported i n both rod and cone synaptic - terminals of monkey photoreceptors (Cohen, 1961b), but are absent i n • synaptic terminals of rabbit (de Robertis and Franchiy 1956) , guinea-pig and opossum (Ladman, 1958). Rod synapses usually have.only one synaptic ribbon (a half-moon shaped lamellar structure) associated with the pene- trating bipolar and horizontal c e l l processes at the synaptic junction (Cohen, 1961b; de Robertis and Franchi, 1956; Ladman, 1958; Sjostrand, 1958), Missotten (1965a) reported that several synaptic ribbons may be present i n the rods of man. Ladman (1958) and Cohen (1961b) described in rat synaptic tentujials another lamellar structure related to the 17 synaptic ribbon, the "rod arciform density". Synaptic vesicles are present i n the rod spherules and are thought to contain neuro-transmitter substance (de Robertis and Franchi, 1956; de Robertis, 1958). Each synaptic ribbon i n both the rods and cones i s usually surrounded by a halo of synaptic vesicles. Gray and Pease (1971) postulated that the synaptic ribbon possibly serves to direct the synaptic vesicles down to the presynaptic membrane. The function of the arciform density might be two fold; f i r s t l y , to anchor the synaptic ribbon to the presynaptic mem- brane and secondly, to guide the synaptic vesicles o f f the ribbon on to the presynaptic membrane after they have passed down along the surface of the ribbon (Gray and Pease, 1971). •-— . --. The rod spherules make synaptic contacts with a cluster of nerve " endings penetrating the spherule i n a single invagination (Sjostrand, 1958, 1961; de Robertis and Franchi, 1956; Ladman, 1958; Cohen, 1963, 1964; Villegas, 1960, 1964; Missotten, 1965b; Evans, 1966). The deeply inserted terminal nerve fibers, situated i n a l a t e r a l position, have been identified as horizontal c e l l axon terminals i n man (Missotten, 1965b). In goldfish the l a t e r a l l y placed terminal fibers are identified as horizontal c e l l dendrites ( S t e l l , 1967). The less deeply inserted terminal fibers which are located centrally have been traced to bipolars i n man (Missotten, 1965b) and i n goldfish ( S t e l l , 1967). Rod superficial contacts have been reported i n the mudpuppy (Dowling and Werblin, 1969) and i n the newt (Dickson and Hollenberg, 1971). They are formed by slight indentations i n the receptor ternuLnal surface produced by the synapsing neuronal processes. Superficial contacts are found i n association with synaptic ribbons i n the mudpuppy, but i n the newt, synaptic ribbons have never been identified with superficial contacts. 18 Cone p e d i c l e s Cone p e d i c l e s o f v e r t e b r a t e s have been s t u d i e d i n d e t a i l by s e v e r a l i n v e s t i g a t o r s (de R o b e r t i s and F r a n c h i , 1956; Cohen, 1963, 1964; P e d l e r and T a n s l e y , 1963; K a l b e r e r and P e d l e r , 1963; P e d l e r and T i l l y , 1964; V i l l e g a s , 1960; S t e l l , 1967/ Evans, 1966; Hol l e n b e r g and B e r s t e i n , 1966; Dov/ling and W e r b l i n , 1S69; Lasansky, 1972). M i s s o t t e n and Dooren (1966) r e c o n s t r u c t e d p e d i c l e s o f human photoreceptors w i t h s e r i a l s e c t i o n s and grouped c o n t a c t s i n t o 3 ty p e s ^ i n v a g i n a t i o n s , s u r f a c e c o n t a c t s and i n t e r r e c e p t o r c o n t a c t s , (a) I n v a g i n a t i o n s : F i b e r s from t h e i n n e r n u c l e a r l a y e r i n v a g i n a t e deep i n t o t h e p e d i c l e forming a symmetrical t r i a d . The two l a t e r a l processes o f t e n c o n t a i n endoplasmic r e t i c u l u m , m i c r o t u b u l e s and ribosomes and a r e now w i d e l y accepted as axons o f h o r i z o n t a l c e l l s . The c e n t r a l p r o c e s s can be e i t h e r h o r i z o n t a l c e l l d e n d r i t e o r b i p o l a r c e l l endings (Hogan e t a l . , 1971). Dowling and B o y c o t t (1966), i n con- t r a s t , f e e l t h e c e n t r a l processes belong e x c l u s i v e l y t o b i p o l a r c e l l s and t h e l a t e r a l p r o c e s s e s are n o t c h a r a c t e r i s t i c o f e i t h e r d e n d r i t e s o r axons. I n t h i s way t h e h o r i z o n t a l c e l l s are a b l e t o r e c e i v e and t r a n s m i t s t i m u l i i n a h o r i z o n t a l d i r e c t i o n , thus c r e a t i n g i n t e r r e c e p t o r c r o s s - c o n n e c t i o n s . The s y n a p t i c r i b b o n faces t h e t r i a d a t a r i g h t angle, and t h e a r c i f o r m d e n s i t y l i e s between t he s y n a p t i c r i b b o n and the c e l l membrane. There a r e numerous s y n a p t i c v e s i c l e s around t h e s y n a p t i c r i b b o n and t h e a r c i f o r m d e n s i t y . (b) S u r f a c e c o n t a c t s : In"this s i t u a t i o n b i p o l a r d e n d r i t e s synapse w i t h t h e cone in s h a l l o w i n d e n t a t i o n s on the b a s a l s u r f a c e s o f t h e p e d i c l e s . There i s a s l i g h t t h i c k e n i n g of the cone and b i p o l a r membranes at t h i s r e g i o n b u t s y n a p t i c v e s i c l e s and s y n a p t i c r i b b o n s are absent. 19 (c) I n t e r r e c e p t o r c o n t a c t s : I n t e r r e c e p t o r synapses have been demon- s t r a t e d i n the guinea p i g ( S j o s t r a n d , 1958.,) , mouse (Cohen, 1960) , f r o g ( M i l s s o n , 1964) , monkey (Cohen, 1961b, Dowling and B o y c o t t , 1966) , man M i s s o t t e n , 1965b; Uga e t a l . , 1970; Hogan e t a l . , 1971) and t u r t l e (Lasansky, 1972). Each p e d i c l e has s e v e r a l l a t e r a l expansions which extend a c o n s i d e r a b l e d i s t a n c e h o r i z o n t a l l y and make c o n t a c t w i t h l a t e r a l expansions o f an adjacent cone o r the l a t e r a l s u r f a c e o f a r o d spherule. No s y n a p t i c v e s i c l e s are found on e i t h e r s i d e o f the c o n t a c t . I t i s not known whether the i n t e r r e c e p t o r c o n t a c t s are s i t e s o f conduction o r t r a n s m i s s i o n o f nerve i i r p u l e s (Hogan e t a l . , 1971). R e t i n a l pigment e p i t h e l i u m The f i n e s t r u c t u r e o f the r e t i n a l e p i t h e l i u m has been s t u d i e d ex- t e n s i v e l y i n r e c e n t years (Yamada e t a l . , 1958a; B e r n s t e i n , 1961, 1966; Yamada, 1961; Dowling and Gibbons, 1962; B a i r a t i and O r z a l e s i , 1963; Breathnack and W y l l i e , 1966; Leur-duPree, 1968; Leeson, 1971a; B r a e k e v e l t and H o l l e n b e r g , 1970). These authors have shown t h a t the mature r e t i n a l e p i t h e l i u m i s a f a i r l y uniform s i n g l e l a y e r o f r e c t a n g u l a r c e l l s , s i t u a t e d around the o u t e r circumference o f the r e t i n a , extending from the edge o f the o p t i c d i s c t o the o r a s e r r a t a . Tn a t a n g e n t i a l s e c t i o n c u t p a r a l l e l t o the pigment e p i t h e l i u m , these c e l l s are hexagonal i n shape. I n many s p e c i e s , pigment e p i t h e l i a l c e l l s are c h a r a c t e r i z e d by numerous b a s a l i n f o l d i n g s o f the plasma membrane, an e x t e n s i v e smooth endoplasmic r e t i - culum, pigment granules and numerous a p i c a l processes surrounding the photoreceptor o u t e r segments. The b a s a l i n f o l d i n g s and the c l o s e presence o f numerous mitochondria suggest an a c t i v e r o l e i n m e t a b o l i c t r a n s p o r t from 20 the c h o r i o c a p i l l a r i e s (Dowling and Gibbons, 1962; B a i r a t i and O r z a l e s i , 1963). The m e t a b o l i t e s are c a r r i e d across the pigment e p i t h e l i u m and s u p p l i e d t o the o u t e r r e t i n a i n c l u d i n g the photoreceptors. I n the f r o g r e t i n a l e p i t h e l i u m , t h e smooth endoplasmic r e t i c u l u m i s c l o s e l y a s s o c i a t e d w i t h l a m e l l a t e d m y e l o i d bodies. P o r t e r and Yamada (1960) have suggested t h a t the myeloid body w i t h i t s l a r g e s u r f a c e area and a s s o c i a t e d smooth endoplasmic r e t i c u l u m may p l a y an a c t i v e r o l e i n d i r e c t i n g i s c m e r i z a t i o n o f a l l - t r a n s r e t i n a l d e h y d e t o the 1 1 - c i s con- f i g u r a t i o n , and the r e c o n s t i t u t i o n o f rhodopsin from 1 1 - c i s r e t i n a l d e h y d e and o p s i n . The smooth endoplasmic r e t i c u l u m a l s o may be s i g n i f i c a n t i n the i n t e r c o n v e r s i o n between r e t i n a l d e h y d e and v i t a m i n A, as w e l l as the t r a n s p o r t o f these compounds t o the photoreceptor c e l l s . I n the newt pigment e p i t h e l i u m , Dickson and Hollenberg (1971) observed l a r g e l i p i d i n c l u s i o n s c l o s e l y a s s o c i a t e d w i t h the smooth endoplasmic r e t i c u l u m and they have suggested t h a t the l a t t e r i s i n v o l v e d i n l i p i d metabolism. The presence o f a l i m i t e d amount o f rough endoplasmic r e t i c u l u m and f r e e ribosomes i s a l s o a common f e a t u r e o f most v e r t e b r a t e r e t i n a l e p i t h e l i a . Leure-duPree (1968) has suggested t h a t d u r i n g development the rough endoplasmic r e t i c u l u m may c o n t r i b u t e t o p r o t y r o s i n a s e and t y r o s i n a s e b i o s y n t h e s i s . A f t e r t h e i r p r o d u c t i o n , these enzymes are concentrated i n the G o l g i apparatus and then i n v e s i c l e s which sub- sequently form pro-pigment granules. I n the pigment e p i t h e l i u m o f the • a d u l t a l b i n o r a t , pigment granules are absent due t o a g e n e t i c b l o c k t o m e l a n i z a t i o n (Dowling and Gibbons, 1962). I n the human a l b i n o melanocypte , the g e n e t i c d e f e c t has been suggested t o be due t o the l a c k o f f r e e 1 - t y r o s i n e a v a i l a b l e t o the raelanosome (Mishima and Loud, 1963). 21 Pigment granules are concentrated i n the apical cytoplasmic processes of the pigmented epithelium. Meyer (1969) has suggested that they may function i n absorbing l i g h t and preventing scatter. This mechanism helps i n refinement of the photoreceptor stimulus thereby increasing visual acuity. The pigment granules of certain submammalian species move outwards away from the apical e p i t h e l i a l processes i n times of low illumination, thus allowing maximal visual sensitivity at the expense of reduced visual acuity (Meyer., 1969). That the pigment epithelium i s indispensable for the visual process was f i r s t noted by Kuhne (1878). He reported that a frog retina taken out of the eye can no longer regenerate rhodopsin and that i t regains this capacity i f l a i d back upon the pigment epithelium. He was convinced that intimate contact between the neural retina and the pigment epithelium was necessary for rhodopsin to be synthesized i n the rods. Retinal detachment results i n blindness i n human subjects but when contact i s re- established, vision i s restored. This, Wald (1958a) f e l t , provided further evidence that the pigment epithelium makes an active contribution to the visual process. In the frog eye, retinene isomerase, which i s responsible for the conversion of 11-trans vitamin A back to 11-cis vitamin A, has been shown to be present i n the retinal pigment epithelium (Hubbard, 1956). The vitamin A liberated by the bleaching of visual pigments i s rapidly esterified i n the eye (Krinsky, 1958). In amphibia, the enzymatic system concerned with this process i s present i n the pig- ment epithelium (Krinsky, 1958). The pigment epithelium also plays a major role i n the turn over of the photoreceptor outer segments. Young and Bok (1969) have shown 22 by r a d i o a u t o g r a p h i c s t u d i e s t h a t i n the f r o g , newly formed r a d i o a c t i v e p r o t e i n i s i n c o r p o r a t e d i n t o d i s c membranes a t the base o f the rod o u t e r segment. These l a b e l l e d d i s c s are p r o g r e s s i v e l y d i s p l a c e d along the o u t e r segments as new d i s c s are formed a t the base. When the l a b e l l e d d i s c s reach the end o f the o u t e r segment, they are detached from- i t and subsequently can be i d e n t i f i e d i n the pigment e p i t h e l i u m . Phagocytosis by t h e pigment e p i t h e l i u m i s accomplished by the a p i c a l e p i t h e l i a l processes. The processes surround'...' the d i s c s a t the outermost ends o f the rod photoreceptors. The d i s c s are then taken i n t o the e p i t h e l i a l c e l l as phagosomes which are subsequently broken down by lysosomal action- (Spitznas and Hogan, 1970; Young, 1971a). F a i l u r e o f t h i s p hagocytic f u n c t i o n r e s u l t s i n an overaccumulation o f r e d o u t e r segment m a t e r i a l and subsequently, v i s u a l c e l l death and b l i n d n e s s . T h i s has been c l e a r l y demonstrated i n the case o f i n h e r i t e d r e t i n a l dystrophy i n the r a t (Dowling and Sidman, 1962; Bok and H a l l , 1971; Herron e t a l . , 1 9 5 9 ) . Herron e t a l . (1969), u s i n g r a d i o a c t i v e amino a c i d , noted t h a t the r e t i n a l d y s t r o p h i c r a t shows a normal r a t e o f o u t e r segment growth u n t i l t h e age o f 18 days. T h e r e a f t e r t h e growth o f l a m e l l a r d i s c s towards the pigment e p i t h e l i u m slews down and the p i g - ment e p i t h e l i u m shows no a b i l i t y t o phagocytose the rod o u t e r segments. Dowling and Sidman ( 1 9 5 2 ) found t h a t the e l e c t r o r e t i n o g r a m (ERG) o f the r e t i n a l d y s t r o p h i c r a t i s normal u n t i l the age o f 18 days. T h e r e a f t e r , a gradual d e t e r i o r a t i o n i n the ERG begins and complete l o s s i s observed by two months of age. The l a m e l l a r m a t e r i a l o f the photoreceptor o u t e r segments g r a d u a l l y b u i l d s up a t t h e i r a pices w h i l e the rhodopsin content i n the r e t i n a o f t h e . d y s t r o p h i c r a t t e m p o r a r i l y i n c r e a s e s . F i n a l l y , a l l v i s u a l c e l l s are l o s t . 'These i n v e s t i g a t i o n s suggest t h a t death o f the 23 photoreceptors may be due t o a primary d e f e c t i n the pigment e p i t h e l i u m (Herron e t a l . , 1969; Bok and H a l l , 1971). These r e c e n t f i n d i n g s have s t r o n g l y reemphasized the c l o s e c o r r e l a t i o n between the r e t i n a and the pigment e p i t h e l i u m i n the maintenance o f v i s u a l f u n c t i o n . I n a d d i t i o n t o the above f u n c t i o n s , the pigment e p i t h e l i u m a l s o s y n t h e s i z e s and s e c r e t e s p a r t o f the mucopolysaccharide m a t e r i a l which f i l l s the spaces between the v i s u a l c e l l o u t e r segments (Bermans, 1964; Moyer, 1969). H i s t o c h e m i c a l s t u d i e s have r e v e a l e d t h a t the e p i t h e l i a l cytoplasm c o n t a i n s h i g h a c t i v i t y o f g l y c o l y t i c dehydrogenase, a c i d phosphatase, ATPase, AMPase, and a l k a l i n e phosphatase ( L e s s e l l and Kuwabara, 1964). 2A N i g h t B l i n d n e s s and i t s A s s o c i a t i o n w i t h V i t a m i n A The a f f l i c t i o n o f n i g h t b l i n d n e s s ( n y c t a l o p i a o r d i f f i c u l t y i n dark adaptation) and i t s cure by l i v e r o r l i v e r o i l s , v/as known t o the a n c i e n t s , l o n g b e f o r e v i t a m i n A was d i s c o v e r e d . Aykroyd (1944) mentioned t h a t Eber's Papyrus, an a n c i e n t Egyptian medical t r e a t i s e o f about 1500 B.C., recommended r o a s t ox l i v e r o r the l i v e r o f a b l a c k cock as c u r a t i v e agents f o r n i g h t b l i n d n e s s . The famous Greek p h i l o s o p h e r and "Father o f M e d i c i n e " , Hippocrates a l s o p r e s c r i b e d ox l i v e r f o r c u r i n g n i g h t b l i n d n e s s , b u t suggested t h a t i t should be eaten i n a raw s t a t e a f t e r d i p p i n g i n honey (Moore, 1957). Modern knowledge, o f course, i n d i c a t e s t h a t l i v e r s o f almost a l l animals are r i c h i n v i t a m i n A. The r e l a t i o n s h i p of v i t a m i n A t o dark a d a p t a t i o n was not r e a l i z e d u n t i l c o n s i d e r a b l e i n f o r m a t i o n about the d i s t r i b u t i o n and chemical n a t u r e o f v i t a m i n A became a v a i l a b l e from other sources. I n 1876, Franz B o l l made the p i o n e e r i n g d i s c o v e r y o f v i s u a l p u r p l e i n the r e t i n a . He n o t i c e d t h a t , i n the f r o g , the pigment e p i t h e l i u m o f the r e t i n a c o n t a i n e d golden c o l o u r e d o i l d r o p l e t s which faded when the eye was b r i g h t l y i l l u m i n a t e d f o r l o n g p e r i o d s . The v i s u a l p u r p l e obtained frcm the f r o g ' s r e t i n a turned y e l l o w on treatment w i t h a c i d . He i n f e r r e d from t h i s change t h a t the v i s u a l p u r p l e was d e r i v e d from the y e l l o w pigment which abounded i n the pigment e p i t h e l i u m . T h i s l e d h i s c o l l e a g u e Capranica (1877) t o conclude t h a t the pigment was l u t e i n , a term which a t t h a t time covered both carotene and x a n t h o p h y l l . The presence o f v i s u a l p u r p l e i n dark adapted r e t i n a s and i t s absence from r e t i n a s adapted t o b r i g h t sunshine was another p o i n t e s t a b l i s h e d i n the e a r l y experiments. Kuhne (1878) demonstrated t h a t , i n the f r o g ' s eye, 25 visual purple reappears slowly i n the dark after i t i s bleached by li g h t . He also noted that, at the same time, the eye becomes more photo-sensitive during a stay i n the dark. Since the photochemical effect of a given amount of ligh t was found to be proportional to the concentration of the light-sensitive substance, Parinaud (1881) supplemented the "duplicity theory" with the assumption that twilight vision was dependent on visual purple. Night blindness was therefore, presumed to be correlated with abnormal function of visual purple. In 1913, McCollum and Davis noted that certain mixtures of fats of animal origin or fats extracted from internal organs, e.g. kidney.or l i v e r , contained a factor absolutely indispensable for survival and growth. The substance, also present i n abundance i n leaves of plants and a few seeds, was designated fat-soluble vitamin A (McCollum and Davis, 1913). When a diet was inadequate i n i t s content of this substance, animals become emaciated and suffered edema of the eyes. Blindness resulted i f the animals were permitted to go without this dietary essential or with an jjiacequate supply for a sufficient time (Holm, 1925). An important step was then taken by F r i d e r i c i a and Holm (1925) who demonstrated that dark adaptation was defective i n vitamin A deficient rats and that the pigment "visual purple" could be formed only slowly i n their retinas. Early i n this century, Stern (1905), u t i l i z i n g platinum chloride fixation i n the dark, was able to produce a visual purple platinum complex which was stable i n the light. Tansley (1931) applying the same technique demonstrated that the retina of vitamin A deficient rat contained sub- normal amounts of visual purple. Two years later, working on the h i s t o l o g i - c a l changes of the retinas of rats and dogs i n avitaminosis A, Tansley (1933) came to the folia-Ting conclusions: 1) blood circulation was important 2b i n t h e r e g e n e r a t i o n of v i s u a l p u r p l e 2) both i n l i v i n g and i n prepared r e t i n a s , s u i t a b l y s t a i n e d v i s u a l p u r p l e was always found t o be present i n the o u t e r limbs o r segments o f the rods and nowhere e l s e i n the r e t i n a 3) v i s u a l p u r p l e and t h e outer limbs o f rods appeared simultane- o u s l y i n developing r e t i n a 4) i n the v i t a m i n A d e f i c i e n t c o n d i t i o n , poor r e g e n e r a t i o n o f v i s u a l p u r p l e was accompanied by changes i n the o u t e r l i m b o f the r o d photoreceptor and 5) i n case o f extreme v i t a m i n A de- p r i v a t i o n , the r e t i n a was unable t o form any v i s u a l p u r p l e . I n develop- i n g r e t i n a s the primary e f f e c t o f v i t a m i n A d e f i c i e n c y was the absence o f v i s u a l p u r p l e formation. L a t e r , r o d s t r u c t u r e was a f f e c t e d (Tansley, 1936). So f a r , i t had been recognized t h a t the formation o f v i s u a l p u r p l e was i n f l u e n c e d by v i t a m i n A, but t h e r e was no evidence t o i m p l i c a t e the v i t a m i n d i r e c t l y i n v i s u a l processes. Evidence o f the d i r e c t p a r t i c i p a - t i o n o f v i t a m i n A i n dark a d a p t a t i o n , however, was subsequently o b t a i n e d i n the c l a s s i c a l r e s e a r c h o f Wald (1935a, 1936). I n e x t e n s i v e s t u d i e s on r e t i n a s o f f r o g s , p i g s , sheep and c a t t l e , Wald found v i t a m i n A i n the n e u r a l r e t i n a and the combined pigment e p i t h e l i a l and c h o r o i d l a y e r (Wald, 1935a). The v i t a m i n was i d e n t i f i e d by i t s a b s o r p t i o n a t 328 ma i n the u l t r a - v i o l e t and a t 620 mu by the antimony t r i c h l o r i d e t e s t (Wald, 1935a). Wald found t h a t the dark adapted r e t i n a s of the b u l l f r o g , Eana ca t e s b i a n a , contained o n l y a t r a c e o f v i t a m i n A which c o u l d be e x t r a c t e d w i t h benzene i n the dark without i n j u r i n g the v i s u a l p u r p l e (Wald, 1936 ). I f the r e t i n a s were then exposed f o r s h o r t time t o l i g h t , e x t r a c t i o n w i t h benzene now produced a y e l l o w pigment which he named " r e t i n e n e " . I f the r e t i n a s a f t e r b l e a c h i n g by l i g h t were allowed t o stand f o r an hour a t 25°C the y e l l o w c o l o u r seen immediately a f t e r b l e a c h i n g disappeared and e x t r a c t i o n w i t h benzene now produced a s u b s t a n t i a l amount o f v i t a m i n A. 27 From these f i n d i n g s , Void formulated a c y c l e (see below) which became a landmark i n the h i s t o r y o f re s e a r c h on v i t a m i n A. v i s u a l p u r p l e v i t a m i n A + protean < r e t i n e n e + p r o t e i n ( " v i s u a l white") ( " v i s u a l yellow") Meanwhile, experiments were c a r r i e d o u t by a number o f r e s e a r c h - e r s on human s u b j e c t s who were de p r i v e d o f v i t a r n i n A i n t h e i r d i e t f o r p e r i o d s which ranged from a few weeks t o over two y e a r s . (Hecht and Mandelbaum, 1939; Wald e t a l . , 1938; Booher e t a l . , 1939; Steven, 1943; Hume and Krebs, 1949). These s u b j e c t s showed a d e t e r i o r a t i o n i n t h e i r c a p a c i t y f o r dark a d a p t a t i o n sooner o r l a t e r i n t h e course o f the v i t a m i n A d e f i c i e n c y . The d e f e c t was c o r r e c t e d by the a d m i n i s t r a t i o n o f v i t a m i n A. Haig e t a l . (1938) noted t h a t n i g h t b l i n d n e s s was a l s o a s s o c i a t e d w i t h c h r o n i c l i v e r d i s e a s e s . 28 The V i s u a l C y c l e The r o l e o f \dtamin'A i n v i s i o n was e l u c i d a t e d i n 3 main st a g e s . F i r s t l y , a v i t a l c l u e was pro v i d e d by Morton ( 19/+/+ ). who demonstrated t h a t r e t i n e n e was the aldehyde o f v i t a m i n A. Secondly, i m p o r t a n t s t u d i e s were made by Wald and o t h e r s (Wald, 1935a, 1935b, 1950; Wald and Hubbard, 1949; B l i s s , 1951; Futterman, 1963) on t h e enzyme systems i n v o l v e d i n the o x i d a t i o n s and r e d u c t i o n s between v i t a m i n A and r e t i n e n e . T h i r d l y , t h e d i s c o v e r y o f Hubbard and Wald (1952) o f t h e c i s - t r a n s isomerism o f r e t i n e n e d u r i n g t h e p r e p a r a t i o n o f r e t i n e n e f o r i t s combination w i t h o p s i n i n the f o r m a t i o n o f v i s u a l p u r p l e . Tne s t r u c t u r a l formalae f o r a l l - t r a n s and 1 1 - c i s v i t a m i n are shown below: CH H CH, H CH, i 3 i i 3 i i 3 x c < * . C H 2 0 H E_C C C C N C C * r e t i n o l ^i I i i i i P'C C-CH.. H H E H 2 C ' 2 N : ' * o I r e t i n a l H ( r e t i n a l d e h y d e ) A l l - t r a n s v i t a m i n PH3 CE, % C ^ i 2 N W ~ \S - 1 ~y C y 1 — i CL . "3 N X C " i H * c " I C H ^ ^ c ' H CH OH 29 All-trans r e t i n a l would not combine with opsin to form rhodopsin (Hubbard and Wald, 1952). Combination took place, however, after the retinene has been isomerized to the 11-cis state. Thus, a cycle of isomerization i s an i n t r i n s i c component of the visual purple or rhodopsin system. Cn the basis of the above additional knowledge, and subsequent research, Wald (1968) proposed his revised version of the visual cycle: Rhodopsin Tf /fl Pre-lumirhodopsin <r- -3 -P c 0) -H U Bl W O -H O + I retinene . , . , isomerase 11-cis r e t i n a l •<• j + opsin DPN + alcohol dehydrogenase 11-cis vitamin A ± esterifying enzymes 11-cis vitamin A esters Lumirhodopsin i Metarhodopsin I \ Metarhodopsin II 11 All-trans retinal + free opsin 1 All-trans vitamin A (retinol) All-trans vitamin A esters Wavy arrows indicate regeneration; straight arrows indicate thermal photoreactions. When a molecule of rhodopsin containing 11-cis r e t i n a l absorbs a single photon, i t i s changed to the more stable all-trans form and separated from opsin. Opsin i s a l i p o p r o t e i n w i t h a m o l e c u l a r w e i ght o f 30,000 t o 40,000 (K r i n s k y , 1958) . Tne f i r s t s t e p o f the l i g h t r e a c t i o n r e s u l t s i n the f o r m a t i o n o f p r e - l u m i r h o d o p s i n which i s h i g h l y u n s t a b l e a t o r d i n a r y temperature (Yoshizawa and Wald, 1964) . F o l l o w i n g t h i s , t h e breakdown proceeds spontaneously. Once a l l - t r a n s r e t i n a l i s r e l e a s e d from o p s i n , i t may be isomer!zed t o 1 1 - c i s r e t i n a l to form rhodopsin o r may be reduced by a dehydrogenase and DPN t o a l l - t r a n s r e t i n o l and then e s t e r f i e d . The e s t e r i s s t o r e d i n the pigment e p i t h e l i u m u n t i l needed f o r dark a d a p t a t i o n . D u r i n g dark a d a p t a t i o n , v i t a m i n A e s t e r i s d e e s t e r i f i e d , o x i d i z e d and i s o m e r i z e d t o the 1 1 - c i s c o n f i g a r a t i o n and once a g a i n a v a i l a b l e f o r r e g e n e r a t i o n o f rhodopsin. I t i s s t i l l n o t known e x a c t l y where i n the i n n e r o r o u t e r segment o f the photoreceptor c e l l 1 1 - c i s r e t i n a l and o p s i n recombine t o form rhodopsin. The pigment e p i t h e l i u m has been found t o s t o r e c o n s i d e r a b l e amounts o f 1 1 - c i s v i t a m i n A (Hubbard and Dowling, 1962; K r i n s k y , 1958). I n c a t t l e eyes, r e t i n e n e isomerase has been found p r i m a r i l y i n the n e u r a l r e t i n a and i n f r o g eyes i t i s found m a i n l y i n the pigment e p i t h e l i u m (Hubbard, 1956). V i t a m i n A e x i s t s i n tv,o d i s t i n c t forms (Wald, 1958b), A^ ( r e t i n a l ^ o r r e t i n o l ^ ) and A^ ( r e t i n a l 2 o r r e t i n o L ^ ) . I f t h e bond on t h e r i n g (at C) o f 1 1 - c i s v i t a m i n A molecule i s s a t u r a t e d , i t i s c a l l e d A^. I f t h i s bond i s u n s a t u r a t e d i t i s A^. V i t a m i n A^ i s the ciiromophore o f the rhodopsin found i n i n v e r t e b r a t e s and most v e r t e b r a t e s . T h i s v i s u a l pigment has a maximum a b s o r p t i o n a t 500 mu. I n f r e s h water and some amphibia, t h e chromcphore i s v i t a m i n A^ which combines w i t h o p s i n t o form p o r p h y r o p s i n having a maximum a b s o r p t i o n a t about 525 mu (Brown e t a l . , 1963) . Wald (1937) r e p o r t e d t h e d e t e c t i o n o f a f u r t h e r pigment, i o d o c s i n i n c h i c k e n r e t i n a . I t absorbes a t 565 mu and i s bleached by 31 r e d l i g h t . I t i s presumed t o be the pigment f o r the cones. V i s u a l pigments o f the human r e t i n a share the same b a s i c s t r u c t u r e o f a l l 2<nown v i s u a l pigments (Wald, 1969). Four types o f v i s u a l pigments have been found i n the human r e t i n a . The rod rhodopsin has a maximal absorbance a t about 500 mu (Brown and Wald, 1964). Cones have t h r e e d i f f e r e n t v i s u a l pigments w i t h d i f f e r e n t a b s o r p t i o n peaks (435 mu, 540 mu, and 565 mu) correspcnding t o b l u e , green and r e d s e n s i t i v e pigments i n the eye. I t i s b e l i e v e d t h a t o n l y one v i s u a l pigment i s present i n each photoreceptor c e l l . Therefore, according t o t h i s h ypothesis t h e r e are th r e e types o f cones, one absorbing maximally a t 435 mu and the ot h e r s a t 540 mu and 565 mu (Wald, 1969) r e s p e c t i v e l y . A l l f o u r possess 1 1 - c i s r e t i n a l d e h y d e as the chromophore but are u n i t e d w i t h f o u r d i f f e r e n t o p s i n s i n the d i s c s o f the r e c e p t o r outer segments (Wald, 1969). Human rhodopsin e x t r a c t e d i n t o aqueous s o l u t i o n bleaches i n the u s u a l way t o o p s i n and a l l - t r a n s r e t i n a l d e h y d e and can be r e - generated i n s o l u t i o n from o p s i n and 1 1 - c i s r e t i n a l d e h y d e (Wald and Brown, 1958). D i f f e r e n c e s p e c t r a on the red and g r e e n - s e n s i t i v e pigments o f cones have been measured by d i r e c t microspectrophotometry o f human and monkey foveas (Brown and Wald, 1963). Human cones which are b l u e - s e n s i t i v e have a l s o been found (Brown and Wald, 1964). A l l three pigments are regenerated on adding 1 1 - c i s r e t i n a l d e h y d e t o the medium, showing t h i s t o be t h e i r carmen chromophore, j o i n e d t o d i f f e r e n t opsins (Wald, 1969). 32 I I I w&TERIALS AND METHODS A l b i n o r a t s o f the w i s t a r s t r a i n (Woodland Farm, Ohio, U.S.A.) were used throughout the s t u d i e s . The animals were kept under l a b - o r a t o r y c o n d i t i o n s v/ith 12 continuous hours o f l i g h t and 12 hours o f darkness p er day. The i n t e n s i t y o f l i g h t i n s i d e - the cages w i t h the l i g h t s cn i n the roan was no more than 10 f o o t candles a t the f r o n t o f the cages and 1 f o o t candle a t the back. The animals were exposed t o an i n t e r v a l o f l e s s than 1 1/2 hours o f l i g h t o u t s i d e the cages a t an i n t e n s i t y o f 25 f o o t candles before they were k i l l e d . The temperature o f the roan i n which the animals were caged was h e l d con- s t a n t a t 20° C. The room i n which the animals were caged was i l l u m i n a t e d v/ith 4, 40 wa t t f l o u r e s c e n t l i g h t s (General E l e c t r i c F40, CW) s i t u a t e d 9 f e e t above the f l o o r . 1) Animals and d i e t s Weanling r a t s , 5 weeks o l d o f both sexes, weighing on the average 73 - 10 gm. v/ere housed i n i n d i v i d u a l w i r e cages. The r a t s were maintained on a v i t a m i n A " f r e e " d i e t (prepared by General B i o c h s n i c a l s , Chagrin F a l l s , Ohio, U.S.A., acco r d i n g t o Roels e t a l . , 1964) supplemented v/ith v i t a m i n A a c i d . Water was a v a i l a b l e ad l i b i t u m . L i t t e n r a t e s v/ere f e d w i t h a complete s t o c k d i e t , P u r i n a Laboratory Chow and water ad l i b i t u m . 33 Composition of the vitamin A free diet The major ingrdients of the basal diet were ( g A g ) : Casein, vitamin free (heat treated) 180.00 Glucose 677.56 Cellulose 50.00 Peanut O i l 50.00 Salt mixture, USP XIV (Biological :Research Pro- ducts, General Biochemicals cat. No. 170800) 40.00 The following vitamins were added to the basal diet (g/kg): Thiamine HC1 0.002 Riboflavin 0.004 Pyridoxine HC1 0.004 Choline Choride 1.000 Inositol 1.000 p-Aminobenzoic acid 0.300 Nicotinamide 0.100 Fo l i c acid 0.0025 Vitamin (crystalline) 0.00005 Biotin 0.0001 Ergocalciferol (40,000,000 units/g) 0.000042 Vitamin K 0.010 Vitamin A acid 0.00172 Calcium pantothenate 0.010 34 Composition of normal - diet (Purina Laboratory Chow)! PROTEIN % 23.4 Arginine % 1.38 Cystines % .32 Glycine % 1.26 Histidine % .62 Isoleucine % 1.22 Leucine % 1.52 Lysine % 1.41 Methionine % .43 Phenylalanine % 1.03 Threonine % .94 Trytophan % .28 Valine 1.24 FAT % 4.5 FIBER % 5.2 TON % 75 NFE (by difference) % 50.8 Gross Energy, KCal/gm 4.25 ASH % 7.3 Calcium % 1.20 Phosphorus % .86 Potassium % .82 Magnesium % .26 Sodium % .49 Chlorine % .51 Fluorine, ppm 35.0 35 I r o n , ppm 198.0 Z i n c , ppm 58.0 Manganese, ppm 51.0 Copper, ppm 18.0 Co b a l t , ppm .4 Io d i n e , ppm 1.7 VITAMINS Carotene, ppm 6.5 Thiamin, ppm 17.7 R i b o f l a v i n , ppm 8.5 N i a c i n , ppm 110.3 Pant o t h e n i c A c i d , ppm 24.8 C h o l i n e , ppm X100 24.0 F o l i c A c i d , ppm 5.9 P y r i d o x i n e , ppm 3.8 B i o t i n , ppm >07 B-12, mcg/lb. 10.2 V i t a m i n A, IU/gm 12.0 V i t a m i n D, IU/gm 5.3 Alp h a - t o c o p h e r o l , I U / l b . 29.8 Experimental and c o n t r o l animals were weighed i n d i v i d u a l l y f o r a t o t a l o f 25 weeks. F o r v i t a m i n A d e f i c i e n c y s t u d i e s , two 'experimental animals w i t h a c o n t r o l were k i l l e d f o r each sampling. 3b 2) Determination of Blood Plasma Vitamin A Level Experimental animals 5 weeks old ,were put on the vitamin A free diet and bleed samples were collected at 3, 4, 6 and 8 weeks later. The animals were anaesthesized with ether and blood collected by puncturing the descending aorta with a 18-gauge needle attached to a syringe containing 0.2 ml of heparin. Blood was collected similarly frcm control animals of 5 weeks old and also 2 and 8 weeks later. Five to six ml cf blood was collected frcm each animal and then centri- fuged to obtain the plasma which v/as frozen immediately and then thawed . before use. The plasma was shielded frcm ligh t by wrapping i t s container with aluminium f o i l . Bleed plasma vitamin A and carotene analyses were carried out using Carr-Price's colorimetric method (Neeld and Person, 1963; Freed, 1966), with a Beckrran DK-2A recording spectrophotometer, the c e l l chamber of which was maintained at 25°C. Carotene and vitamin A v/ere extracted from the blood plasma by the following procedure: 1. 2 ml cf plasma was transferred into a 10 ml glass test tube. 2. 2 ml of 95% ethanol was then added and followed by 3 ml of petroleum ether. 3. The mixture was shaken vigorously for two minutes and centri- fuged at low speed for three minutes to separate the emulsion. 4. 1 ml of petroleum ether extract (upper layer) was pippetted off and read for carotene at 450 mu against a petroleum ether blank. 5. 2 ml of petroleum ether extract was transferred to a 10 ml test tube which was then placed i n a water bath at 45°C and the extract evaporated to dryness under a stream of nitrogen. 37 6. The r e s i d u e was d i s s o l v e d i n 0.1 ml c h l o r o f o r m (CHCl^), and a drop o f a c e t i c anhydride from a No. 25 needle and 1 ml t r i f l o r o a c e t i c a c i d (TFA) mixture (1 TFA : 2 CHC1 3) were added. 7. V i t a m i n A was read immediately a t 620 mu a g a i n s t a blank o f 0.1 ml chl o r o f o r m and 1 ml o f TFA mi x t u r e . The above r e a c t i o n gave a c l e a r b l u e c o l o u r which faded r a p i d l y . The time when the TFA mixture was added (T Q) , the i n i t i a l (T^) and f i n a l (T2) r e c o r d i n g s o f the c o l o u r absorbed were noted. The maximum absorbance was ob t a i n e d by e x t r a p o l a t i n g the slop e o f absorbance t o the T Q time. An example graph showing how the maximum absorbance f o r v i t a m i n A was obtained i s shown i n f i g . 2a. The standard curve f o r carotene was obt a i n e d by d i s s o l v i n g 50 jag o f B-carotene (General B i o c h e m i c a l , U.S.A.) i n a few ml of reagent grade chloroform. Petroleum e t h e r was added t o a f i n a l volume o f 100 ml i n a v o l u m e t r i c f l a s k . T h i s s o l u t i o n was then d i l u t e d 1 t o 100 w i t h petroleum e t h e r t o prepare the inte r m e d i a t e standard. T h i s i n t e r m e d i a t e s o l u t i o n was f u r t h e r d i l u t e d w i t h petroleum e t h e r t o g i v e s o l u t i o n s c o n t a i n i n g 0.5, 1.0, 2.0 and 4.0 jag of B-carotene p e r ml r e s p e c t i v e l y . The o p t i c a l d e n s i t y (OD) o f carotene was read a t 450 mu a g a i n s t a petroleum blank. Thus a standard curve made up a t O D ^ Q a g a i n s t the c o n c e n t r a t i o n o f B-carotene was obtained. The B-carotene s o l u t i o n s were again read a t 620 mu a g a i n s t the petroleum b l a n k . The r a t i o o f the two s e t s o f readings a t the two d i f f e r e n t wavelengths was determined as OD45Q/ODg2Q=0.3. Since the petroleum e x t r a c t o f b l o o d plasma contained both carotene and v i t a m i n A, the r a t i o was used t o c o r r e c t the i n t e r - 38 ference caused by carotene at O D ^ Q i n order to estimate the concentration of vitamin A accurately. " The standard curve for vitamin A was obtained by using transretinol (Sigma Chemical Company). Five iriilligrams of transretinol was dissolved in a few ml of reagent grade chloroform and diluted to 5 0 ml i n a volumetric flask. Vitamin A standards were prepared from this stock containing 1 0 , 2 0 , 3 0 , 4 0 and 5 0 Aig/ml respectively. To prepare the standard curve, 0 . 1 ml of each of the above standards was placed i n a 1 ml capacity cuvette for reaction with 1 ml of TFA mixture. The procedures that followed were the same as described above for vitamin A determination i n blood plasma. From the standard curve of vitamin A at O D ^ , . , the factor F, which i s the correlation between concentration of vitamin A per tube and i t s optical density at 6 2 0 mu, was determined as: „ _ Aig vitamin A/ tube _ 3 _ „ , optical density at 6 2 0 mu 0 . 4 2 Therefore, vitamin A level i n 1 0 0 ml blood plasma: [ O D 6 2 Q - ( O D 4 5 0 X 0 . 3 ) ] ( 7 . 1 X 7 5 ) = ng vitamin A / 1 0 0 ml plasma 3 ) iDeteimdnation of feed vitamin A content. Three samples of Purina Laboratory Chow and 3 samples of the vitamin A free diet were analysed for vitamin A content. Twenty grams of each sample, finely ground, was extracted for 2 hours with petroleum ether i n an extraction apparatus. The ether was evaporated off and the residue saponified. After saponification was completed, 39 c a r o t e n e and v i t a r n i n A v/ere e x t r a c t e d by e t h e r . The e t h e r e x t r a c t was used to. r e a d carotene and v i t a m i n A a t 450 mu and 620 mu r e s p e c t i v e l y f o l l o w i n g t h e same method as d e s c r i b e d above. P u r i n a L a b o r a t o r y Chow was found t o c o n t a i n 3.97/ag o f v i t a m i n A/g o f f e e d and the v i t a m i n A " f r e e " d i e t , 0.21 pg o f v i t a m i n A/g o f f e e d , i . e . 5% o f the v i t a m i n A c o n t e n t o f the normal d i e t . 4) L i g h t microsoopy The animals were a n a e s t h e s i z e d w i t h sodium p e n t o b a r b i t a l (nembutal) i n j e c t e d i n t r a p e r i t o n e a l l y and the eyes removed w i t h a p a i r o f s c i s s o r s . The e n u c l e a t e d eyes were punctured a t the o r a s e r r a t a w i t h a sharp r a z o r blade t o f a c i l i t a t e p e n e t r a t i o n o f the f i x a t i v e , and then f i x e d i n a c o l d (4°C) s o l u t i o n o f 2% paraformaldehyde and 2.5% g l u t a r a l d e h y d e i n 0.1 M sodium c a o o d y l a t e b u f f e r a t pH 7.3 f o r 4-5 hours. The cornea and l e n s o f each eye were removed. The eyes were then r i n s e d i n 3% sucrose and 0.1 M sodium c a c o d y l a t e b u f f e r a t 4°C o v e r n i g h t , and p o s t - f i x e d i n 2% osmium t e t r o x i d e i n 0.1 M sodium c a c o d y l a t e b u f f e r f o r 2 hours at'room temperature. Dehydration was c a r r i e d o u t i n e t h a n o l , s t a r t i n g w i t h 15%, then,30%, 50%, 70%, 80%, 90% and 100% e t h a n o l , each f o r 15 minutes. A f t e r g o i n g through the l a s t change o f e t h a n o l , the eyes were t r a n s f e r r e d t o u n d i l u t e d propylene o x i d e , two changes, each f o r 30 minutes. Each eye was hemisected a n t e r o - p o s t e r i o r l y b e f o r e i t v/as t r a n s f e r r e d t o an e q u a l volume o f propylene o x i d e and epon 812 m i x t u r e . B e f o r e embedding, each h a l f o f the eye was f u r t h e r c u t m e r i d i o n a l l y i n t o 3 t o 4 s m a l l e r p i e c e s . The t i s s u e s were embedded i n epon 812 m i x t u r e and p o l y m e r i z e d a t 60°C o v e r n i g h t . The epon 4o embedded material was sectioned at 0.5-1.0 p. on a Reichert Cm U2 ultramicrotome. Sections were mounted on glass slides and stained with 0.25% alkaline toluidine blue at 65°C for 1-2 minutes. A l l conventional l i g h t micrographs were taken with a Leitz Orhthoplan li g h t microscope and recorded on Kodak Panatcmic X f i l m with an orange or green f i l t e r . The f i l m was developed for 9 minutes i n Kodak Microdol-X at room temperature and prints were made using Kodak Kodabromide photographic paper. 5) Electron microscopy For transmission electron microscopy, epon embedded material, prepared as above, was sectioned with a diamond knife on a Reichert Cm U2 ultramicrotome. Silver to gray sections were placed on 0.25% formvar coated 75/300 or 150/150 mash copper grids and then doubly stained with saturated uranyl acetate and lead citrate (Reynolds, 1963). They were examined by a Philips 300 electron microscope operated at 60 kv. A l l electron micrographs v/ere recorded on Kodak fine grain positive 35imi film. They were developed i n Kodak D-19 for 6 minutes at room temperature and printed on Kodak Kodakbromide photographic paper. 6) Acid phosphatase histochemistry Acid phosphatase i s generally accepted as a lysosomal enzyme (De Duve, 1963) and i s the most widely used histochemical marker for the demonstration of lysosomes. Portions of the posterior retina, hemisected antero-posteriorly 41 from both experimental and control animals were used for study of lysosomal a c t i v i t y i n the r e t i n a l epithelium. After f i x i n g i n cold (4°C) sodium cacodylate buffered solution of paraformaldehyde and glutaraldehyde as described i n section 4, the hemisected retinas were cut antero-posteriorly into small size, and washed in cold 0.1 M sodium cacodylate buffer containing 3% sucrose for 1 hour. During washing, the re t i n a l epithelium with choroid was detached from the retina proper and the sclera by peeling slowly with a pair of fine forceps (Ishikawa and Yamada, 1969). To demonstrate acid phosphatase ac t i v i t y , the method of M i l l a r and Palade (1964) was used. The r e t i n a l epithelium with choroid was incubated i n Gomori medium with sodium beta-glycerophosphate as substrate at 37°C for 30-40 minutes. The control for the acid phosphatase reaction was run by incubating some tissues i n Gomori medium without sodium beta-glycerophosphate. The tissues were rinsed b r i e f l y i n cold 0.05 M sodium acetate buffer (pH 5.0) containing 75% sucrose, postfixed i n 2% osmium tetroxide for 1 hour, then dehydrated rapidly with 70%, 90% and 100% ethanol, each change for 10 minutes. They were transferred to propylene oxide, two changes, each for 20 minutes and then to an equal volume of propylene oxide and epon 812 mixture and sectioned and photographed as described under section 5. A black precipitate of lead phosphate indicates the presence of acid phosphatase i n the tissue. Note: Gomori medium i s prepared by dissolving 0.12 g. lead nitrate [Pb(N0 3) 2] in 100 ml 0.05 M sodium acetate buffer (pH 5.0) containing 7.5% sucrose. Then 10 ml of a 3% solution of sodium beta-glycerophosphate i s slowly added. Before use, the mixture 42 i s wanned a t 60 WC f o r 1 hour, cooled t o room temperature and f i l t e r e d t o e l i m i n a t e the s l i g h t p r e c i p i t a t e which, u s u a l l y develops. 7) Radioautography-light microscopy Animals t h a t had been on the v i t a m i n A f r e e d i e t f o r 2.5, 8 and 10 months and a p a i r o f IQ month o l d c o n t r o l animals were l a b e l l e d 3 i n t r a v i t r e a l l y xn each eye w i t h 100 uC o f H -methionine Csp. a c t i v i t y : 4.02 C/mM, New England Nuclear, U.S.A.).. Before i n j e c t i o n , t h e a l c o h o l s o l v e n t o f the t r i t i a t e d methionine was evaporated o f f and r e p l a c e d w i t h Kreb's s o l u t i o n . The animals were l a b e l l e d f o r ' 4" and 24 hours. The enucleated eyes were processed as d e s c r i b e d under s e c t i o n 4. For l i g h t m i c r o s c o p i c radioautography, 0.5-1.0 u s e c t i o n s from the epon- embedded m a t e r i a l were c u t on a P e i c h e r t Cm U2 ultrainicrotome and p l a c e d on c l e a n g l a s s s l i d e s . The s l i d e s were dipped i n Kodak NTB2 l i q u i d emulsion kept a t 45°C i n a water bath. Dipping was done under a Wratten #1 s a f e l i g h t , kept a t l e a s t 6 f e e t away from the emulsion. The s l i d e s ware allowed t o dry i n t o t a l darkness and a t 75-80% r e l a t i v e humidity. The d r y s l i d e s were kept i n b l a c k p l a s t i c boxes w i t h s e v e r a l grams of " D r i e r i t e " and each box v/as wrapped i n aluminium f o i l . F o l l o w i n g an exposure o f 2 months, the s l i d e s were developed i n Kodak D-170 f o r 6 minutes and f i x e d i n 24% sodium t h i o s u l f a t e f o r 3 minutes a t 18°C (Kopriwa, p e r s o n a l ooirrmrdcation). The developed s l i d e s were, s t a i n e d w i t h a l k a l i n e t o l u i d i n e blue f o r 5 minutes and d e s t a i n e d i n a c i d a l c o h o l . L i g h t micrographs v/ere taken and developed as d e s c r i b e d i n s e c t i o n 4. 43 I V OBSERVATIONS I n t r o d u c t o r y Note In the p r e p a r a t i o n o f t h i s t h e s i s p a r t i c u l a r care has been taken t o p r o v i d e conprehensive f i g u r e legends t o f a c i l i t a t e review o f the micrographs. The f i g u r e legends, i n t h i s case, p r o v i d e a complete r e c o r d o f the r e s e a r c h which can be f o l l o w e d independently o f the s e c t i o n on o b s e r v a t i o n s i f d e s i r e d . T h i s approach has, o f n e c e s s i t y , produced a c e r t a i n redundancy between t he f i g u r e legends and the ob s e r v a t i o n s s e c t i o n . I t i s suggested t h a t the f i g u r e s and f i g u r e legends be perused p r i o r t o read i n g the observations s e c t i o n . 44 Notes on Obse r v a t i o n s : 1) An i r r a l s were put on the v i t a m i n A f r e e d i e t when they were 5 weeks o l d . Therefore an animal d e s i g n a t e d as a 5 week v i t a m i n A d e f i c i e n t animal i s a c t a u l l y 10 weeks o l d . 2) D e f i n i t i o n o f terms: a) " P o s t e r i o r r e t i n a " means the a r e a behind t h e equator c l o s e t o t h e o p t i c nerve o f the eye and " p e r i p h e r a l r e t i n a " means the a r e a i n t h e r e g i o n o f the equator and towards t h e o r a b) The terms " i n n e r " and " a p i c a l " used i n a s s o c i a t i o n w i t h t h e r e t i n a and r e t i n a l e p i t h e l i u m r e s p e c t i v e l y mean toward t h e v i t r e o u s o r c a n t e r o f the eye w h i l e " o u t e r " and " b a s a l " mean towards the s c l e r a . c) The term " d i s t a l " used i n a s s o c i a t i o n w i t h p h o t o r e c e p t o r o u t e r and i n n e r segments means f a r t h e s t s c l e r a l l y from the photoreceptor n u c l e u s , and " p r o x i m a l " means n e a r e s t t o the photoreceptor n u c l e u s . 45 A) Growth Curves Weanling r a t s 5 weeks o l d o f both sexes, weighing approximately 73 g. were put on the .vitamin A f r e e d i e t , supplemented w i t h v i t a m i n A a c i d . T h e i r growth was compared w i t h l i t t e r - m a t e s a l s o 5 weeks o l d , f e d P u r i n a Laboratory Chow and water ad l i b i t u m ( F i g . 1 ) . Each p o i n t on the curves r e p r e s e n t s the mean weight o f 10-19 c o n t r o l animals o r 14-45 v i t a m i n A d e f i c i e n t a n i mals, recorded a t v a r i o u s times d u r i n g the course o f the experiment. The v e r t i c a l b a r a t each p o i n t r e p r e s e n t s p l u s o r minus the standard d e v i a t i o n o f t h e mean. Both t he v i t a m i n A d e f i c i e n t and c o n t r o l animals grow a t approximately the same r a t e u n t i l t h e 4th week. A p e r c e p t i b l e d i f f e r e n c e i n t h e i r r a t e o f growth i s then apparent. The c o n t r o l animals c o n t i n u e t o g a i n weight r a p i d l y u n t i l t h e 15th week when they assume a slower r a t e o f growth. The v i t a m i n A d e f i c i e n t animals d i s p l a y a more g r a d u a l growth r a t e u n t i l the 21st week when t h e i r growth r a t e reaches a p l a t e a u . None o f the v i t a m i n A d e f i c i e n t o r c o n t r o l animals d i e d d u r i n g t h e course o f the s t u d i e s . The v i t a m i n A d e f i c i e n t animals appeared i n good and h e a l t h y c o n d i t i o n , except f o r a few which showed c l o u d i n e s s i n the cornea and exudate around t he e y e l i d s . B) Plasma V i t a m i n A L e v e l s i n C o n t r o l and V i t a m i n A D e f i c i e n t Animals Weanling r a t s about 5 weeks o l d were d i v i d e d i n t o two groups: one group was f e d P u r i n a Laboratory Chow and water ad l i b i t u m and served as c o n t r o l s ; t h e o t h e r group was put on the v i t a m i n A f r e e d i e t . The experimental animals were k i l l e d a t 3, 4, 6 and 8 weeks a f t e r they were 46 on t h a v i t a m i n A f r e e d i e t . The c o n t r o l animals were sampled a t 0 (5 weeks o l d ) 2, and 8 weeks. I t was assumed t h a t the. b l o o d v i t a m i n A l e v e l o f t h e c o n t r o l animals would remain s t a b l e and t h e r e f o r e they were not sampled c o n c u r r e n t l y w i t h t h e experimental animals. " The b l o o d - c o l l e c t e d was a n a l y s e d f o r i t s v i t a m i n A content and the r e s u l t s a r e shown i n F i g u r e 2. Each p o i n t i n t h e curves r e p r e s e n t s t h e mean v i t a m i n A content/lOCml o f b l o o d plasma o f 1-2 animals. The s i z e o f each samnle was n o t s u f f i c i e n t l y b i g t o account f o r the b i o l o g i c a l v a r i a t i o n , however, t h e de t e r m i n a t i c r . was merely done t o c o n f i r m what Dowling and .Wald (1958) had done e a r l i e r ."for t h e p r e s e n t system,;. The v i t a m i n A content per 100 ml o f b l o o d plasma from t h e 3 samples of the c o n t r o l animals was found t o be 58.5, 46.5 and 52.5 jog r e s p e c t i v e l y . The b l o o d v i t a m i n A content o f the v i t a m i n A d e f i c i e n t animals d e c l i n e d r a p i d l y a f t e r t h e animals were on t h e v i t a m i n A f r e e d i e t for 3 weeks. By t h e 4 t h week t h e i r v i t a m i n A l e v e l f e l l from 57.5 j j g t o 30.0 p g per 100 ml b l o o d plasma. By the 6 t h week t h e v i t a m i n A l e v e l f e l l f u r t h e r t o 9.5 jog/100 ml b l o o d plasma. By t h e 8 t h week when t h e experiment t e r m i n a t e d , t h e v i t a m i n A d e f i c i e n t animals had a mean v i t a m i n A c o n t e n t of 7.0 jug p e r 100 ml of b l o o d plasma, about 13% o f the v i t a m i n A l e v e l of t h e c o n t r o l animals. C) L i g h t Microscopy 1. R e t i n a l e p i t h e l i u m and photoreceptors i n c o n t r o l r a t s . The v e r t e b r a t e r e t i n a i s c o n v e n t i o n a l l y d i v i d e d i n t o 10 l a y e r s r e p r e s e n t i n g , i n t h e main, v a r i o u s p o r t i o n s o f f o u r d i f f e r e n t types o f c e l l s , r e t i n a l e p i t h e l i a l c e l l s , p h o t o r e c e p t o r s , b i p o l a r c e l l s and g a n g l i o n c e l l s . The l a y e r s are from o u t s i d e inwards ( F i g . 3 ) : 1, r e t i n a l 47 epithelium; 2, layer of reels and cones; 3, outer limiting membrane; 4, outer nuclear layer; 5, cuter plexiform layer; 6, inner nuclear layer; 7, inner plexiform layer; 8, ganglion c e l l layer; 9, nerve fiber layer; 10, inner limiting membrane. The r e t i n a l epithelium consists of a single layer of c e l l s . Each e p i t h e l i a l c e l l has 1-2 nuclei which are large and oval i n shape (Fig. 4). The layer of rods and cones contains the photoreceptor outer and inner segments. In a 2 month control animal i t measures about 9.6 „u thick (Fig. 3). The segments are closely packed together posteriorly (Fig. 4) but those from the peripheral retina l i e further apart (Fig. 7 ) . Both the photoreceptor outer and inner segments are elongated and c y l i n d r i c a l (Fig. 8). The outer limiting membrane forms a l i n e of demarcation between the outer nuclear layer and the photo- receptor inner segments (Figs. 4, 8). The outer nuclear layer, comprising the c e l l bodies of the photoreceptors, contains about 9-11 rows of nuclei in the posterior retina. Two types of nuclei are found i n the rat retina, rod and cone nuclei (Fig. 6) but rod nuclei predominate i n this nocturnal animal. The rod nucleus i s characterized by a large block of centrally located chromatin whereas i n the cone nucleus the chromatin i s more diffuse and usually divided into several lobes. No appreciable difference i n the photoreceptor nuclei i s observed between 2 month old and 12 month old animals. In Figure 5, a photoreceptor nucleus i s seen lying among the photoreceptor inner segments. At higher magnification, three displaced photoreceptor nuclei are identified between the retinal epithelium and the photoreceptor inner segments (Fig. 6). Nucleus No. 1 i s at the junction of the photoreceptor inner and outer segments, nucleus No. 2 at the junction of the photoreceptor outer segments and r e t i n a l 48 epithelium and nucleus No. 3 l i e s partly witiiin the r e t i n a l epithelium.. The photoreceptor c e l l s diiainish i n number perceptibly towards the periphery. Figure 5 shows the peripheral retina from a 9 month old control animal. In the retinal periphery there i s a reduction i n the thickness of the outer nuclear layer to 5-6 rows of nuclei. The outer plexiform layer i s the synaptic zone between the receptors and bipolar and horizontal neurons. I t has a reticular structure under the l i g h t microscope (Figs. 3, 5). The inner nuclear layer consists of the c e l l bodies of four types of c e l l s : bipolar c e l l s , horizontal c e l l s , Muller c e l l s and amacrine c e l l s . The inner plexiform layer marks the junction between the c e l l s of the inner nuclear layer and the ganglion c e l l s of the retina. It also has a finely reticular appearance and contains large blood vessels and capillaries (Figs. 3, 5). The c e l l bodies of the ganglion c e l l s are found i n the ganglion c e l l layer. They are round and closely spaced i n a single row (Fig. 3). The nerve fiber layer i s composed of the axons of the ganglion c e l l s . Processes of the Muller c e l l s are known to form the inner liirdting membrane. The retina has a blood supply from two sources (Fig. 5). The outer retina, the r e t i n a l epithelium and the photoreceptors are nourished mainly by choriocapillaries from the choroidal circulation. The inner retina i n most mammals i s nourished mainly by capillaries leading from branches of the central retinal artery (Polyak, 1957). 2. Retinal epithelium and photoreceptors i n vitamin A deficiency The following observations refer to the posterior (this term has been defined i n the notes on observations) retina unless otherwise indicated. Animals v/ere placed on the vitomin A free diet.when they 49 were about 5 weeks o l d . After 3.5 months on the vitamin A free diet, the ptotoreceptor outer and inner segments frcm the posterior retina appear fragile and broken (Fig. 9 ) . At higher magnification, many of the photoreceptor outer segments show evidence of disintegration. The photoreceptor inner segments are shorter than normal and some appear slig h t l y swollen (Fig. 1 0 ) . Above the outer nuclear layer the outer limiting membrane i s v i s i b l e . The outer nuclear layer contains 9-11 rows of nuclei. The outer plexiform layer i s l i g h t l y stained while the inner neural retina appears normal (Fig. 9 ) . The layer of photoreceptor outer and inner segments decreases i n thickness after 6 months of vitamin A deficiency (Fig. 1 1 ) . The layer measures 4.4 ja in thickness compared to 8 ju i n a control animal. At higher magnification (Fig. 1 2 ) , severe breakdown of photoreceptor outer segments i s evident. They lose their highly ordered orientation, appear irregular and display many empty spaces. The photoreceptor inner segments are shorter than their normal counterparts. The outer limiting membrane i s present. The outer nuclear layer now has only 3-5 rows of photoreceptor nuclei (Fig. 1 1 ) . The l i g h t l y stained outer plexiform layer appears thinner. The inner neural retina i s unchanged. In the reti n a l epithelium many small dark granules are present along i t s inner surface (Fig. 1 2 ) . An overall reduction i n the retinal thickness i s noted i n the posterior retina after 9 months of vitamin A deficiency (Fig. 1 3 ) . The photoreceptor outer segments have almost disappeared. The inner segments are round and short (Fig. 1 4 ) . The layer of inner and outer segments now measures 3.2 ; j i n thickness. The outer limiting membrane i s clearly v i s i b l e . There are now 2-3 rows of photoreceptor nuclei. 30 remaining. I n F i g u r e 13, t h r e e photoreceptor n u c l e i are seen o u t s i d e the o u t e r n u c l e a r l a y e r and i n Fi g u r e 14 one nucleus l i e s o u t s i d e t h e ou t e r l i m i t i n g membrane. The outer p l e x i f o r m l a y e r i s t h i n and no lo n g e r forms a d i s t i n c t zone between the o u t e r and i n n e r n u c l e a r l a y e r s . Numerous s m a l l dark granules are seen w i t h i n the i n n e r s u r f a c e o f the r e t i n a l e p i t h e l i u m ( F i g . 14). I n a 10 month v i t a m i n A d e f i c i e n t animal, t h e p o s t e r i o r r e t i n a i shows f u r t h e r degeneration. There i s g r e a t e r approximation o f the r e t i n a l e p i t h e l i u n and the n e u r a l r e t i n a l l a y e r ( F i g s . 15, 16). The photoreceptor o u t e r and i n n e r segments have disappeared. The' o u t e r l i m i t i n g membrane i s v i s i b l e and now demarcates the o u t e r border of the n e u r a l r e t i n a . Cnly 1-2 rows o f photoreceptor n u c l e i are ob- served. U n i d e n t i f i e d c e l l s are v i s i b l e among the photoreceptor n u c l e i . The o u t e r p l e x i f o r m l a y e r i s no longer v i s i b l e i n some re g i o n s but the i n n e r n e u r a l r e t i n a i s e s s e n t i a l l y unchanged ( F i g . 15). The p o s t e r i o r r e t i n a from an 11 month v i t a m i n A d e f i c i e n t animal i s f u r t h e r reduced i n t h i c k n e s s ( F i g . 17). Some photoreceptor n u c l e i are present between t he r e t i n a l e p i t h e l i u m and the n e u r a l r e t i n a ( F i g . 17, 18). The r e t i n a l e p i t h e l i u m l i e s immediately n e x t t o t h e n e u r a l r e t i n a . The o u t e r n u c l e a r l a y e r c o n t a i n s o n l y one i r r e g u l a r row o f photoreceptors w i t h l i g h t l y s t a i n e d cytoplasm. C e l l s from the i n n e r n u c l e a r l a y e r appear between photoreceptor n u c l e i ( F i g . 17). The outer l i m i t i n g membrane forms a d i s t i n c t boundary above t he o u t e r n u c l e a r l a y e r . Tne c u t e r p l e x i f o r m l a y e r i s obscure although the i n n e r n e u r a l r e t i n a appears almost normal ( F i g . 17). 51 D) Electron Microscopy 1. Normal retinal morphology (a) The retinal epithelium The retinal epithelium consists of a single layer of c e l l s which appear rectangular i n cross-section. The fine structure of two adjacent e p i t h e l i a l c e l l s i s shown i n Figure 19 and Figure 22 which are from the posterior retinas of 1.5 month and 9 month old control animals respective- l y . The basal surface of each c e l l i s infolded. A basement membrane i s present beneath the e p i t h e l i a l surface which forms part of Bruch's mem- brane. Bruch's membrane also has a core of collagen and dense f i b r i l l a r material and an outer layer formed by the basement membrane of the chorio- ca p i l l a r i e s . The inner e p i t h e l i a l surface displays both long and slender, and short and broad processes. The former often form a palisade around the photoreceptor outer segments. Both the basal infoldings and the long apical processes are usually devoid of identifiable subcellular structures. However, in a 1.5 month old control animal (Fig. 19), premelanosomes are present i n the broader inner processes. They are absent i n older animals (Figs. 20, 21, 22) . Near the inner or apical surfaces, e p i t h e l i a l c e l l s are joined by junctional complexes consisting in each case, of a zonula occludens and zonula adherens (Figs. 19, 22). The zonula adherens l i e s just sclerad to the zonula occludens. Both of these portions of the junctional complex have the typical structure described originally by Farquhar and Palade (1963). The ep i t h e l i a l nucleus i s large.and oval, containing diffuse chromatin. Nuclear pores are often observed (Fig. 19). The e p i t h e l i a l cytoplasm i s characterized by a predominance of sirooth endoplasmic reticulum (Figs. 19, 52 20, 21, 22). Rough endoplasmic reticulum i s also present scattered in the cytoplasm, but more often, aggregated close to the e p i t h e l i a l inner surface (Figs. 19, 21). Polysomes are dispersed sparingly i n the cytoplasm. A single row of mitochondria i s present inside the basal infoldings and along the l a t e r a l c e l l border. Some mitochondria are also found close to the e p i t h e l i a l inner surface. The mitochondria are usually rod-shaped i n longitudinal section and round i n cross- section. One or more well-developed Golgi complexes are found near the nucleus i n each epi t h e l i a l c e l l (Figs. 19, 21). Microtubules can sometimes be observed i n the cytoplasm (Fig. 19). Several types of dense bodies are also v i s i b l e i n the cytoplasm. The type most commonly observed has a dense homogeneous matrix surrounded by a disti n c t clear space and then an outer single membrane. These dense bodies vary from round to oval i n shape and vary i n size from 0.2 û to 0.5 AI i n diameter (Figs. 19, 22). They have been found to be acid phosphatase positive (Yamada, 1969} and hence are thought to be lysosomes. Lamellar-like structures enclosed within a membrane closely resembling the lamellar discs of the photoreceptor outer segments are frequently observed i n the cytoplasm as well (Fig. 20). They are discarded portions of the photoreceptor outer segments which have been phagocytosed by the r e t i n a l epithelium. These cytoplasmic inclusion bodies have been termed "phagosomes" (Young, 1967). Dense bodies that contain undigested lipofusin-like materials (Fig. 20) are found i n addition and have been designated "residual bodies" (De Duve et a l . , 1966). Lastly, l i p i d droplets are present occasionally i n the cytoplasm and can be easily recognized by their homogenous but less dense matrix. They are larger than the lysosomes and the c e l l membranes surrounding the l i p i d droplets are indistinct (Fig. 34). Pinocytosis i s often observed along the 53 plasma membrane o f the b a s a l e p i t h e l i a l s u r f a c e and coated v e s i c l e s are seen s c a t t e r e d i n the e p i t h e l i a l cytoplasm ( F i g . 22). I t i s important t o note t h a t the s t r u c t u r e o f the pigment e p i t h e l i a l c e l l d e s c r i b e d above does not change v/ith aging from 1.5 t o 12 months o f age (Fig s . 19, 20, 21, 22). (b) The photoreceptor o u t e r segments Rats are n o c t u r n a l -animals and t h e i r photoreceptors are predominant- l y rods. I n the f o l l o w i n g d e s c r i p t i o n , u n l e s s otherwise s t a t e d , t h e term photoreceptor r e f e r s t o rods. The photoreceptor o u t e r segments are c y l i n d r i c a l i n shape and each i s composed o f a st a c k o f f l a t t e n e d s a c c u l e s o r d i s c s surrounded by a c e l l membrane continuous w i t h the plasma membrane o f the i n n e r segment ( F i g . 24) . The d i s c s are r e g u l a r l y arranged and stacked a t r i g h t angles t o the l e n g t h o f the o u t e r segment. Each d i s c i s composed o f two membranes t h a t are continuous a t the edges. The membranes enc l o s e a l e s s dense narrow space, the i n t r a d i s c space. The d i s t a l p o r t i o n o f each o u t e r segment i s surrounded o r e n c i r c l e d by a p i c a l processes o f the r e t i n a l e p i t h e l i u m ( F i g . 21) and the pr o x i m a l o u t e r segment i s continuous w i t h the i n n e r segment through a connecting c i l i u m ( F i g . 24). Each l a m e l l a r d i s c i n v a g i n a t e s a t the same p o i n t forming a l o n g i t u d i n a l groove o r i n c i s u r e which extends the whole l e n g t h o f the o u t e r segment. P o r t i o n s o f the l o n g i t u d i n a l groove o f the o u t e r segment are seen i n Fig u r e 24. F r e q u e n t l y , i r r e g u l a r l y arranged s a c c u l e s are seen a t the bases o f the outer segments ( F i g . 25). Fur t h e r up the o u t e r segments the i r r e g u l a r s a c c u l e s g i v e way t o the h i g h l y ordered l a m e l l a r d i s c s . Other s u b c e l l u l a r s t r u c t u r e s are absent i n the o u t e r segments. 54 The connecting c i l i u n o r i g i n a t e s i n the b a s a l body which i s l o c a t e d i n the cytoplasm o f the d i s t a l i n n e r segment s l i g h t l y t o the s i d e o f i t s c e n t r a l a x i s , f a c i n g the ou t e r segment. I t ' s f i n e s t r u c t u r e was f i r s t d e s c r i b e d by de R o b e r t i s (1956). I t c o n t a i n s n i n e doublets o f p e r i p h e r a l t u b u l e s . Tne c e n t r a l p a i r o f tubules which a r e character-^ i s t i c o f m o t i l e c i l i a (Fawcett, 1958) are absent here ( F i g s . 23, 24, 26). (c) The plootoreceptor i n n e r segments The i n n e r segments are elongated, c y l i n d r i c a l s t r u c t u r e s c o n t a i n i n g f i n e l y g r a n u l a r cytoplasm. Long, s l e n d e r mitochondria w i t h w e l l developed t r a n s v e r s e c r i s t a e are arranged around the p e r i p h e r y o f each i n n e r segment. In both the 1.5 and 9 month o l d c o n t r o l animals, the m i t o c h o n d r i a are observed t o extend down t o the proximal ends o f the i n n e r segments (F i g s . 23, 26, 27). O c c a s i o n a l l y , m i t o c h o n d r i a c o n t a i n i n g glycogen granules are observed i n i n n e r segments from o l d e r but not younger c o n t r o l r a t s ( F i g s . 24, 27). I t i s important t o note t h a t t h i s i s t he o n l y s i g n i f i c a n t change t h a t separates the morphology o f photoreceptors from ages 1.5 t o 12 months i n the c o n t r o l r a t s s t u d i e d . From the b a s a l body o f the c i l i u m , a s t r i a t e d r o o t l e t extends some d i s t a n c e down the i n n e r segment ( F i g s . 23, 26) . Polysomes are abundant and they are evenly d i s t r i b u t e d i n the cytoplasm. A G o l g i apparatus surrounded by numerous s m a l l v e s i c l e s i s present i n the b a s a l r e g i o n o f each i n n e r segment (Fig s . 26, 27). Rough endoplasmic r e t i c u l u m i s abundant and a few smooth c i s t e r n a e can be observed (Figs. 26, 27). The i n n e r segments are not i n d i r e c t c o n t a c t w i t h adjacent photoreceptors but are separated from each, o t h e r by t h i n v i l l o u s extensions o f M i i l l e r c e l l s ( F i g . 26) . 55 (d) The outer limiting msrbrane By l i g h t microscopy, the outer limiting membrane appears to be a thin l i n e separating the cuter nuclear layer from the photoreceptor inner segments. Electron microscopy shows that the outer limiting membrane i s formed by a single row of c e l l junctions between the photoreceptor inner segments and Muller c e l l s , and also between adjacent Muller c e l l s (Figs. 26, 27). These c e l l junctions have often been named "terminal bars" or "desmosomes". Cohen (1965), i n a study of primate eyes, correctly interpreted the c e l l junctions that form the "membrane" as "Zonulas adhsrentes". His work has been confirmed by Spitznas (1970) i n a study of the human eye. (e) The photoreceptor synaptic processes The synaptic processes of .the photoreceptor extend a short distance inward and make synaptic contact with neuronal processes derived from c e l l s i n the inner nuclear layer (Figs. 28, 29). Two types of synaptic terminals are observed. The rod synaptic process ends i n anoval structure known as a "spherule" (Fig. 28). The cone synaptic process ends i n a broad swelling called-a "pedicle" (Fig. 30). The basal surface of each rod spherule i s invaginated by two neuronal processes forming a dyad (Fig. 29). Sometimes, i n addition to the two latera l processes, a third central process i s present forming a tri a d . In the study of monkey and human retinas, Dowling and Boycott (1966) have indicated that the central process of the t r i a d i s derived exclusively from a bipolar c e l l and the lateral processes from horizontal c e l l s . If the rat outer plexiform layer, i s similar i n structure to that i n monkey 5b and human (Dowling and Boycott, 1966), then the two l a t e r a l processes i n rat would be horizontal c e l l processes and the central one bipolar c e l l process',^ The c e l l wall of the spherule encloses the terminations of the neuronal processes. The rod spherule and i t s enclosed neuronal processes comprises the^synaptic unit or synaptic complex. I t consists of three parts) presynaptic (the spherule), synaptic (spherule-bipolar and spherule-horizontal c e l l contacts), and post synaptic (bipolar dendrite, horizontal c e l l processes) (Hogan et a l . , 1971). The presynaptic membrane i s separated from the post synaptic membrane by a synaptic c l e f t . The spherule contains a large number of presynaptic vesicles which are evenly distributed and one or two mitochondria and some polysomes (Figs. 28, 29, 31). Within the spherule, an osmiophilic lamellar structure, the synaptic ribbon i s found at right angle to the dyad or the triad. I t i s a crescent shaped structure (Ladman, 1958) containing five layers, three dense layers separated from each other by two less electron-dense layers (Fig. 28). Each synaptic ribbon i s surrounded by a halo of synaptic vesicles. Between the synaptic ribbon and the presynaptic membrane l i e s a dense structure, the arciform density (Ladman, 1958). At the base of the spherule, superficial contact with other neuronal processes originating frcm c e l l s i n the inner nuclear layer i s marked by a slight indentation and increased density of the presynaptic membrane (Fig. 28). In this area, no synaptic ribbon i s present. The most common type of synapse observed i s between the spherules, the horizontal c e l l processes and the bipolar c e l l dendrites (Figs. 28, 29, 31). An additional type of rod synapse where the post synaptic processes make direct contact with the body of the rod c e l l and the synaptic ribbon l i e s near the receptor nucleus (Fig. 29) i s also present. This synapse has been termed "somato dendritic synapse" by de Robertis and Franchi (1956). The cone pedicle, i n contrast t o the rod spherule, has a complex structure. Each cone pedicle makes contact with a number of processes o r i g i n a t i n g i n the inner nuclear layer. These processes possibly from horizontal and bipolar c e l l s invaginate the cone pedicles to es t a b l i s h contact and synaptic ribbons are present i n the cone cytoplasm at these s i t e s (Figs. 30, 32). Hence cone pedicles t y p i c a l l y contain a large number of synaptic ribbons. Other processes from the inner nuclear layer make contact with the surface of the pedicle forrning what have been termed s u p e r f i c i a l contacts (Fig. 30). The cone pedicle t y p i c a l l y contains large numbers of synaptic v e s i c l e s which are usually evenly d i s t r i b u t e d . Seme synaptic v e s i c l e s , however, are always present congregated at the s i t e of the synaptic ribbons. 2. R e t i n a l morphology i n v i t a m i n A d e f i c i e n c y (a). R e t i n a l changes a f t e r 1 month o f v i t a n i n A d e f i c i e n c y Change i n r e t i n a l s t r u c t u r e i s f i r s t observed i n the p o r t i o n o f p h o t o r e c e p t o r o u t e r segments n e a r e s t t o t h e r e t i n a l e p i t h e l i u m . Some l a m e l l a r d i s c s become s w o l l e n and break down i n t o v e s i c l e s ( F i g u r e 33). A d i s t o r t e d o u t e r segment i n which the l a m e l l a r d i s c s have opened up t o form an almost o v a l l a m e l l a r s t r u c t u r e surrounded by the a p i c a l p r o cesses o f the r e t i n a l e p i t h e l i u m i s seen i n F i g u r e 33. Some v e s i c l e s a r e v i s i b l e w i t h i n the d i s t o r t e d l a m e l l a r s t r u c t u r e . The r e s t o f t h e p h o t o r e c e p t o r c e l l s , t h e i n n e r r e t i n a and t h e r e t i n a l e p i t h e l i u m a r e normal. (b) R e t i n a l changes a f t e r 1.5 months o f v i t a m i n A d e f i c i e n c y S t r u c t u r a l breakdown of t h e p h o t o r e c e p t o r o u t e r segments i s g r a d u a l and occurs s p o r a d i c a l l y among the o u t e r segments. Some o u t e r segments show more severe breakdown w h i l e o t h e r s a r e l e s s a f f e c t e d ( F i g . 35). A l a r g e number o f v e s i c l e s and some abnormally arranged s a c c u l e s can be observed i n the o u t e r segments undergoing t h e s e v e r e s t d e g e n e r a t i o n . I n t h e r e t i n a l e p i t h e l i u m , many lysosorres a r e now p r e s e n t i n the cytoplasm c l o s e t o i t s i n n e r s u r f a c e and a l s o i n the broad a p i c a l o r i n n e r processes ( F i g . 34). D i s i n t e g r a t i n g phagosomes, t o o , are seen near t h e e p i t h e l i a l i n n e r s u r f a c e ( F i g . 34). The l o n g and narrow a p i c a l p rocesses which surround the degenerating o u t e r segments do n o t c o n t a i n o r g a n e l l e s . 59 (c) R e t i n a l changes a f t e r 2 months o f v i t a m i n A deficiency- tore severe d i s i n t e g r a t i o n o f the photoreceptor o u t e r segments can now be observed. Most o f the l a m e l l a r d i s c s i n the o u t e r segments are unable t o m a i n t a i n t h e i r s t r u c t u r a l i n t e g r i t y and break down i n t o t u b u l e s o r v e s i c l e s ( F i g . 36). Near the base o f the outer segments, some l a m e l l a r d i s c s appear i n t a c t . The connecting c i l i a , photo- r e c e p t o r i n n e r segments and the i n n e r n e u r a l r e t i n a remain normal. (d) R e t i n a l changes a f t e r 2.5 months o f v i t a m i n A d e f i c i e n c y The breakdown o f the photoreceptor o u t e r segments continues. The o u t e r segments c o n t a i n many v e s i c l e s , t u b u l e s and d i s o r d e r e d s a c c u l e s . Many l a m e l l a r d i s c s have disappeared completely l e a v i n g i n t r a c e l l u l a r spaces ( F i g . 38). The photoreceptor i n n e r segments begin t o show some morphological changes a t t h i s stage o f v i t a m i n A d e f i c i e n c y . The d i s t a l p o r t i o n s o f the i n n e r segments f o r the f i r s t time are s l i g h t l y s w o l l e n and t h e i r cytoplasm i s almost devoid o f polysomes. Large vacuoles can be observed i n a f f e c t e d r e g i o n s o f the i n n e r segments ( F i g . 39). At t h i s stage, the a p i c a l o r i n n e r processes o f the r e t i n a l e p i t h e l i u m are i r r e g u l a r l y o r i e n t e d and appear a c t i v e l y engaged i n e n g u l f i n g degenerating o u t e r segments ( F i g . 37). The smooth endoplasmic r e t i c u l u m , rriitochondria and the G o l g i complexes i n the r e t i n a l e p i t h e l i u m remain unchanged. 6o (e) R e t i n a l changes a f t e r 4-5 months o f v i t a m i n A d e f i c i e n c y - The photoreceptor o u t e r segments show f u r t h e r d e t e r i o r a t i o n . V e s i c l e s , t u b u l e s and d i s o r d e r e d s a c c u l e s r e s u l t from breakdown o f the l a m e l l a r d i s c s . Due t o l o s s o f o u t e r segment m a t e r i a l many i n t r a - and e x t r a c e l l u l a r spaces are formed ( F i g s . 40, 41) . The l a m e l l a r d i s c s t h a t a r e s t i l l p r e s e n t w i t h i n the o u t e r segments are f r e q u e n t l y l o o s e l y arranged o r o r i e n t e d d i f f e r e n t l y from t h e i r normal p o s i t i o n p e r p e n d i c u l a r t o t h e l o n g axes of o u t e r segments ( F i g . 40). The photoreceptor i n n e r segments l o s e t h e i r e l o n gated and c y l i n d r i c a l s t r u c t u r e . They appear s h o r t e r than normal and are b a r r e l - s h a p e d . I n p a r t i c u l a r , t he i n n e r segments are r e t r a c t e d towards the photoreceptor ' n u c l e i ( F i g . 41). The s w o l l e n ends o f the i n n e r segments, observed p r e v i o u s l y , have disappeared. The mitochondria i n each i n n e r segment appear l a r g e l y u n a f f e c t e d . Polysomes gather mainly i n the i n n e r h a l v e s o f t h e i n n e r segments. I n t h i s r e g i o n , rough endoplasmic r e t i c u l u m and a few l a r g e vacuoles can a l s o be i d e n t i f i e d . I n t a c t c o n n e c t i n g c i l i a and s t r i a t e d r o o t l e t s are p r e s e n t ( F i g s . 41, 42). Degeneration o f e n t i r e p hotoreceptor i n n e r segments can a l s o be observed ( F i g . 41). I n F i g u r e 42, a degenerating i n n e r segment w i t h v e r y dense cytoplasm can be seen.. I t c o n t a i n s several' mdtcchondria, two o f which are g r e a t l y e n l a r g e d and c o n t a i n glycogen granules. . The o u t e r l i m i t i n g membrane i s s t i l l i n t a c t at t h i s stage ( F i g . 41) . V i l l o u s e x t e n s i o n s o f the M u l l e r c e l l s p r o j e c t through the i n t e r c e l l u l a r spaces s e p a r a t i n g adjacent photoreceptor i n n e r segments ( F i g . 42). The a p i c a l processes o f the r e t i n a l e p i t h e l i u m show s l i g h t p r o l i f e r a - t i o n and t h e r e i s an i n c r e a s e i n number o f lysosomes near t h e e p i t h e l i a l oX inner surface [Fig. 43). The basal infoldings and other subcellular structures i n the r e t i n a l epithelium appear unchanged. (f) Retinal changes after 6 months of vitamin A deficiency The deterioration of the photoreceptors has progressed and the inner segments now l i e close to the r e t i n a l epithelium (Fig. 44). Few of the outer segments appear intact. Some outer segments and their contained discs have been l o s t (Fig. 44) and others have broken down into vesicles (Fig. 45). S t i l l other discs have lost their compact and transverse arrangement within the c e l l membranes (Fig. 46). Many i n t r a - and extracellular spaces are observed. The photoreceptor inner segments are marked by different stages of shortening (Fig. 47). Those that are not shortened too extensively show a perceptible difference i n the polysome distribution between the d i s t a l and basal (inner) halves of the cytoplasm. In the d i s t a l halves of these inner segments, the cytoplasm i s less granular as most of the polysomes aggregate close to the photoreceptor nuclei (Figs. 44, 47). The cisternae of the smooth endoplasmic reticulum that remains are dilated and rough endoplasmic reticulum i s absent (Fig. 47). An inner segment, almost oval i n shape, i s seen i n Figure 47. It contains a large number of polysomes, an i l l - d e f i n e d Golgi apparatus, several short mitochondria and a small amount of rough endoplasmic reticulum. The outer limiting membrane i s present, composed of c e l l junctions between photoreceptor inner segments and Muller c e l l s and between adjacent Muller c e l l s (Fig. 47). Between adjacent photoreceptor inner segments, the intercellular spaces are wider and more Muller c e l l processes extend through them (Fig.. 47). The photoreceptor synaptic processes show vi s i b l e morphological changes for the f i r s t time at this stage of vitaim A deficiency. In each synaptic process there are fewer synaptic vesicles, many lying immediately adjacent to synaptic sites (Fig. 48). Plasma membranes between adjacent synaptic processes have broken down and cytoplasm merges freely across the sites of breakage (Fig. 48). The c e l l membranes of mitochondria within the synaptic processes are also affected. Ih Figure 48, a mitochondrion which displays swelling of part of i t s outer membrane i s seen. More seriously affected mito- chondria are observed i n Figure 49. The mitochondrial membranes appear to lose t h e i r structural integrity and most of the transverse cristae disappear. At the synaptic sites, the synaptic ribbons surrounded by a cluster of vesicles persist. The postsynaptic processes containing closely packed synaptic vesicles are unchanged. An accumulation of lysosomes along the inner surface of the r e t i n a l epithelium i s again observed. The apical processes of the epithelium are numerous but are shorter than normal and appear to be active i n engulfing degenerating outer segments (Figs. 45, 46). A phagosome- li k e body i s seen i n the r e t i n a l epithelium i n Figure 45. (g) Retinal changes after 7-8 months of vitamin A deficiency Approximation of the photoreceptor inner segments and the r e t i n a l epithelium i s again evident (Fig. 51). As before, degeneration of the photoreceptor outer segments involves breakdown of discs (Figs. 50, 51) and abnormal arrangement of the remaining discs (Figs. 51, 52). Intra- and extracellular spaces are abundant. Some outer segments do not seem to be as badly damaged as those observed in animals at 6 months of vitamin A 63 deficiency (Fig. 44). Morphological changes observed i n the photoreceptor inner segments resemble those described at the 6th month stage of vitamin A deficiency. The inner segments have shortened to different degrees. The d i s t a l halves of the inner segments are less granular and contain dilated p r o f i l e s of smooth endoplasmic reticulum (Figs, 51, 53). Polysomes gather mainly i n the basal halves of the inner segments. In Figure 52, a structure resembling a discarded inner segment l i e s close to the r e t i n a l epithelium. I t contains lysosomes and aggregated dense material. The photoreceptor synaptic processes are now shorter and display fewer synapses (Figs. 54, 55). Each photoreceptor synaptic process contains only a few synaptic vesicles. The breakdown of c e l l membranes between adjacent synaptic processes and of mitochondria within the synaptic processes i s again evident. Occasionally, unidentified c e l l s containing large numbers of lysosomes and undigested debris can be identified (Fig. 55). The r e t i n a l epithelium i s marked by a very large increase i n lysosomes aggregated close to the inner e p i t h e l i a l surface and i n the broad apical processes. Other lysosomes l i e near the Golgi apparatus (Fig. 50). There appears to be a proliferation of apical e p i t h e l i a l processes (Figs 50, 51) . In the 8 month vitamin A deficient animal, the apical processes of the retinal epithelium are short and irregularly oriented (Fig. 51). Ch). Retinal changes after 9 months of vitamin A deficiency At this stage, striking morphological changes are evident. The photoreceptor outer segments have disappeared except for a few sporadic clusters of disordered saccules (Figs. 56, 58). The photoreceptor inner segments are shortened considerably (Figs. 56, 58, 59, 60). The inner segments contain, vacuoles of various sizes, several unusually short mitochondria and some polysomes (Figs. 56, 58, 59). In the longer inner segments, polysomes and some rough endoplasmic reticulum can be found in basal regions close to the c e l l nucleus. Within the inner segments, some mitochondria show evidence of membrane degeneration (Figs. 56, 59). The extensively retracted inner segments s t i l l contain polysomes and rough endoplasmic reticulum (Fig. 60) but mitochondria, normally a characteristic feature, are no longer present. C i l i a i n cross-section can be identified lying between adjacent inner segments (Fig. 59). The outer limiting membrane i s now formed more by c e l l junctions between Muller c e l l s than between photoreceptors and Muller c e l l s (Figs. 56, 58). The approximation of neural retina and the retinal epithelium continues. Many vi l l o u s processes of the Muller c e l l s extend through the interce l l u l a r spaces between adjacent inner segments (Figs. 58, 59). The photoreceptor nuclear chromatin appears unchanged, but for the f i r s t time the nuclear membranes show evidence of degeneration (Fig. 60). At this stage the shortened synaptic processes of the photoreceptors contain very few synaptic vesicles. Synaptic ribbons, each surrounded by a cluster of vesicles s t i l l persist, however, i n some photoreceptors (Fig. 60). As before, lysosomes are abundant i n the epithelium close to inner epithelial surface but the other subcellular structures of the retinal epithelium are unchanged (Figs. 56, 57, 58). The apical e p i t h e l i a l processes are now numerous and prominent but the infoldings on 65 the basal epithelial surface are unchanged from the normal. The apical processes are regularly arranged and point towards the neural retina (Figs. 55, 57, 58). (i) Retinal changes after 10 months of vitamin A deficiency The most striking feature characterizing retinal structure at this stage of vitamin A deficiency i s the close association between the r e t i n a l epithelium and the neural retina (Fig. 61). The accumulation of lysosomes i n the inner r e t i n a l epithelium and i n the broader e p i t h e l i a l processes i s s t i l l present (Figs. 61, 64). Also some lysosomes can be found i n central regions of the epithelium near the Golgi apparatus (Fig. 64). The prominent apical processes are regularly oriented and abut directly on the neural retina or interdigitate with outer processes of Muller c e l l s (Figs. 61, 63). The photoreceptor outer segments have completely disappeared except for a few degenerating fragments, often oval i n shape (Figs. 62, 63). These fragments, oontaining a large number of vesicles and tubules and seme saccules, are found among the processes of retinal epithelium and the Muller c e l l s . In Figure 64, remnants of two photoreceptor inner segments are present betv;een Muller c e l l processes. One of them has an intact connecting cilium and a basal body. Its cytoplasm contains numerous polysomes and a couple of irutochondria. Remnants of a photo- receptor outer segment are also present lying transversely next to the inner epithelial surface (Fig. 64). Also i n Figure 64, a degenerating cone photoreceptor c e l l situated i n a niche of the neural retina, can be identified by i t s characteristic nuclear chromatin which i s less bb dense and more diffuse than that of the rod. The cone nucleus seems intact and a few mitochondria, some polysomes and an i l l - d e f i n e d Golgi apparatus are v i s i b l e i n the cytoplasm. The outer limit i n g membrane i s now composed almost entirely of c e l l junctions between adjacent Muller c e l l s (Fig. 63). The photo- receptor c e l l s (mainly rods) that remain have prominent nuclei but display only a narrow rim of cytoplasm containing a few polysomes and other i l l - d e f i n e d subcellular structures (Fig. 61). Synapses have almost completely disappeared. The photoreceptors and other unidentified c e l l s l ying next to them are now surrounded by several layers of membranes prob- ably of a g l i a l nature (Fig. 61). (j) Petinal changes after 11 months of vitamin A deficiency Retinal structure at t h i s stage i s marked by a close structural association between r e t i n a l epithelium and neural retina. Large numbers of lysosomes l i e beneath the inner e p i t h e l i a l surface and the apical e p i t h e l i a l processes remain prominent (Figs. 65, 66, 67). Frequently, there are regions where the apical processes are deflected sideways by the neural retina (Figs. 66, 67). There are also areas where segments of the r e t i n a l epithelium which do not possess apical processes are i n d i r e c t contact with the neural retina (Fig. 66). Remnants of photorecep- to r outer and inner segments are s t i l l scattered among the processes of the r e t i n a l epithelium and Muller c e l l s . The c e l l junctions between adjacent Muller c e l l s persist (Fig. 69). At some areas close to the outer border of the neural retina, Muller c e l l processes bend sideways and inwards, possibly contributing to g l i a l membrane formation (Fig. 67). 67 Synapses of photoreceptors v/ith nerve processes of c e l l s of the inner nuclear layer and synaptic ribbons are now absent. Unidentified c e l l s appear between the photoreceptors. These c e l l s , l i k e the photo- receptors are surrounded by several layers- of membranes (Figs. 65, 68). Capillaries now can be found lying close to the retinal epithelium due to the absence of photoreceptors. The endothelial cytoplasm of the capillaries demonstrates active pinocytosis (Fig. 66). 68 E) Acid Phosphatase Localization i n the Retinal Epithelium Tne r e t i n a l e p ithelia from the posterior retinas of animals, which were 6 months vitamin A deficient, were used to locate sites' of acid phosphatase a c t i v i t y as a test for the presence of lysosomes. The inner surface of the r e t i n a l epithelium i s distorted because the r e t i n a l epithelium with the choroid layer was detached from the retina proper by means of a p a i r of forceps during tissue preparation. Fragments of photoreceptor outer segments adhere to apical e p i t h e l i a l processes. When tissues are incubated i n Gomori medium with sodium beta-glycerophosphate as substrate, acid phosphatase a c t i v i t y causes the lead nitrate [Pb(NC<3)2] contained i n the Gomeri medium to react with the substrate producing lead phosphate. The reaction product, lead phosphate, i s seen as black precepitate within the c e l l u l a r structures indicating the si t e s of acid phosphatase reaction. In the r e t i n a l epithelium, reaction product i s present around the periphery of lysosomes lying beneath the inner e p i t h e l i a l surface (Fig. 70), i n phagosomes (Figs. 70, 71) and i n cisternae of the Golgi apparatus (Fig. 71). In the tissues that were incubated i n Gomori medium without the sodium beta-glycerophosphate, reaction product was completely absent i n the r e t i n a l epithelium. 3 F) ^fethionine-H Incorporation i n the Retina i n Vitamin A Deficiency 3 I-fethionine-H was injected intravxtreally into the eyes of control and vitamin A deficient animals to test for the presence or absence of amino acid uptake and protein synthesis i n the photoreceptors. In retinas of 10 month old control animals, s i l v e r grains are evenly distributed i n the r e t i n a l epithelium, photoreceptor outer 69 segments, inner segments and the outer nuclear layer (Fig. 72) 4 hours after injection. After 24 hours, there i s a marked increase i n labelling over the r e t i n a l epithelium, the photoreceptor inner segments and the outer nuclear layer. An accumulation of the label i s v i s i b l e at junctions between photoreceptor inner and outer segments and also in the basal portions of the outer segments. In the outer nuclear layer, s i l v e r grains are found primarily i n the c e l l cytoplasm (Fig. 73). In 2.5 month vitamin A deficient animals, 4 hours after injection, the label i s evenly dispersed over the r e t i n a l epithelium, the photo- receptor outer and inner segments and the outer nuclear layer (Fig. 74). After 24 hours, there i s a sli g h t increase of the label over the photo- receptor inner segments and the outer nuclear layer. Silver grains are especially concentrated at the junctions between the photoreceptor inner and outer segments. In the outer nuclear layer, s i l v e r grains are ob- served i n the nuclei and i n the cytoplasm (Fig. 75). In the 8 month vitamin A deficient animals, 4 hours after labelling 3 . with methionine-H , sparse labelling i s v i s i b l e over the r e t i n a l epithelium, photoreoeptor inner segments and the outer nuclear layer (Fig. 76). After 24 hours, there i s a marked increase i n labelling over the r e t i n a l epithelium, photoreceptor outer segments, inner segments and outer nuclear layer. Silver grains are again observed concentrated at the basal portions of the outer segments. In the outer nuclear layer, the label i s mainly localized around the periphery of the nuclei and i n the cytoplasm (Fig. 77): In 10 month vitamin A deficient animals, only 1-2 rows of photo- reoeptor c e l l s remain. In this retina, 4 hours after injection with 3 methionine-H , there i s l i t t l e incorporation of radioactive material 70 i n t o the neural r e t i n a and none over the few photoreceptor c e l l s observed (Fig. 78). A f t e r 24 hours, there i s a v i s i b l e increase i n l a b e l l i n g over the r e t i n a l epithelium, the photoreceptor c e l l s and the neural r e t i n a . In the photoreceptor c e l l s , s i l v e r grains are d i s t r i b u t e d mainly around the periphery of the n u c l e i (Fig. 79).. Thus, despite the presence of severe s t r u c t u r a l changes in:.the 3 r e t i n a due t o viatmin A deficiency, incorporation of methionine-H p e r s i s t s i n the remnants of photoreceptor c e l l s and i n the r e t i n a l epithelium. 71 FIGURE LEGENDS Notes on Figure Legends: . 1) Animals were put on the vitamin A free diet when they were 5 weeks old. Therefore an animal designated as a 5 week vitamin A deficient animal i s actually 10 weeks old. 2) In the following electron micrographs a l l horizontal bars represent 1 u unless otherwise specified. 3) Definition of terms: a) "Posterior retina" means the area behind the equator close to the optic nerve of the eye and "peripheral retina" means the area i n the region of the equator and towards the ora serrata. b) The terms "inner" and "apical" used i n association with the retina and r e t i n a l epithelium respectively mean towards the vitreous or center of the eye while "outer" and "basal" mean towards the sclera. c) The term " d i s t a l " used i n association with photoreceptor outer and inner segments means farthest s c l e r a l l y from the photoreceptor nucleus and "proximal" means nearest to the photoreceptor nucleus. 72a Figure 1. Graph shotting the growth rate of the control and vitamin A deficient animals. Each point represents the mean weight of 14-45 animals i n the curve of vitamin A deficient animals. In the curve representing the weights of control animals, each point represents the mean weight of 10-19 animals. The standard deviation (- S.D.) of each mean i s represented by the v e r t i c a l bar at each point. The experiment began when the animals were 5 weeks old and weighing on the average 73.0 g. Both the vitamin A deficient and control animals grew at approximately the same rate u n t i l the 4th week. The control animals continue to gain weight rapidly u n t i l the 15th week when they assume a slower rate of growth. The vitamin A deficient animals display a more gradual growth rate u n t i l a plateau i s reached at the 21st week.. None of the control or vitamin A deficient animals died during the course of the studies. The vitamin A deficient animals appeared i n good healthy condition, except for a few which showed cloudiness i n the cornea and red exudate around the eyelids. 72 b F i g u r e 1.. Growth r a t e o f c o n t r o l and v i t a m i n A d e f i c i e n t a n i m a l s . 50 0 I — J : L » » » 1 1 ! 8 I j s 0 2 4 6 8 10 12 14 16 18 20 22 2< Weeks on d i e t 73a Figure 2a. Graph shewing how the maximum absorbance of vitamin A i s obtained. T Q represents the time when the TFA mixture i s added to vitamin A solution; T^ represents the time when the i n i t i a l optical density of the vitamin A solution i s noted; 1^ represents the time of the f i n a l absorbance of the vii^min A solution which i s zero optical density. The maximum optical density of the solution at T Q i s obtained by extrapolating the slope of absorbance of T^ and T 2 to T N (dotted l i n e ) . 73 b Time i n seconds. 74a Figure 2b. Graph shewing the bleed plasma vitamin A levels i n the control and vitamin A deficient animals. Weanling rats, about 5 weeks old were placed on a vitamin A free diet and litter-mates also 5 weeks old were used as controls. Each point on the curves represents the mean vitamin A ccntent/lOCml of blood plasma of 1-2 animals. The vitamin A deficient animals were sampled at 3, 4, 6 and 8 weeks after they were cn the vitamin A free diet. The control animals v/ere sampled at 0 (5 weeks old), an&.;2 and 8 weeks, • The vitamin A content/lOOml of the three samples of the control animals was found to be 58.5, 46.5 and 52.5 ug respectively. The blood plasma vitamin-A content of the vitamin A deficient animals declined rapidly after 3 weeks on the vitamin A free diet. By the 4th week, the vitamin A le v e l f e l l frcm 57.5 jug to 32D,ug per 100ml blood plasma. By the 6th week, the vitamin A l e v e l f e l l to.9.5 ;ag/100ml blood plasma and by the 8th week, the vitamin A deficient anirnls had a vitamin A content of 70jug/100ml of blood plasma. That was about 13% of the vitamin A l e v e l of the control animals. 74 b Weeks on diet. 7 5 a F i g u r e 3. L i g h t mioDograph from a 2 month o l d c o n t r o l animal showing t h e o v e r a l l s t r u c t u r e o f the normal r e t i n a p o s t e r i o r t o the equator o f the eye. From o u t s i d e inwards: 1) r e t i n a l e p i t h e l i u m , 2) photo- r e c e p t o r o u t e r and i n n e r segments, 3) o u t e r l i m i t i n g membrane, 4) c u t e r n u c l e a r l a y e r , 9-11 n u c l e i t h i c k , 5) o u t e r p l e x i f o r m l a y e r , 6) i n n e r n u c l e a r l a y e r , 7) i n n e r p l e x i f o r m l a y e r , 8) g a n g l i o n c e l l l a y e r , 9} nerve f i b e r l a y e r , 1 0 ) i n n e r l i m i t i n g membrane. bv=blcod v e s s e l . x 1 , 1 9 0 F i g u r e 4. L i g h t micrograph a t h i g h e r m a g n i f i c a t i o n showing the p o s t e r i o r r e t i n a o f a 2 month o l d c o n t r o l animal. The r e t i n a l e p i t h e l i u m (RE) c o n s i s t s o f a s i n g l e l a y e r o f c e l l s . Each e p i t h e l i a l c e l l has 1-2 n u c l e i (N) which are o v a l i n shape. The t h i c k n e s s o f t h e l a y e r o f photoreceptor o u t e r (OS) and i n n e r segments (IS) i s about- 9.6 u. The segments are c l o s e l y packed together. The o u t e r l i m i t i n g membrane (OEM), formed mainly by cell j u n c t i o n s between the M u l l e r cells and the photoreceptor i n n e r segments i s v i s i b l e . The photo- r e c e p t o r n u c l e i form the o u t e r nuclear l a y e r (ONL). x 4,750 75b Figure 5. . Light micrograph showing the retina peripheral to the equator of the eye from a 7 month old control animal. The choriocapillaries with many red blood c e l l s are seen above the retina proper. The outer nuclear layer i s about 5-6 nuclei thick. A displaced photo- receptor nucleus (arrow) i s seen lying among the photoreceptor inner segments. A re t i n a l blood vessel l i e s i n the inner nuclear layer. x 1,190 Figure 6. Light micrograph showing the peripheral outer retina from the same specimen as Figure 5. Three displaced photoreceptor nuclei are seen lying between the retinal epithelium (RE) and the photoreceptor inner segments (IS) : nucleus No. 1 i s at the junction of the photoreceptor inner and outer segments; nucleus No. 2 at the junction of the photo- receptor outer segments and retinal epithelium and nucleus No. 3 i s seen partly within the retinal epithelium. Two types of photoreceptor nuclei are found i n the outer nuclear layer (ONL). Rod nuclei pre- dominate over the cone nuclei in the rat. The rod nucleus (RN) has a dense block of centrally located cnrcmatin whereas the cone nucleus (CN) has more diffuse chromatin usually divided into several lobes. x 4,750 75o 76a Figure 7. Light micrograph shewing the peripheral retina from a 12 month old control animal. The layer of choriocapillaries can be seen above the retina proper! The photoreceptor outer segments are less closely packed together than i n the posterior retina. The outer nuclear layer i s about 5-6 nuclei thick. Several capillaries (CP) are present i n the inner nuclear layer. x 1,100 Figure 8. Light micrograph at higher magnification showing the outer retina frcm the same specimen as Figure 7. Two epit h e l i a l nuclei and some dark granules (arrows) are seen i n the ret i n a l epithelium. Both photoreceptor outer (OS) and inner segments (IS) are elongated structures and cyl i n d r i c a l i n shape. The outer limiting membrane (OIM) demarcates the photoreceptor inner segments and the outer nuclear layer (ONL). x 4,750 76b Figure 9. Light macrograph of the posterior retina from an animal which was on a vitamin A free d i e t for 3.5 months. The choroid layer i s seen above the re t i n a l epithelium. The photoreceptor outer and inner segments appear f r a g i l e and broken and the layer measures about 10 u thick. The outer liirdting membrane i s v i s i b l e above the outer nuclear layer which i s about 9-11 nuclei thick. The outer plexiform layer, about 4.7 u thick, i s l i g h t l y stained. The inner neural retina appears normal. x 1,190 Figure 10. Light micrograph at higher magnification showing the outer retina from the same specimen as Figure 9. The retinal epithelium displays three oval e p i t h e l i a l nuclei (N). The photoreceptor outer segments (OS) are not well defined and show evidence of disintegration (single arrows). The photoreceptor inner segments (IS) are shorter than they normally are and sore appear sl i g h t l y swollen (double arrows). Above the outer nuclear layer (ONL) the outer limiting membrane i s not v i s i b l e due to the plane of the section. x 4,750  77a Figure 11. Light irdcrcgraph showing the posterior retina from a 6 month vitamin A deficient animal. The layer of photoreceptor inner and outer segments measures about 4.4 u i n thickness compared to 8 u in a control animal of about the same age. The outer limiting membrane i s v i s i b l e above the outer nuclear layer which i s about 3-5 nuclei thick. The l i g h t l y stained outer plexiform layer measures 1.8 u in width while the inner neural retina appears unchanged. A blood vessel i s seen in the inner neural retina on the l e f t . x 1,600 Figure 12. Light micrograph at higher magnification showing the same specimen as Figure 11. Severe breakdown of the photoreceptor outer segments CCS) i s evident. The outer segments are irregular in shape and there are many empty spaces (x) between them. The cuter limiting membrane (GLM) i s present and the photoreceptor nuclei i n the outer nuclear layer (ONL) appear normal. In the retinal epithelium, many small dark granules (arrows) are v i s i b l e along i t s inner surf ace. x 4,750 77b Figure 13. Light micrograph showing the posterior retina from a 9 month vitamin A deficient animal. The outer segments have almost disappeared and the inner segments are markedly reduced i n thickness. The layer of inner and outer segments measures 3.2 u i n thickness. The outer limiting membrane i s distinct. There are about 2-3 layers of photo- receptor nuclei remaining. Three photoreceptor nuclei are seen out- side the outer limiting membrane. The re t i n a l epithelium i s darkly stained. Thinning of the outer plexiform layer, which now measures 0.9 u i n thickness, causes the inner nuclear layer to appear closer to the outer nuclear layer. x 1,600 Figure 14. Light micrograph at higher magnification showing the same specimen as Figure 13. The inner segments (IS) that are discernable are short and round and l i g h t l y stained. Empty spaces above the inner segments are coiimonly seen. A photoreceptor nucleus i s v i s i b l e between the r e t i n a l epithelium (RE) and the outer nuclear layer (ONL). The inner nuclear layer (INL) appears intact. The outer plexiform layer (white arrows) no longer forms a disti n c t zone between the outer and inner nuclear layers. Small dark granules (black arrows) are s t i l l observed along the inner surface of the retinal epithelium. x 3,880  78a Figure 15. Light micrograph shewing the posterior retina of a 10 month vitamin A deficient animal. The retinal epithelium, which i s darkly stained, i s closely apposed to the neural retinal layer. The outer nuclear layer contains only 1-2 rows of photoreceptor nuclei. The outer limiting membrane appears to l i e nearly adjacent to the re t i n a l epithelium. The outer plexiform layer has disappeared i n some regions (arrows) while the inner neural retina appears unchanged. x 1,600 Figure 16. Light micrograph at higher magnification showing the same specimen as Figure 15. Further approximation of the retinal epithelium (RE) and the remaining photoreceptor nuclei i n the outer nuclear layer (ONL) i s evident. The outer limiting membrane (OLM) i s s t i l l present despite the severe loss of photoreceptors. Unidentified c e l l s (arrows) appear between the few remaining photoreceptor c e l l s . The outer p l e x i - form layer i s discemable only at some regions. x 4,750 78b Figure 17. Light micrograph, shewing the posterior retina from an 11 month vitamin A deficient animal. Only one irregular row of photoreceptor nuclei i s l e f t . Seme photoreceptor nuclei are again observed be- tween the retinal epithelium and the neural r e t i n a l layer. The outer limiting membrane i s present. Occasionally, c e l l s from the inner nuclear layer are seen i n the outer nuclear layer (arrow). The outer plexifrom layer has almost disappeared. The inner neural retina appears unchanged. x 1,600 Figure 18. Light micrograph at higher magnification showing the same specimen as Figure 17. A cone photoreceptor nucleus i s seen close to the r e t i n a l epithelium (arrow). The outer limiting membrane (OLM) i s clearly v i s i b l e . The photoreceptor nuclei i n the outer nuclear layer (ONL) appear to be each surrounded by a narrow rim of cytoplasm which i s very l i g h t l y stained. I t i s possible, however, that the clear zone outside the photoreceptor nuclei represents a shrinkage artefact. The ce l l s i n the inner nuclear layer (INL) appear largely unchanged. x 4,750  79a Figure 19. Electron rnicrograph shewing a portion of the r e t i n a l epithelium from a 1.5 month old control animal. The fine structure of two adjacent ep i t h e l i a l c e l l s i s shown. The basal surface (B) of each c e l l i s i n - I folded. A layered basement merrbxane which forms part of Bruch's membrane (Bi-l) i s present beneath the basal surface. The epi t h e l i a l inner surface displays numerous long and slender processes (AP). Premelano- somes (Bit) are present i n the processes. Apical c e l l junctions (ACJ) between adjacent e p i t h e l i a l c e i l s are composed of a zonula occludens and zonula adherens. The zonula adherens, marked by dense plasma membranes and oondensation of subjacent cytoplasmic matrix, l i e s just sclerad to the zonula ccciodens. The e p i t h e l i a l nucleus (N) contains diffuse chjxmatin. Nuclear pores (NP) are present. The e p i t h e l i a l cytoplasm i s characterized by a preckDminance of smooth endoplasmic reticulum (SER). Rough endoplasmic reticulum (PER) i s also present, scattered i n the cytoplasm and aggregated along the inner surface of the retina"! epithelium. A single row of mitochondria (M) l i e s inside the basal infoldings and along l a t e r a l c e l l borders. One or more well developed Golgi complexes (G) are found near the nucleus i n each ep i t h e l i a l c e l l . Polysomes (?) are sparsely distributed i n the cytoplasm and microtubules (MT) can be observed. Photoreceptor outer segments (OS) can be identified lying between the apical processes of the epithelium. x 17,500 79b 80a Figure 20. FAectrcn micrograph, showing the inner r e t i n a l epithelium and photoreceptor outer segments from a 7 month old control animal. An abundance of smooth erxioplasmic reticulum (SER) i s present i n the r e t i n a l epitJ>elium. lamellar-like structures or phagosomes (Ph) present i n the e p i t h e l i a l c e l l cytoplasm, probably represent discarded portions of photoreceptor outer segments. Residual bodies (R), which probably represent debris from disintegrating phagosomes are observed. A mitocixxidrion i s seen near the ingested fragments and a lysosome (L) above the phagosome. Long, slender apical processes (AP) extend inward and surround each photoreceptor outer segment (OS). The apical processes are devoid of identifiable subcellular structures. x 32,700 8) b 81a Figure 21. Electron micrograph showing the retinal epithelium and the photo- receptor outer segments from the same specimen as Figure 20. Rough endoplasmic reticulum (RER) i s v i s i b l e just inside the e p i t h e l i a l inner surface. The bulk of the e p i t h e l i a l cytoplasm contains smooth endoplasmic reticulum (SER). A well developed Golgi complex (G) and several mitochondria (M) are v i s i b l e . E p i t h e l i a l apical processes (AP) form a palisade around the orderly arranged photoreceptor outer segments (OS). The photoreceptor outer segments are c y l i n d r i c a l structures and each i s composed of a series of discs or flattened saccules surrounded by a plasma membrane (PM). The discs are regularly arranged and stacked at right angles to the length of the outer segment. x 28,080 81b 82a Figure 22. Electron mcrograph. showing the fine structure of two adjacent retinal e p i t h e l i a l c e l l s from a 9 month old control animal. The fine structure of the retinal epithelium does not change with aging i n control animals. Bruch's membrane (BM) overlies the r e t i n a l epithelium and part of the fenestrated endothelium (E) of the choriocapillary can be seen. Basal infoldings (B), and apical junctional complexes are present. The zonula occludens (Zo) i s clearly v i s i b l e and the zonula adherens (Za), i s marked by prominent plasma membranes and ' condensation of subjacent cytoplasmic matrix. Smooth endoplasmic reticulum (SER) predcminates i n the cytoplasm. Several lysosomes' ' (L) and residual bodies (R) are present. Mitochondria l i e close to the basal infoldings and along the l a t e r a l c e l l borders. Pinocytosis (Pi) occurs at the basal surface and coated vesicles (CV) can be seen scattered i n the cytoplasm frcm the basal to the apical surface. x 21,060 •i~ b 83a Figure 23. Electron nacrograph. showing photoreceptor inner (IS) and outer segments (OS) from a 1.5 ircnth old control animal. Each outer segment consists of stacks of regularly arranged lamellar discs enveloped with- i n a plasma membrane. The outer segment i s connected to the inner segment by a cilium (C). The inner segment contains numerous polysomes (P) . Long, slender irdtc<±^dria (M) are arranged mainly around the periphery of the inner segment. A striated rootlet (SR) extends from the basal body (Bb) of the ciliuia down the inner segment. ;; x 17,320  «4a Figure 24. Electron micrograph showing the photoreceptor outer segments (OS), connecting cilium (C) and inner segments (IS) from a 7 month old control animal. The regularly arranged lamellar discs of the outer segment are surrounded by a single plasma membrane (PM) which i s continuous with the plasma membrane of the inner segment. The discs appear identical to those seen i n the 1.5 month old control animal. Each lamellar disc contains an invagination at the same point forming a longitudinal groove or incisure extending the whole length of the outer segment. Portions of such longitudinal grooves are indicated by arrows. A nutochondrion containing glycogen granules (SM) i s seen at the lower right hand corner. x 29,720 Figure 25. Electron micrograph showing photoreceptor outer (OS) and inner segments (IS) from the same specimen as Figure 24. At the base of the outer segment, irregularly arranged saccules, are seen. Next to them, the discs have a more regular appearance and further up, the lamellar discs are precisely arranged. The inner segment contains mitochondria (M) , polysomes (P), smooth (SER) and rough endoplasmic reticulum (PER). x 35,760 84b 85a Figure 26. Electron micrograph showing photoreceptor inner segments (IS) from a 9 month old control animal. The inner segments are long and cyli n d r i c a l i n shape. Elongated mLtochondria (M) with well developed transverse critae are arranged mainly around the periphery of the inner segments. A Golgi apparatus (G) surrounded by numerous vesicles i s present i n the basal region of the inner segment. A few smooth surfaced cistemae (SER) are v i s i b l e i n the cytoplasm. Polysomes (P) are evenly distributed. The connecting cilium (C) originates i n a basal body i s located i n the cytoplasm of the d i s t a l inner segment sl i g h t l y to the side of i t s central axis. The striated rootlet (SR) extends from the basal body (Rb). The inner segments are closely packed together. Nevertheless, they usually are separated at their bases from each other by thin, v i l l o u s extensions of Muller c e l l s (MP). The outer limiting membrane (arrows) i s formed mainly by c e l l junctions between photoreceptors and Muller c e l l s . Portions of two rod photoreceptor nuclei (PN), each characterized by a large dense block of centrally located chromatin are shown in the lower part of the micrograph. x 12,090 85b 86a Figure 27. Electron micrograph at higher magnification showing photoreceptor inner segments and the outer limiting membrane from the same specimen as Figure 26. Mitochondria are seen disposed l a t e r a l l y i n the inner segments. Two senile mitochondria (SM) each containing a large number of glycogen granules are seen i n the inner segments. The Golgi complexes (G) are surrounded by numerous vesicles. Smooth surfaced cisternae (SER) and rough endoplasmic reticulum (RER) are v i s i b l e i n the inner segments. The cytoplasm i s r i c h i n polysomes(R>.The closely packed inner segments are separated by Muller c e l l processes (MP), Zonulae adherentes (arrows) between the inner segments and the Muller c e l l s form the outer limiting membrane. Portions of three photoreceptor nuclei (PN) are seen at the bottom of the micrograph. x 18,650  87a Figure 28. Electron micrograph shewing i n i t s center a rod synaptic process . (R) from a 1.5 month old control animal. The process ends i n an oval structure- known as a "spherule". The basal surface of the spherule i s invaginated by two l a t e r a l processes probably from horizontal c e l l s (H) and one central process probably from a bipolar c e l l (B) of the inner nuclear layer. The c e l l wall of the spherule encloses the terminations of these neuronal processes. The rod spherule and i t s invaginating neuronal processes comprise the synaptic unit. The cytoplasm of the rod synaptic processes contains a large number of synaptic vesicles (SV), some polysomes (P) and one or two mitochondria (M). A synaptic ribbon (Sr) i s t y p i c a l l y found at the invaginating type of synapse. I t i s a crescent shaped structure composed of three dense layers separated from each other by two l i g h t e r layers. Below the synaptic ribbon l i e s condensed material forming the arciform density (Ad). Superficial contacts with neuronal processes from the inner nuclear layer are marked by a prominenoe of the presynaptic membrane of the rod spherule (long arrow). At the s i t e of su p e r f i c i a l synapses the synaptic ribbon i s not present. The rod spherule shown i n the center of the micrograph i s surrounded by portions of several other rod spherules. x 46,700 87 b 88a Figure 29. Electron ipicrograph. shewing the outer plexiform layer from a 1.5 month old control animal. Two photoreceptor nuclei (PNL are shown at the top of the micrograph. Synapses and synaptic ribbons (Sr)_ are numerous. Each synaptic process i s f u l l of synaptic vesicles (SV). and contains one or more mibocix)ndria(MlAt the synaptic s i t e , each, synaptic ribbon i s surrounded by a cluster of synaptic vesicles. The most carmen type of synapses observed are contacts between rod spherules and processes of horizontal (H) and bipolar c e l l s . Less commonly, post-synaptic processes, instead of penetrating into a spherule, make direct contact with the surface of the c e l l body. In t h i s situation the synaptic ribbon l i e s near the receptor nucleus (arrow head). This type of contact has been described as a somatodendritic synapse. x 18,650 88 b 89a Figure 30. Electron micrograph showing a cone synaptic process (C) from a 1.5 month, old control animal. The synaptic process ends i n a broad swelling wm.cn has been ca"!led a "pedicle". The cone pedicle i s characterized by i t s complex base into which other neuronal processes from c e l l s of the inner nuclear layer penetrate and make synaptic contact. In the cytoplasm of the pedicle, synaptic vesicles (SV) are evenly distributed and one or more irdtochondria (M) are often present. There are more synapses of the invaginating type and therefore more synaptic ribbons (Sr) than i n the rod spherule. Each synaptic ribbon i s seen to be surrounded by a cluster of synaptic vesicles. Superficial synapses can also be observed at the basal surface of the pedicle (arrow). x 19,750 Figure 31. Electron micrograph showing rod spherules (R) from a 1.5 month old control animal. The spherule makes synaptic contact with the neuronal processes from the inner nuclear layer. Synaptic vesicles (SV) are evenly distributed in each spherule. Note that the horizontal c e l l processes also contain vesicles which resemble the synaptic vesicles of the spherule. Synaptic ribbons (Sr) are found at the invaginating type of synapses. x 22,290 39 b 90a F i g u r e 32. E l e c t r o n micrograph, sinewing the o u t e r p l e x i f o r m l a y e r f r c m a 12 month o l d c o n t r o l a n i m a l . A cone p e d i c l e (C) i s shown p a r t i a l l y surrounded by s e v e r a l r o d s p h e r u l e s (R). S y n a p t i c v e s i c l e s (SV) a r e numerous and e v e n l y d i s t r i b u t e d i n b o t h t h e r o d and cone s y n a p t i c p r o c e s s e s . The r o d synapse t y p i c a l l y r e c e i v e s d e n d r i t e s f r o m one b i p o l a r c e l l and u s u a l l y o n l y one s y n a p t i c r i b b o n (Sr> i s seen a t t h e s y n a p t i c s i t e i n a c r o s s - s e c t i o n . The cone s y n a p t i c p e d i c l e i s more complex than t h a t o f t h e r o d , and i t p r o b a b l y makes c o n t a c t w i t h s e v e r a l b i p o l a r and h o r i z o n t a l c e l l s . T h e r e f o r e , more s y n a p t i c s i t e s and more s y n a p t i c r i x j b c n s are observed i n a c r o s s - s e c t i o n o f a cone p e d i c l e t h a n a r o d s p h e r u l e . On t h e l o w e r l e f t o f t h e m i c r o g r a p h a mi t o c h o n d r i o n i n one o f t h e s y n a p t i c p r o c e s s e s has accumulated g l y c o g e n g r a n u l e s . A t t h e t o p o f t h e f i g u r e , p o r t i o n s o f f o u r p h o t o r e c e p t o r n u c l e i a r e seen. x 21,060 90 b yia Figure 33. Electron micrograph showing the photoreceptor outer segments from a 1 month vitamin A deficient animal. The d i s t a l portions of the outer segments (OS) are the f i r s t portions of the photoreceptors to show signs of change. Some of the lamellar discs break down into vesicles (arrows). A distorted outer segment i n which the lamellar discs have opened up to form longitudinally arranged lamellar structures, appears to be p a r t i a l l y engulfed by apical processes (AP) of the r e t i n a l epithelium (RE). Part of the r e t i n a l epithelium containing a Golgi apparatus (G) i s shown on top of the micrograph. Note the absence of many lysosomes i n the retinal epithelium at this stage. x 21,060 I b Figure 34. Electron itiicrograph showing the retinal epithelium and photoreceptor outer segments from a 1.5 month vitamin A deficient animal. The outer segments (OS) are now more severely affected. More lamellar discs have broken down into vesicles (V). Seme disordered saccules (arrow heads) are also observed. The disintegrating outer segments are closely surrounded by apical processes of the retinal epithelium (RE). In the retinal epithelium, lysosomes (L) have gathered within i t s inner surface. Ph^ and Pix, are two p a r t i a l l y disintegrating phagosomes. A l i p i d droplet (LD) i s seen on the right hand side of the micrograph in the ret i n a l epithelium. Polysomes are scattered i n the e p i t h e l i a l cytoplasm. Rough endoplasmic reticulum i s found close to the inner surface of the epithelium. x 28,080 92 b F i g u r e 3o. E l e c t r o n micrograph sinewing photoreceptor o u t e r segments (OS) from the same specimen as F i g u r e 34. A f f e c t e d o u t e r segments de- generate t o d i f f e r e n t degrees. The more s e v e r e l y a f f e c t e d o u t e r segments are almost completely surrounded by a p i c a l e p i t h e l i a l p r o c e s s e s , which appear t o be i n the a c t o f i n g e s t i n g them. P a r t o f the r e t i n a l e p i t h e l i u m (RE) w i t h lysosomes (L) along i t s i n n e r s u r f a c e i s seen a t the top r i g h t hand c o m e r . Note the a p i c a l c e l l j u n c t i o n (ACJ). x 21,060  94a Figure 36. Electron micrograph, showing photoreceptor outer and inner segments from a 2 month vitamin A deficient animal. Most of the lamellar discs i n the outer segments are affected and are i n various stages of disintegration. However, near the base of each outer segment, some lamellar discs appear intact. The inner segments (IS) contain large numbers of polysomes. Mitochondria (M), c i l i a (C) and basal bodies (Bb) are s t i l l present and are unchanged i n appearance. x 21,060 94 b 95a Figure 37. Electron micrograph showing the junction between the re t i n a l epithelium and phaboreceptor outer segments from a 2.5 month vitamin A deficient animal. The section i s cut s l i g h t l y obliquely. The apical processes (AP) of the retinal epithelium (RE) appear to have undergone a sl i g h t proliferation and are irregularly oriented. They appear to be active i n engulfing degenerating outer segments (OS). The smooth endoplasmic reticulum (SER), mitochondria (M) and Golgi complexes (G) i n the ret i n a l epithelium remain unchanged. x 18,650 9 5 b 96a Figure 38. Electron micrograph sixy^ing disintegrating photoreceptor outer segments (OS) from the same specimen as Figure 37. More outer segments are affected and the destruction i s more extensive than i n the 1.5 month vitamin A deficient animals. The disintegrating outer segments contain vesicles and tubules. Many discs have d i s - appeared completely leaving behind large intracellular spaces (x). Apical processes (AP) of the retinal epithelium i n irregular array are seen at the top l e f t hand corner of the micrograph. , x 14,040 96 b 97a Figure 39. Electron micrograph shewing the photoreceptor inner segments (IS) of the same specimen as Figure 37. At this stage of vitamin A deficiency the d i s t a l portions of the inner segments also show morphological change. The d i s t a l portions are slightly swollen and their cytoplasm i s almost devoid of polysomes. Vacuoles (V) which may result from fusion and dilation of the smooth endoplasmic reticulum are observed within inner segment cytoplasm. Cross- sections of two connecting c i l i a (C) are present and appear normal Some obliquely cut outer segments (OS) which have yet to undergo severe degeneration are present between and above the affected inner segments. x 21,060 97b 98a Figure 40. Electron micrograph shewing photoreceptor outer segments (OS) from a 4 month vitamin A deficient animal. Within the outer segments, some of the lamellar discs have broken down into vesicles (V) while others are either loosely arranged or oriented i n an abnormal directions. Many lamellar discs have completely disappeared leaving empty spaces (x). Apical processes (AP) of the retinal epithelium (RE) are seen surrounding the degenerating outer segments. x 28,080 98 b 99a figure 41. Electron micrograph showing photoreceptor outer segments (OS) , inner segments (IS) and the outer limiting membrane from the same specimen as Figure 40. Many outer segments have now disappeared leaving gaps (x). The inner segments appear shorter than normal. Basal bodies (Bb) and striated rootlets (SR) are s t i l l present i n the inner segments. Polysomes (P) have gathered i n the inner halves of the inner segments. Rough endoplasmic reticulum (RER) and large vacuoles (V) can also be identified i n inner segment cytoplasm. Some photoreceptor inner segments have undergone almost complete degeneration and can be seen as three dense structures i n the micrograph (arrows). The outer limiting membrane appears intact (double arrows). Portions of tvo photoreceptor nuclei are seen at the bottom of the micrograph. x 14,700 99 b IS wry +4 • ** us 100a Figure 42. Electron micrograph shewing photoreceptor inner segments (IS) from the same specimen as Figure 40. Smooth endoplasmic reticulum and vacuoles (V) are v i s i b l e i n the short and plump inner segments. Mitochondria (M) , connecting c i l i a (C) and basal bodies (Bb) i n the inner segments appear intact. A degenerating inner segment (IS) i s seen i n the center of the micrograph. Its dense cytoplasm contains several initochondria, two of which (SM) are swollen with glycogen granules. Many Muller c e l l processes (MP) extend between adjacent inner segments. x 18,650 I .»• b l o i a Figure 43. Electron micrograph showing two adjacent r e t i n a l e p i t h e l i a l c e l l s (RE) from a 5 month vitamin A deficient animal, Bruch's membrane (BM) appears normal. Basal infolding (B), apical c e l l junctions (ACJ) and mitochondria (M) i n the r e t i n a l epithelium are intact. The smooth endoplasmic reticulum (SER) of the r e t i n a l epithelium has not been altered either quantitatively or structurally. The major change i s a large increase i n lysosomes (L) near the inner e p i t h e l i a l border. The apical or inner processes (AP) of the r e t i n a l epithelium have lost t h e i r t y p i c a l regular arrangement. Portion of an outer segment (OS) i s seen at the bottom of the micrograph. x 21,060 101b Figure 44. Electron nucrograph shelving the retinal epithelium, photoreceptor outer segments and inner segments from a 6 month vitamin A deficient animal. More severe destruction of the photoreceptors i s i n evidence. The outer segments (OS) have lost their normal highly ordered orientation. None of them appear intact. Many in t r a - and extracellular spaces (x) are observed. The inner segments now are closer to the retinal epithelium. The inner segments display shorter mitochondria than normally and the cytoplasm i n their d i s t a l halves contains few polysomes. The r e t i n a l epithelium, contains an elongated nucleus (N) and displays a marked increase i n lysosomes (L) along i t s inner surface. The apical processes (AP) of the retinal epithelium appear to be engulfing the degenerating outer segments (OS). x 12,090 102 b 103a Figure 45. FJLectron nucrograph shewing the re t i n a l epithelium (RE) and the photoreceptor outer segments (OS) from the same specimen as Figure 44. A phagosome (Ph) i s seen i n the middle of the epithelium. An increase of lysosomes (L) along the ep i t h e l i a l inner surface i s evident. The apical processes (A?) are short and randomly oriented. The d i s i n - tegrating photoreceptor outer segments have lost their normal regular orientation and many i n t r a - and extracellular spaces (x) have formed. x 16,470  104a Figure 46. Electron micrograph shewing the photoreceptor outer segments frcm the same'specimen as Figure 44. Most of the outer segments (OS) show degenerative changes. Some outer segment discs have lost their normal compact and horizontal arrangement and as a result the outer segments appear badly damaged. Other groups of discs have a surpris- ingly normal appearance. The entire layer of photoreceptor outer segments i s greatly reduced i n thickness. Parts of two photoreceptor inner segments (IS), one with an intact oomecrting cilium, are seen at the lewer right hand corner. At the top l e f t i s part of the r e t i n a l epithelium with several lysosomes (L). x 21,060 104 b Figure 47. Electron micrograph shewing mainly photoreceptor inner segments (IS) from the same specimen as Figure 44. Above the inner segments, severe destruction of the outer segments i s observed. The inner segments have undergone different stages of shortening. In the less contracted inner segments to the right, there i s a perceptible difference i n the poly- some distribution between the d i s t a l and basal halves of the cytoplasm. The d i s t a l halves of the inner segments (towards the ret i n a l epithelium) contain fewer polysomes (P). The smooth cisternae (SER) are slightly; dilated but the basal bodies (Bb) and striated rootlets (SR) are intact. The basal portions of the inner segments close to the photoreceptor nuclei contain some polysomes and smooth endoplasmic reticulum. Mitochondria (M) appear shorter than they normally are, though they are structurally unchanged otherwise. An almost oval inner segment i s seen to the l e f t of the micrograph. It contains several polysomes and a few mitochondria. There are wide intercellular spaces between adjacent inner segments and many I'ul1er c e l l processes (MP) extend freely through them. Cross sections of ci. 1 ia (C) are found near the inner segments. Ce l l junctions between photoreceptors'and Muller c e l l s (single arrow) and between Muller c e l l s and 2-iiller c e l l s (double arrows) are observed. x 21,060 105 b 106a Figure 48. Electron macrograph showing the outer plexiform layer from the same specimen as Figure 44. The synaptic processes show several signs of degeneration i n vitamin A deficiency. In each synaptic process, there are fewer synaptic vesicles (SV) than normal and many are found lying iirmediately adjacent to the synaptic sites. Plasma membranes between adjacent synaptic processes have broken down and the c e l l cytoplasm appears to merge freely across the processes (arrows). A ndto<±ondrion (M) with a slight swelling of part of i t s outer membrane i s observed (double arrows). Synaptic ribbons are present at the synaptic sites, and each i s surrounded by a cluster of synaptic vesicles. The subjacent horizontal c e l l processes (H) contain post-synaptic vesicles and appear normal. x 21,060 106 b i o y a Figure 49.. Electron micrograph shewing -the photoreceptor synaptic processes from the same specimen as Figure 48. Breakdown of the plasma membranes of synaptic processes i s i n evidence (arrows). Each synaptic process contains sparsely dispersed synaptic vesicles (SV). Mtochcndria (M) within the synaptic processes are also affected i n vitamin A deficiency and appear markedly swollen. Most of the transverse cristae have disappeared. The synaptic ribbon (Sr) as well as the horizontal c e l l processes (H) containing closely packed post-synaptic vesicles are essentially unchanged. x 32,700 107 b 108a Figure 50. Electron micrograph showing the re t i n a l epithelium (RE) and photoreceptor outer segments (OS) from a 7 month vitamin A deficient animal. Bruch's membrane (BM) i s intact. In the retinal epithelium the basal iiofoldings (B), rrdtochcridria and other subcellular structures appear unchanged. A remarkable increase of lysosomes (L) i s observed deep to the inner surface of the retinal epithelium. There i s some proliferation and disorganization of the apical processes (AP) of the epithelium. The lamellar discs of the outer segments have broken • down into vesicles (arrows) and groups of discs have disappeared (double arrows). •x 15,700 108 b 109a Figure 51. Electron iricrograph shewing the r e t i n a l epithelium (RE), photo- receptor outer segments (OS) and inner segments (IS) from an 8 month vitamin A deficient animal. The photoreceptor inner segments and the retinal epithelium are now closer together. Degeneration of the outer segments involves loss of discs (x) and abnormal orientation of many of the remaining discs (arrows). The d i s t a l portions of the inner segments shown in the niicrograph contain few polysomes (P^ numerous short mitochondria (M) and a few dilated smooth cisternae (SER). ; The r e t i n a l epithelium i s marked by an increase of lysosomes (L) which aggregate iriainly beneath the inner ep i t h e l i a l surface and i n the wider apical processes (AP). The apical processes of the epithelium are irregularly oriented and appear to have increased i n number. x 13,300 1 0 9 b 110a Figure 52. Electron rrucrograph shewing inner segments, photoreceptor outer segments (OS) and part of the r e t i n a l epithelium from the same specimen as Figure 51. At the top l e f t hand corner, an outer segment i s seen with i t s lamellar discs i n disarray. Some of the lamellar discs have also broken down into vesicles. The few outer segments seen at the lower right are also distorted. In this zone of the retina, the photoreceptor outer segments have disintegrated to such an extent that some inner segments now l i e almost adjacent to the retinal epithelium. The large structure i n the center of the micrograph i s probably a degenerating inner segment (IS) undergoing autolysis. Its cytoplasm i s f u l l of lysosomes and aggregates of dense material. This structure i s almost i n contact with the apical e p i t h e l i a l processes (AP). Part of an e p i t h e l i a l nucleus i s seen at the top right hand corner. The apical processes of the r e t i n a l epithelium are short but appear to have increased i n number. Several lysosomes (L) are found i n the thicker processes of the retinal epithelium and i n the inner e p i t h e l i a l cytoplasm. x 21,060 110b 111a Figure 53. Electron micrograph shoving photoreceptor inner segments from the same specimen as Figure 51. The segments show different degrees of shortening. A less contracted inner segment (IS), i n the center of the micrograph, shows s l i g h t swelling of i t s d i s t a l end. The d i s t a l portion of the inner segment contains an elongated vacuole (V) but very few polysomes CP). Jtost of the polysomes are confined to the inner portions of the inner segments shown. Other photoreceptor inner segments have shortened further. A well developed Golgi apparatus (G) i s seen i n one of the inner segments. Degenerating outer segments are seen above the inner segments. (M, mitochondrion) • x 21,060 - • " s 112a F i g u r e 54. E l e c t r o n micrograph, shewing the o u t e r p l e x i f o r m l a y e r from the same specimen as F i g u r e 51. The s y n a p t i c processes (SP) are now s h o r t e r b u t a few synapses p e r s i s t . Each s y n a p t i c p r o c e s s c o n t a i n s a few s y n a p t i c v e s i c l e s (SV) ^ many l y i n g immediately a d j a c e n t t o the s y n a p t i c s i t e s . T h i s i s r e m i n i s c e n t o f those seen i n the specimens from 6 month v i t a m i n A d e f i c i e n t a n i m a l s . Breakdown o f plasma membranes (arrow heads) between a d j a c e n t s y n a p t i c processes i s a g a i n observed. A t the s y n a p t i c s i t e s y n a p t i c r i b b o n s (Sr) a r e s t i l l p r e s e n t and processes o f h o r i z o n t a l c e l l s (H) c o n t a i n i n g s y n a p t i c v e s i c l e s appear i n t a c t . S e v e r a l p b o i x i r e c e p t o r n u c l e i (FN) a r e seen i n the upper h a l f o f t h e m icrograph and an u n i d e n t i f i e d c e l l , p o s s i b l y a h o r i z o n t a l c e l l , i s v i s i b l e i n the lower h a l f . x 14,040 112b 113a Figure b5. Electron micrograph shewing the outer plexiform layer frcm the same specimen as Figure 54. 3reai<r]own of the plasma membranes (arrow) between adjacent synaptic processes and also of the iidtochondria (M) within the synaptic processes i s evident. The synaptic processes only contain a few dispersed synaptic vesicles. Synaptic ribbons are not observed i n th i s secrtion. An unidentified c e l l (U) undergoing autolysis i s seen. This c e l l contains lysosomes and a large mass of dense rraterial. Portions of two unidentified c e l l s i n the inner nuclear layer are seen at the bcttcm of the micrograph. x 16,900 113b 114a Figure 56. Electron micrograph showing the outer retina from a 9 month vitamin A deficient animal. The photoreceptor outer segments have disappeared except for occasional remnants of discs (CS). The micrograph shows two photoreceptors with markedly altered inner segments (IS). Although comecting c i l i a are not observed, basal bodies (Bb) s t i l l p e r sist i n the inner segments. Polysomes and short, c y l i n d r i c a l inutochondria are s t i l l found-in the retracting inner segments. Some mitochondria v/ith partly (lisintegrating plasma membranes (double arrows) are present i n one of the inner segments. The photo- receptor nuclei (PN) that remain do not show any variation frcm the normal i n their chromatin distribution. The outer limi t i n g membrane i s now formed mainly by c e l l junctions between Muller c e l l s (arrows) . In the r e t i n a l epithelium (RE) , lysosomes (L) aggregate close to i t s inner surface. Proliferation of the apical e p i t h e l i a l process (AP) i s prominent. The basal infoldings (B) and other subcellular structures i n the r e t i n a l epithelium appear unchanged. (MP. Muller c e l l processes) x 12,090 114b 115a Figure 57. Electron roicrograph showing the retinal epithelium (RE), remnants of photoreceptor outer segments (OS) and protions of photoreceptor inner segments from a 9 month vitamin A deficient animal." A loosely arranged lamellar structure representing a degenerating and distorted outer segment (OS) i s seen to the right of the micrograph. Another disintegrating outer segment which consists only of an aggregation of tubules and vesicles i s seen to the l e f t surrounded by e p i t h e l i a l apical processes (AP). The photoreceptor inner segments'(IS) have retracted further and Muller c e l l processes (MP) are now seen extending above them. The apical processes of the epithelium have proliferated markedly. Many lysosomes (L) are s t i l l found beneath the inner surface of the retinal epithelium. (N, re t i n a l e p i t h e l i a l nucleus) x 18,650 115 b 116a Figure 53. Electron irdcrograph shewing the outer retina from a 9 month vitamin A deficient animal. Sporadic clusters of saccules (arrows) are a l l that remains of the photoreceptor outer segments.. The photoreceptor inner segments (IS) are closer to the r e t i n a l epithelium than before. Seme inner segments have retracted more than others but a l l have undergone severe degeneration. In the d i s t a l or s c l e r a l halves of the inner segments, only a few polysomes remain and vacuoles (V) are present. The remaining mitochondria observed i n each inner segment are mostly round and short. Numerous polysomes persist i n the basal halves of the inner segments. Photoreceptor nuclei are seen at the bottom of the micrograph. Their nuclear chromatin appears normal. Junctions between Muller cells now comprise almost a l l of the outer limiting membrane (double arrows) . As photoreceptor outer and inner segments are discarded, Muller c e l l processes (MP) move i n to occupy the tissue gap. Lysosomes (L) s t i l l aggregate close to the surface of the r e t i n a l epithelium. The apical e p i t h e l i a l processes (AP) have markedly proliferated and now l i e adjacent to the inner segments i n some regions. x 1 4 , 0 4 0 1 1 6 b 117a Figure 59. Electron micrograph showing at higher magnification, photo- receptor inner segments and the. outer limiting membrane from the same specimen as Figure 58. In tine retracting inner segments (IS) shown, subcellular structures such as the Golgi apparatus and rough endoplasmic reticulum have disappeared. A few polysomes (P), some mitochondria (M) and smooth endoplasmic reticulum dilated to form vacuoles (V) are l e f t . A mitcx±ondrion i n one of the inner segments shows detachment and elongation' of part of i t s maiibrane (double arrows). An almost ovoid ; inner segment containing an intact basal body (Bb) i s seen i n the center of the micrograph. Clusters of saccules which are remnants of the outer segments (OS) are seen above the inner segments. Some apical e p i t h e l i a l processes (AP) are v i s i b l e at the top right hand corner of the micro- graph. The outer lindting membrane i s now formed mainly by c e l l junctions between Muller c e l l s (arrows). Several Muller c e l l processes (MP) extend through the extracellular.space between the inner segments. A cilium (C) i n cross-section i s v i s i b l e lying freely among the Muller c e l l processes. x 29,720 117 b 118a Figure 60. Electron micrograph showing degenerating photoreceptors from a 9 month vitamin A deficient animal. The photoreceptors show marked retraction of both inner segments (IS) and synaptic processes (SP). In the inner segments, the mitochondria have entirely degenerated and disappeared. Some polysomes (P) and rough endoplasmic reticulum (RER) are s t i l l present. The chromatin of the photoreceptor nuclei (PN) appears normal but the nuclear membranes show evidence of breakdown (thick arrows). There are few synaptic vesicles (SV) l e f t i n the degenerating synaptic process shown i n the micrograph. The synaptic ribbon (Sr) surrounded by some synaptic vesicles s t i l l persists. x 18,650 118 b uga Figure 61. Electron micrograph showing the posterior outer retina from a 10 month vitamin A deficient animal. There i s a closer association between the ret i n a l epithelium and the neural retina than observed at earlie r intervals. Two adjacent r e t i n a l e p i t h e l i a l c e l l s (RE) are shown. The retinal epithelium contains a large number of lysosomes close to the inner e p i t h e l i a l surface and i n some of the broad apical processes. The apical processes (AP) are numerous and now regularly oriented. The e p i t h e l i a l nucleus (N) and other subcellular structures i n the r e t i n a l epithelium are essentially unchanged. Remnants of photoreceptor outer (CS) and inner segments (IS) are scattered among the apical e p i t h e l i a l processes. Niiller c e l l junctions (arrows) are now widely spaced and mark the outer l i m i t of the neural retina. A displaced photoreceptor c e l l i s seen at the upper right of the micro- graph (double arrows). The photoreceptor c e l l s (PC) that remain have very l i t t l e cytoplasm containing only a few polysomes and an occasional mitochondrion. Each of the remaining photoreceptor c e l l s i s surrounded by several layers of membranes (Gm) probably of g l i a l origin. x 12,090 119 b 120a Figure 62. Electron micrograph showing the close association between the retinal epithelium (RE) and the neural retinal layer at the 10th month of vitamin A deficiency. The retinal epithelium contains numerous lysosomes (L) mainly aggregated close to i t s inner surface. Lysosomes are also present i n more central regions of the epi t h e l i a l c e l l s . An oval structure consisting of saccules, tubules and vesicles, the remnants of a photoreceptor outer segment (OS), i s seen p a r t i a l l y surrounded by apical processes (AP) of the retinal epithelium. The outer limiting membrane now appears to be formed by c e l l junctions between processes of Muller c e l l s (arrows) although the presence of c e l l processes from other types of g l i a l c e l l s cannot be excluded. Bruch's membrane (BM) overlying the r e t i n a l epithelium appears unchanged. x 18,650 120 b 121a Figure 63. Electron micrograph showing at higher magnification the close association between the apical processes (AP) of the r e t i n a l epithelium (RE) and the processes of the Muller c e l l s (MP) from the same specimen as Figure 61. Some apical process of the epithelium are displaced side- ways by the processes of the Muller c e l l s and others interdigitate with the l a t t e r . The outer limiting membrane i s well defined i n this region (arrows). A disintegrating photoreceptor outer segment (OS) with part of the inner segment (IS) i s seen at the lower l e f t of the micrograph. A pair of centrioles (c) i s v i s i b l e i n the cytoplasm of a Muller c e l l . Parts of two r e t i n a l e p i t h e l i a l c e l l s with several lysosomes are seen at the top l e f t hand comer. Apical c e l l junctions (ACJ) of the r e t i n a l epithelium are s t i l l intact. A portion of a photoreceptor c e l l remnant i s present at the lower right. x 25,730 121 b 122a Figuxe 64. Electron micrograph shaving the outer retina from the same specimen as Figure 61. In the r e t i n a l epithelium (RE) some lysosomes (L) are found i n the v i c i n i t y of the Golgi apparatus (G) but most of them are close to the inner surface of the ret i n a l epithelium. The apical e p i t h e l i a l processes (AP) are numerous and prominent. A photoreceptor c e l l (PC) , possibly a cone, i s seen i n the neural retina. The photo- receptor nucleus appears intact while a few degenerating mitochondria. (M), some polysomes and an il l - d e f i n e d Golgi apparatus (G) are discernable i n i t s cytoplasm. A couple of photoreceptor inner segments (IS) are present. One of them has an intact connecting cilium (C) with a basal body (Bb). Tne cytoplasm of this c e l l contains numerous polysomes and a couple of mitochondria. Remnants of a photoreceptor outer segment (OS) can also be distinguished lying next to the inner surface of the re t i n a l epithelium. x 21,060 122 b 123a Figure 65. Electron micrograph showing the outer retina from an 11 month vitamin A deficient animal. As usual, the inner cytoplasm of the r e t i n a l epithelium (RE) i s marked by the presence of a large number of lysosomes (L) while the e p i t h e l i a l nucleus (N) and the other subcellular structures i n the epithelium appear normal. The apical processes (AP) of the epithelium remain numerous and prominent. Remnants of some photoreceptor inner segments (IS) and outer segments (OS) can be seen among the apical processes of the retinal epithelium. One of the inner segments l i e s inside the outer limiting membrane and i s surrounded by g l i a l membranes (Gm). Two unidentified c e l l s each v/ith scanty cytoplasm are seen i n the center of the itacrograph. They are each surrounded by several layers of membranes. x 12,090  124a F i g u r e 66. E l e c t r o n micrograph shewing the r e t i n a l e p i t h e l i u m and the o u t e r r e t i n a from the same specimen as F i g u r e 65. The p r o l i f e r a t i o n o f lysosomes (L) c l o s e t o the i n n e r s u r f a c e o f the r e t i n a l e p i t h e l i u m i s s t r i k i n g . A t one r e g i o n , t h e r e t i n a l e p i t h e l i u m l i e s immediately adjacent t o the n e u r a l r e t i n a . A t t h a t s i t e , t h e a p i c a l e p i t h e l i a l processes have disappeared (arrows) but on bot h s i d e s o f t h i s r e g i o n , a p i c a l e p i t h e l i a l processes (A?) remain prominent. A r e t i n a l c a p i l l a r y (CP) l i e s c l o s e t o the r e t i n a l e p i t h e l i u m . Note t he l a y e r s o f g l i a l : membranes (Gm) above t h e c a p i l l a r y . x 13,700 124 b 125a Figure 67. Electron micrograph showing the outer retina from an 11 month vitamin A deficient animal. Lysosomes CL) lying close to the inner surface of the r e t i n a l epithelium and numerous, apical processes (AP) of the epithelium are again noted. Remnants of photoreceptor outer segments (OS) can s t i l l be seen above the neural retina. In this micrograph, what appear to be Muller c e l l processes (MP) bend l a t e r a l l y and contribute to the array of g l i a l membranes (Gm) v i s i b l e above and to the sides of the photoreceptor c e l l (PC) seen at the lower l e f t oomer. The scanty cytoplasm of the photoreceptor contains l i t t l e or no recognizable subcellular structures. C e l l junctions between Muller c e l l s can be ciistinguished (arrow). x 18,650 1 2 5 b 126a Figure 68. Electron micrograph showing the outer retina from an 11 month vitamin A deficient animal. In the retinal epithelium a well developed Golgi apparatus (G), numerous lysosomes (L) and prcminent apical processes (AP) are v i s i b l e . Fragments of photoreceptor inner segments (IS) l i e scattered among Muller c e l l processes (MP). The l a t t e r , a majority of which appear i n cross-section, are seen i n abundance i n this region. In the neural retina, areas not occupied by photoreceptors ; or unidentified c e l l s are replaced by numerous layers of g l i a l membranes (Gm). x 18,650 196 b 127a Figure 69. Electron micrograph from the same specimen as Figure 68 showing prominent apical processes (AP) of the re t i n a l epithelium and layers of membranes (Gm) probably of a g l i a l nature surrounding each of the remaining photoreceptor c e l l s (PC). Numerous well marked c e l l junctions probably between M i l l e r c e l l s (MT) are seen forming a limiting membrane at the outer edge of the neural retina. Above the neural retina, many Muller c e l l processes (MP), most of them cut i n cross-section, are present and show a tendency to bend sideways. x 23,000 127 b 128a Figure 70. Electron micrograph, showing acid phosphatase localization i n the ret i n a l epithelium from the posterior retina of a 6 month vitamin A deficient animal. After fixation, the tissue i s prepared by detaching the r e t i n a l epithelium and choriocapillaries from the retina proper. This procedure causes seme structural distortion of the inner r e t i n a l epithelium particularly the apical processes. The detached tissue i s then incubated i n Gomori medium with sodium-glycerophosphate as sub- strate and postfixed i n 2% osmium tetroxide. The tissue i s sectioned and double stained with uranyl acetate and lead citr a t e . A black, precipitate of lead phosphate indicates the presence of the enzyme acid phosphatase. In the itucrograph, black precipitate of lead phosphate can be observed around the periphery of the lysosomes (L) and i n the phagosomes (Ph)., but nowhere else i n the ep i t h e l i a l cytoplasm. (B, basal infolding of re t i n a l epithelium; M, mitochondria.) x 32,700 128 b 129a Figure 71. Electron micrograph showing acid phosphatase localization i n the ret i n a l epithelium from the same specimen as Figure 70. A black precipitate of lead phosphate i s present i n the lysosomes (L), phagosomes (Ph) and Golgi apparatus (G). Ph^ indicates a newly ingested phagosome which i s just beginning to be subjected to lysosomal enzyme activity. (AP, apical processes of re t i n a l epithelium.) x 32,700 129 b 130a F i g u r e 72. L i g h t m i c r o s c o p i c radioautograph showing the p o s t e r i o r o u t e r r e t i n a from a 10 month o l d c o n t r o l animal, 4 hours a f t e r i n t r a - 3 v i t r e a l i n j e c t i o n o f H -methionine. The s e c t i o n has been exposed f o r 2 months and p o s t s t a i n e d w i t h t o l u i d i n e b l u e . Sparse, evenly d i s t r i b u t e d r a d i o a c t i v e m a t e r i a l i s present i n the r e t i n a l e p i t h e l i u m (RE), photoreceptor o u t e r segments (OS), i n n e r segments (IS) and t h e o u t e r n u c l e a r l a y e r (ONL). The background i s almost c l e a r o f r a d i o - a c t i v e m a t e r i a l . x 4,750 F i g u r e 73. L i g h t m i c r o s c o p i c radioautograph shewing the p o s t e r i o r o u t e r r e t i n a from a 10 month o l d c o n t r o l animal, 24 hours a f t e r l a b e l l i n g 3 . i n t r a v i t r e a l l y w i t h H -metnionine. The s e c t i o n has been exposed f o r two months and p o s t s t a i n e d w i t h t o l u i d i n e b l u e . There i s a marked accnsnulation o f r a d i o a c t i v e m a t e r i a l over the r e t i n a l e p i t h e l i u m , t h e photoreceptor i n n e r segments and the o u t e r n u c l e a r l a y e r . An accumulation o f t h e r a d i o a c t i v e m a t e r i a l can be observed a t j u n c t i o n s between photoreceptor i n n e r and o u t e r segments and a l s o i n the b a s a l p o r t i o n s o f the o u t e r segments (black arrows). I n the o u t e r n u c l e a r l a y e r , s i l v e r g r a i n s are found p r i m a r i l y i n the c e l l cytoplasm (white arrows) x 4,750 130b Figure 74. Light microscopic radioautograph showing the posterior outer r e t i n a from a 2.5 month vitamin A d e f i c i e n t animal, 4 hours a f t e r i n t r a v i t r e a l l a b e l l i n g with K^-methionine. The section has been exposed for 2 months and poststained with t o l u i d i n e blue. Padioactive material i s evenly d i s t r i b u t e d over the r e t i n a l epithelium, the photoreceptor outer and inner segments and the outer nuclear layer. x 4,750 Figure 75. Light microscopic radioautograph from a 2.5 month vitamin A d e f i c i e n t animal showing the posterior outer r e t i n a , 24 hours a f t e r 3 xntravxtreal l a b e l l i n g with H -methionine. The section has been exposed f o r 2 months and poststained with t o l u i d i n e blue. This oblique section shows a s l i g h t increase i n l a b e l l i n g over the photo- receptor inner segments and the outer nuclear layer. S i l v e r grains are e s p e c i a l l y concentrated at junctions between the photoreceptor inner and outer segments (black arrows). In the outer nuclear l a y e r , s i l v e r grains are observed i n the n u c l e i (white dotted arrows) and i n the cytoplasm between the n u c l e i (white arrows). x 4,750  131a Figure 76. Light microscopic radioautograph from an 8 month vitamin A deficient animal, shaving the posterior outer retina, 4 hours after 3 i n t r a v i t r e a l labelling with H -methionine. The section has been exposed for 2 months and poststailed with toluidine blue. Sparse labelling i s present over the r e t i n a l epithelium, photoreceptor outer segments, inner segments and the outer nuclear layer. In the l a t t e r , s i l v e r grains reside primarily around the periphery of the nuclei. x 4,750 Figure 77. Light microscopic radioautograph from an 8 month vitamin A deficient animal, showing the posterior outer retina, 24 hours after 3 intravitreal labelling witn. H -methionine. The section has been exposed for 2 months and poststained with toluidine blue. A marked increase of radioactive material i s seen over the retinal epithelium, photoreceptor outer segments, inner segments and outer nuclear layer. Silver grains are especially concentrated at junctions between the photoreceptor inner.and outer segments and i n basal portions of the outer segments (black arrows). Marked incorporation of radioactive material i s found around the periphery of each photoreceptor nucleus (white dotted arrows). Some si l v e r grains are also observed i n the cytoplasm (white arrows) around each photoreceptor nucleus. x 4,750 131b Figure 78. Light microscopic radicautograph from a 10 month vitamin A deficient animal shewing the r e t i n a l epithelium and the neural retina, 3 4 hours after intravitreal labelling with H -methionine. The section has been exposed for 2 months and poststained with toluidine blue. L i t t l e radioactive material i s seen over the r e t i n a l epithelium (RE), inner nuclear layer (LNL) and inner plexifrem layer (XPL). None i s present over the few photoreceptor c e l l s (arrows) seen i n the micro^ graph. / x 4,750 Figure 79. Light microscopic radioautograph from a 10 month vitamin A deficient animal shewing the same structures as Figure 78, 24 hours 3 after i n t r a v i t r e a l labelling with H -methionine. The section has been exposed for 2 months and poststained with toluidine blue. There i s an increase i n labelling of the retinal epithelium (RE) , inner nuclear layer (TNL), and inner plexiform layer (IPL). Some s i l v e r grains are found over the few photoreceptors (PC) seen i n the micro- graph. They are distributed mainly around the periphery of the nuclei (black dotted arrows). x 4,750 1 "<tlC V. DISCUSSION A) Resume of the Most Pertinent Results Morphological changes i n the photoreceptor c e l l s and r e t i n a l epi- thelium produced by \dtamin A deficiency were studied i n the albino Wistar rat by l i g h t and electron microscopy. The process of degeneration of the photoreceptors i n vitamin A deficiency noted i n this study can be summarised as follows. F i r s t , the lamellar discs of the photoreceptor outer segments broke down into vesicles and tubules after 1 month of vitamin A deprivation. After 2.5 months, the d i s t a l portions of the inner segments became slig h t l y swollen and the d i s t a l inner segment cytoplasm suffered a loss of polysomes and underwent swelling of the endoplasmic reticulum. By 6 months of vitamin A deficiency, the photo- receptor synaptic terminals were affected. Fewer synaptic vesicles were present i n the cytoplasm and the plasma membranes i n the synaptic region developed large gaps. As deterioration of the photoreceptors continued, only fragments of the outer segments remained while degenerating inner segments and synaptic processes gradually retracted towards the photo- receptor nuclei. Tne nuclear envelopes of the photoreceptors began to disintegrate at 9 months. By 11 months, most of the photoreceptor c e l l s had disappeared and only one irregular row of photoreceptor nuclei remained. Each of the remaining visual c e l l nuclei was surrounded by several layers of g l i a l membranes. The neural cells of the inner r e t i n a l layers appeared unchanged although these were not studied i n detail by electron micro- scopy. In the retinal epithelium, a marked increase of lysosomes close to the inner ep i t h e l i a l surface and a prominent proliferation of the apical 135 processes were observed during the breakdown of the photoreceptors. In addition to the structural changes noted, the vitamin A deficient animals gained weight more slowly than the control animals. Their plasma vitamin A levels declined rapidly after 3 weeks on the vitamin A free diet supplemented with vitamin A acid. The acid phosphatase test confirmed the presence of a large number of lysosomes i n the inner r e t i n a l e p i t h e l i a l cytoplasm of the vitamin A deficient animals. Padioautographic data of 3 . . . H -methionine incorporation indicated that the degenerating photoreceptors were s t i l l capable of amino acid uptake and protein synthesis. B) Storage and Metabolism of Vitamin A According to Moore (1964), i n the human body, about 90% of the vitamin i s stored i n the l i v e r as vitamin A palmitate. The blood contains about 0.45% of the t o t a l body stores i n the form of vii^min A alcohol which i s kept at a remarkably constant l e v e l i n spite of dietary fluctuations. Only 0.005% of the body's vitamin A, i n the form of vitamin A aldehyde i s present i n the retina. The remaining vitamin A i s present i n the other organs and fatty tissues of the body. When animals are placed on a vitamin A free diet, the concentration of vitamin A i n the l i v e r slowly declines, whereas the blood vitamin A l e v e l remains r e l a t i v e l y constant u n t i l l i v e r stores are p r a c t i c a l l y depleted (Olson, 1969). Cowling and Wald (1958) reported that when weanling rats were placed on a vitamin A deficient diet, their l i v e r stores of the vitamin A declined linearly to zero i n about 3 1/2 weeks. After 3 1/2 weeks on "the diet, the blood vitamin A l e v e l f e l l precipitously to zero and rhodopsin content i n the retina ccmmenced a linear decrease, marking the onset of night blindness. By 2 months, opsin levels in the retina also declined and histological deterioration of the retina was observed. In the present study, the blood vitamin A content of the vitamin A deficient rats declined rapidly after 3 weeks of vitamin A deficiency when the f i r s t anatomical changes i n the retina were detected. In this study, although vitamin A content of the l i v e r was not analysed,. the fact that the blood vitamin A began to decline after the 3rd week c f vitamin A deprivation indicates that depletion of l i v e r stores of vitamin A should have occurred before this time according to Da-ling and Wald (1958). The retinas of the experimental animals i n this study began to degenerate about a month ea r l i e r after introduction of the vitamin A deficient diet than the rats observed by Dowling and Wald (1958). Since i n the early stages of this study only a few lamellar discs i n the d i s t a l portions of the photoreceptor outer segments were noted to be affected, i t i s possible that such changes were wer locked by Dowling and Wald. Another p o s s i b i l i t y i s that the room i n which the rats were kept i n Darling and Wald's study was not as brightly lighted or lighted for as long a time as i n the present study. Unfortunately, the lighting ccnditicns were not specified i n the reports of Dowling and Wald (1958). The vitamin A deficient animals i n the present investigation began to show a slower weight gain than the control animals at about the same.: time that their blood vitamin A levels began to decline. This finding implies that the function of vitamin A i n the body was only p a r t i a l l y f u l f i l l e d by the vitamin A acid which supplemented the vitamin A free diet. 135 C) The Photoreceptors i n Vitamin A Deficiency 1. The Outer Segments Tansley (1933,1335) and Johnson (1939, 1943), by l i g h t microscopy, noted that after young rats had been placed on a vitamin A free diet for 7-13 weeks, many photoreceptor outer segments disappeared and those that remained stained abnormally. Dowling and Gibbons (1961), too, reported that when young rats were raised on a vitamin A free diet supple- mented with vitamin A acid the photoreceptor outer segments stained less intensely than normal and appeared fragile and broken after 2 months on the diet. Only fragments of the outer segments remained after 6 months on the diet and they ccnpletely disappeared by 10 months. Dowling and Gibbons (1961), by electron microscopy, observed that degeneration began with a marked swelling of the highly ordered transverse discs of the outer segment which pirjched o f f to form large vesicles and tubules. After a high proportion of the discs had degenerated i n this way, the outer segments lo s t their normal elongated, cylindrical shape and became almost spherical. Most of the interior of the spherical outer segments was f i l l e d with distended vesicles and tubules. Fragments of the outer seg- ments were frequently observed lying free i n the space between the retina and the pigment epithelium. In the present study, morphological changes observed by l i g h t microscopy i n the outer segments cf the cegenerating photoreceptors were similar to those described by Tansley (1936), Johnson (1939, 1943) and Dowling and Gibbons (1961). However, the fine structural changes of the photoreceptor outer segments were studied i n greater detail i n this work and a number of new details of the degenerative process have been uncovered. I t was 136 noted that the breakdown of the lamellar discs into vesicles began from the d i s t a l portions of the photoreceptor outer segments and progressed towards the inner segments. There, v/as same variation i n the degree of outer segment involvement from one animal to another which may be related to differences i n vii^min A stores i n the l i v e r at the time the animals were placed on the special diet. The basal portions of the outer segments were always observed to contain some normal looking lamellar discs during the early stage of degeneration. Radioautographic studies of protein synthesis i n rod photoreceptors by Droz (1963) i n rat and mouse, Young (1967) i n rat, mouse and frog and Young and Droz (1968) i n frog have indicated that the labelled amino acid are concentrated i n i t i a l l y i n the inner segment of the c e l l . Within 24 hours, the radioactive material i s displaced to the base of the outer segment, where i t accumulates as a d i s t i n c t reaction band. The reaction band than gradually moves d i s t a l l y along the outer segment and ultimately disappears at the apex of the c e l l to reappear later i n phagosomes i n the pigment epithelium. This finding indicates that the protein component of the rod photoreceptor outer segment i s continually renewed by repeated apposition of material at the base of the outer segment i n conjunction with a balanced removal of material at the apex. From this work i t seems l i k e l y that the normal looking discs observed at the bases of the degenerating outer segments i n the present study were newly formed. Dowling and Wald (1958) and Dowling and Gibbons (1961) have suggested that i n vitamin A deficiency the cause of the breakdown of the lamellar discs i n the outer segments i s due to a loss of opsin i n the absence of vitamin A aldehyde. Since opsin i s stabilized by combining with vitamin A to form rhodopsin (Radding and Wald, 1956; Hubbard, 1958), opsin may 137 deteriorate i n prolonged vitamin A deficiency when there i s no vitamin A available for i t to combine with. In the present study, incorporation 3 of K -methionine into the inner segments was found to continue i n vitamin A deficiency indicating that there i s s t i l l protein synthesis i n the inner segments of the degenerating photoreceptors.. In the vitamin A. deficient animals, i n this study, the radioactive material was observed to concentrate at the junction of the inner and the outer segments and also at the bases of the outer segments 24 hours after labelling. Presumably, opsin was synthesized, but was not u t i l i z e d i n the absence of vitamin A aldehyde. Therefore, the disc membranes broke down i n spite of the fact that protein synthesis continued i n the inner segments. This finding supports the hypothesis of Dowling and Gibbons (1961), that vitamin A aldehyde, besides i n i t i a t i n g visual excitation, also functions i n maintaining the structural integrity of the disc membranes. Another p o s s i b i l i t y i s that the protein synthesized i n the inner segment i s abnormal or decreased and this w i l l be considered later. 2. The. Inner Segments, Synaptic Processes and Photoreceptor Nuclei Dowling and Gibbons (1961) have observed that the photoreceptor inner segments become greatly reduced i n number and shorter and thicker than normal after the animals have been on the vitamin A free diet for 6 months. However, no changes were noted by these authors i n the fine structure of the components of the inner segments such as the mitochondria, the cytoplasmic granules, and the cytoplasmic membranes. Changes ocoirring in the photoreceptor synaptic processes as a result of vitamin A deficiency 130 have not been previously studied. The results of the present study i n - dicate that, i n contrast to the results of the prior study by Dowling and Gibbons (1961), the photoreceptor inner segments show marked changes during and after the degeneration of the outer segments. The d i s t a l portions of the inner segments become sl i g h t l y swollen and contain only a few polysomes and some large vacuoles after 2.5 months of vitamin A deficiency. later, the inner segments gradually loose their elongated, cy l i n d r i c a l structure and become short and barrel-shaped as they retract towards the nuclei. In the inner segment cytoplasm, the mitochondria also gradually shorten and decrease i n number, the polysomes gather mainly i n the proximal halves and the Golgi complexes become i l l - d e f i n e d . The synaptic processes, also, show retraction towards the nuclei, account- ing for the tMnning of the outer plexiform layer observed by l i g h t microscopy. Each of the shortened synaptic processes contains only a few synaptic vesicles and the plasma membranes between adjacent synaptic processes form gaps. The membranes of the mitochondria both i n the inner segments and the synaptic processes show evidence of degeneration. what causes death of the photoreceptors i n the vitamin A deficient animals? Dowling and Wald (1960) have suggested that disintegration of photoreceptor outer segments i n vitamin A deficiency deprives the photoreceptor c e l l s of stimulation from photochemical excitation. This lack of stimulation might then cause the death of the visual c e l l s . Hans son (1970) has suggested that photoreceptor death i s due to an i n - correct synthesis of protein or other elements necessary for the continuous renewal of the inner and outer segments. In the present study, i t was observed that, prior to photoreceptor c e l l death, the inner segments and the synaptic processes underwent a continuous retraction while the outer segments were gradually disintegrating. Most of the photoreceptors then 139 disappeared l e a v i n g behind o n l y a s i n g l e l a y e r i n which the c e l l s had l o s t most o f t h e i r s u b c e l l u l a r s t r u c t u r e s . I would l i k e t o suggest t h a t the u n d e r l y i n g mechanism i n v o l v e d i n the phenomena operates a t the gene l e v e l . During v i t a m i n A d e p r i v a t i o n , the photoreceptor c e l l s c o u l d be a f f e c t e d somewhere along t h i s pathway: _._ t r a n s c r i p t i o n . -.^ t r a n s l a t i o n v . . DNA £ > mRNA > p r o t e i n where DNA = De o x y r i b o n u c l e i c a c i d mRNA = Messenger r i b o n u c l e i c a c i d Presumably some genes c o u l d be repressed i n v i t a m i n A d e f i c i e n c y so t h a t a l e s s e r amount o f p r o t e i n o r a d i f f e r e n t p r o t e i n i s s y n t h e s i z e d i n t h e degenerating photoreceptors. Data i n support o f t h i s suggestion have been p r o v i d e d by T r y f i a t e s and Krause (1971) who have shown t h a t an a l t e r e d messenger RNA i s s y n t h e s i z e d i n the l i v e r o f t h e v i t a m i n A d e f i c i e n t r a t . They have suggested t h a t v i t a m i n A e x e r t s a c o n t r o l over c e l l u l a r d i f f e r e n t i a t i o n a t the t r a n s c r i p t i o n l e v e l by i n f l u e n c i n g t h e s p e c i e s o f mRNA sy n t h e s i z e d . Johnson e t a l (1969) r e p o r t e d t h a t i n r a t i n t e s t i n a l mucosa and l i v e r , RNA l a b e l l i n g i s i n c r e a s e d a f t e r v i t a m i n A i n j e c t i o n i n v i v o . Kleiriman e t a l (1971) found t h a t v i t a m i n A d e f i c i e n c y 3 l e a d s t o a decreased i n c o r p o r a t i o n o f b o t h u r i d i n e - H and o r o t i c a c i d - 5-C 1 4 i n t o RNA. The a c t i o n o f v i t a m i n A on gene a c t i v i t y has been f u r t h e r i m p l i c a t e d i n the f o l l o w i n g p a t h o l o g i c a l s t u d i e s . I n v i t a m i n A d e f i c i e n t r a t s , bundles o f k e r a t i n appear i n the c e l l s o f p a r o t i d duct and t r a c h e a r e s u l t i n g i n m e t a p l a s i a and h y p e r p l a s i a (Hayes e t a l . , 1970; Wong and Buck, 1971). I n the male r e p r o d u c t i v e t r a c t o f the v i t a m i n A d e f i c i e n t r a t , 140 spermatogenesis stops and the semiferous epithelium degenerates (Thompson et a l , 1964).. In connective tissues of lung, bone and dura mater, the synthesis and/or turnover of collagen and mucopolysaccharide ground substances appears to be disturbed i n vitamin A deficiency result- ing i n measurable excesses of these components. These excesses are thought to be involved i n the thickening of the dura mater and the associated impaired reabsorption of cerebral spinal f l u i d noted i n vitamin A deficiency (Cousins et a l , 1969). They may also account for excessive growth i n periosteal bone also noted i n t h i s condition (Hayes and Cousin, 1970). The above pathological alteration imply that the result of vitamin A deficiency i s to l i m i t differentiation of recently divided c e l l s towards a favoured pathway so that, for example, i n spermatogenesis, the unidirectional differentiation of spermatozoa i s blocked resulting i n testicular degeneration (Hayes, 1971). Similarly i n the studies of parotid duct (Hayes et a l . , 1970) and trachea (Wong and Buck, 1971), the bipotent ep i t h e l i a l basal c e l l i n these organs i s restricted i n vii^min A deficiency to synthesis of fibrous, keratinizing proteins at the expense of mucous c e l l production (Hayes, 1971). In bone and collagen, the periosteal progenitor c e l l and the fibroblast favour the synthesis of collagen fibers and ground substance i n vitamin A deficiency (Hayes, 1971). In the present investigation, the degenerating photoreceptors were 3 capable of protein synthesis as evidenced by the incorporation of H - methionine i n the inner segments. Although the protein synthesized has not been analysed quantitatively or qualitatively, i t has been shown i n radioautographic studies, that production of photoreceptor outer segment material i s decreased i n vitamin A deficient rats (Herron and Riegel, 1974a, 1974b). The reduced amount of protein synthesized may have been i n an altered form, as suggested by Hansson (1970), and could not be used by the visual c e l l s to maintain their normal structure. The visual c e l l s , therefore, began to dedifferentiate along a "retrograde" pathway manifested by retraction of photoreceptor processes and the degeneration of subcellular structures observed i n the present study. Most of the photoreceptor remnants were then discarded by an unknown mechanism and removed by the r e t i n a l epithelium. It has been suggested i n many studies that membranes are major sites of vitamin A alcohol (retinol) action, particularly lysosomal and mitochondrial membranes. Roels et a l . (1969) reported that i n l i v e r of vitamin A deficient rats, lysosomal membranes were more l a b i l e . The membranes of erythrocytes obtained from vitamin A deficient rats were found to be markedly swollen and distorted and subject to faster hemolysis compared to those from control animals (Anderson et a l . , 1967). In e p i t h e l i a l c e l l s of the bulbourethral glands of vitamin A deficient rats, Latalski (1972) observed a great reduction i n the number of cristae of the mitochondria present i n the c e l l cytoplasm. He suggested such changes were due to a disturbance i n permeability and s t a b i l i t y of the lipoprotein membrane of the involved mitochondria. A similar type of disturbance was observed i n the mitochondria of degenerating photoreceptor inner segments and the synaptic processes i n the present study. Moreover, plasma membranes and nuclear membranes of the photoreceptors too, showed evidence of breakdown i n the present study. Roels et a l (1969) have suggested that vitamin A alcohol regulates the binding of the membrane bound ATPase responsible for lipoprotein interaction i n s p e c i f i c areas of certain biological membranes and thereby influences their structure and s t a b i l i t y . I t i s possible therefore that i f vitamin A alcohol i s also 142 present i n the mitccnor-drial, nuclear and plasma membranes of the visual c e l l , prolonged vitamin A deficiency would deplete vitamin A i n these membranes leading to membrane breakdown. . In the vitamin A deficient animals of this study the degenerating photoreceptors were discarded so rapidly that only one irregular row was l e f t by the 11th month. The nuclei of the remaining photoreceptors appeared normal, yet the other parts of the c e l l s such as the outer and inner segments and synaptic processes had disappeared. Whether the nuclear chromatin or gene structures had been altered qualitatively i s not known. Further studies would be necessary to examine t h i s p o s s i b i l i t y . D) The Retinal Epithelium i n Vitamin A Deficiency According to Dowling and Gibbons (1961) the r e t i n a l epithelium of the albino rat remained unchanged after 10 months of vitamin A deprivation. Hansson (1970) observed by scanning electron microscopy that after 6 months of vitamin A deficiency, the apical processes of the r e t i n a l epithelium were thicker and shorter than normal. The results of the present study indicate clearly that marked changes do occur i n the structure of the retinal epithelium of the vitamin A deficient rat. The most notable change was a large increase i n small dark granules with the ultrastructural characteristics of lysosomes beneath the inner e p i t h e l i a l surface after 4-5 months of vitamin A deprivation. A positive reaction for acid phosphatase confirmed that the granules were lysosomes. The lysosomes were sometimes observed near the Golgi apparatus and appeared to stream towards the epithelial inner surface. A positive reaction for acid phosphatase was also present i n the Golgi apparatus, and phagosomes of the e p i t h e l i a l c e l l . This finding tends to confirm previous work that primary lysosomes are formed i n the Golgi apparatus (DeDuve and Wattiaux, 1 9 6 6 ) and that phagosomes are degraded i n the retinal epithelium by the hydrolytic enzymes of the lysosomes (Ishikawa and Yamada, 1 9 7 0 ) . In the present study, i t was found that the increase of lysosomes persisted throughout the photoreceptor degeneration process. The apical processes of the r e t i n a l epithelium proliferated s l i g h t l y at f i r s t and later became very prcminent. These changes were not observed in the retinal epithelium of control animals of the same age. The physiological significance of the close structural relation of the pigment epithelium to the photoreceptors was f i r s t noted by Kuhne ( 1 8 7 8 ) who found that i n v i t r o regeneration of visual purple took place only i f pigment epithelium remained i n contact with the photoreceptors. In recent years, much more has been learned of this intimate relationship between the pigment epithelium and the photoreceptors. For example, experiments u t i l i z i n g horseradish peroxidase as a diffusion tracer have pointed out the importance of the pigment epithelium as a selective f i l t e r , preventing intercellular diffusion of larger molecules from the choriocapillaries into the retina. (Bok and Young, 1 9 7 2 ) . In the frog, i t has been shown that radioactive vitamin A i s rapidly taken into the o i l droplets of the retinal e p i t h e l i a l c e l l s within a few minutes after intravenous injection (Bok and Young, 1 9 7 2 ) . Glycerol and palmitic acid are also avidly taken up from the blood stream and concentrated i n the o i l droplet as well (Bok and Young, 1 9 7 2 ) , The mucopolysaccharides of the interphotoreceptor matrix are believed to be synthesized within the pigment epith e l i a l c e l l s and later secreted extracellularly by the well developed Golgi system of these c e l l s (Berman, 196*+). The enzyme isomerase, responsible for isomerizing vitamin A to the 1 1 - c i s configuration, has 144 been identified i n both rod outer segments and the retinal pigment epithelium i n amphibia (Hubbard, 1956). The pigment epithelium also acts as a scavenger, as i t normally phagocytoses and then destroys packets of outer segment discs. This phagocytic function of the retinal epithelium has been well studied by many investigators (Cowling and Gibbons, 1962; Ba i r a t i and Orzalesi, 1963; Ishikawa and Yamada, 1970; Young, 1967, 1971a; Young and Bok, 1969; Spitznas and Hogan, 1970). In the present study, i n vitamin A deficiency, the apical processes of the retinal epithelium appeared to be more active i n engulfing photoreceptor fragments than i n the control animal. Displaced photo- receptor nuclei were often observed unusually close to the retinal epithelium i n vii^rnin A deficient animals and i t seems l i k e l y that a l l parts of the photoreceptor c e l l including the nuclei are phagocytosed by the pigment epithelium i n the vitamin A deficient rats. The presence of the large number of lysosomes beneath the inner epithelial; surface suggests that the photoreceptor fragments were degraded by the lysosomes as soon as they were phagocytosed. This may explain why i n this study portions of photoreceptor c e l l s aside from outer segment fragments were not seen within the r e t i n a l epithelial cytoplasm. The fact that i n this study the time of proliferation of lysosomal activity i n the retinal epithelium corresponded to the time of breakdo/m of the photoreceptors strongly supports a cause and effect relationship. E) Muller Cells i n Vitamin A Deficiency Muller c e l l s , the most prominent nonnervous elements situated among the retinal neurons, are thought to provide mechanical support and physiological insulation for intervening nerve ce l l s (Polyak, 1957). 14b Fore r e c e n t l y , they have been shown t o be the main s i t e s o f carbohydrate storage i n the r e t i n a (Kuwabara and Cogan, 1961; Magalhaes and Coimbra, 1970). They a l s o o f course are capable o f s y n t h e s i z i n g p r o t e i n (Hodson and M a r s h a l l , 1967). I t has been noted, i n a d d i t i o n , t h a t glycogen s y n t h e s i s (Magalhaes and Coimbra, 1970), as w e l l as c e r t a i n enzymatic a c t i v i t i e s (Cogan and Kuwabara, 1959; Kuwabara and Cogan, I960; L e s s e l l and Kuwabara, 1964) p r e v a i l a t d i f f e r e n t s i t e s i n the M u l l e r c e l l . T h i s suggests t h a t the c e l l might p l a y d i f f e r e n t f u n c t i o n a l r o l e s a t d i f f e r e n t l e v e l s o f the r e t i n a . I t has been p o i n t e d o ut i n s e v e r a l animal sp e c i e s t h a t t h e d i s t r i b u t i o n o f o r g a n e l l e s i n M u l l e r c e l l cytoplasm d i f f e r s as one moves from t h e i n n e r end o f t h e c e l l a t the i n n e r l i m i t i n g membrane, t o t h e ou t e r end a t the o u t e r l i m i t i n g membrane. F o r example, v e s i c l e s and f i b r i l s have been observed i n the cytoplasm o f t h e i n n e r end o f t h e c e l l i n r e p t i l e s (Pedler, 1963), c a t (ladman, 1961) and man (Hogan and Feeney, 1963). Mi t o c h o n d r i a have been found i n the o u t e r end o f the c e l l i n r e p t i l e s (Pedler, 1963) and the r a b b i t (Sjostrand and N i l s s o n , 1964), but i n the i n n e r p o r t i o n , i n man (Fine, 1961). Magalhaes and Coimbra (1972) have suggested t h a t the M u l l e r c e l l o f the r a b b i t can be d i v i d e d i n t o 3 p o r t i o n s , each c h a r a c t e r i z e d by c e r t a i n u l t r a s t r u c t u r a l f e a t u r e s , p o s s i b l y having d i s t i n c t f u n c t i o n s . The i n n e r p o r t i o n i s c h a r a c t e r i z e d by a g r e a t d e n s i t y o f glycogen p a r t i c l e s and m i c r o f i l a m e n t s , a network o f smooth endoplasmic r e t i c u l u m and the presence o f some dense bodies. A h i g h r a t e o f glycogen s y n t h e s i s i s present i n t h i s p o r t i o n (Magalhaes and Coimbra, 1970) although the smooth r e t i c u l u m i s b e l i e v e d t o be the s i t e o f n e a r l y a l l glucose-6-phosphate a c t i v i t y i n the r e t i n a (Magalhaes and Coimbra, 1972). The middle p o r t i o n o f the 14b Muller c e l l i s marked by the presence of ergastoplasm and Golgi complexes, suggesting protein synthesis (Magalhaes and Coimbra, 1972). The Muller c e l l outer portion seems well adapted to absorption and intracellular transport. An absorptive function i s suggested by the presence of long m i c r o v i l l i and microtubules while the presence of mitochondria i n this part of the c e l l suggest that active intracellular transport i s taking place (Magalhaes and Coimbra, 1972). In addition, the outer portion of the Muller c e l l s may play a role i n the pathological condition of photoreceptor cJestructicn as shown i n the present investigation. As degeneration of the photoreceptors i n the vitamin A deficient' animals continued, the terminal processes of the Muller c e l l s became highly conspicuous. In late stages of degeneration (11 months of vii^min A deficiency), the remaining photoreceptors and other unidentified c e l l s of the inner nuclear layer were each surrounded by several layers of membranes probably of a g l i a l nature. The terminal processes of the Muller c e l l s v/ere frequently observed slanting l a t e r a l l y and probably contributing to g l i a l membrane formation and encirclement of the reri^ining outer neurones. I t i s possible that these membranes serve as barriers preventing the remaining photoreceptor nuclei from being phagocytosed by the retinal epithelium. In this study, by light microscopy, the ce l l s of the inner re t i n a l layers appeared unaffected even after a l l the photoreceptors had disap- peared. These c e l l s were not examined i n detail by electron microscopy. The po s s i b i l i t y of alterations i n the inner retinal c e l l s i n vitamin A deficiency therefore, remains to be investigated. 1 4 7 F) Light Damage to Photoreceptors I t i s of interest that animals that have been exposed to continuous illumination show a similar degeneration or destruction of the photo- receptors (Noell, 1966; Noell et a l , 1966; Grignolo, 1969; Weale, 1969; Tso, 1973; Lawwill, 1973) to that observed i n vitamin A deficiency. Kuwabara and Corn (1968) have shown that the photoreceptor outer segments of the albino rat demonstrate severe membranous changes upon exposure to continuous relatively cold l i g h t at a brightness of about 750 foot- candles. In their studies, the l a m e l l a r discs at the outermost tips of the photoreceptors broke down and formed vesicles after 1 hour of exposure to light. When the retinas of the rat were exposed to l i g h t at that intensity for periods from 6 to 24 hours, the discs i n the outer portions of the segments lost their regular lamellar structure and became markedly separated and vacuolated. When the animals were exposed for more than two days, the discs broke down into tubules and became irregularly packed within large, round, or pear-shaped outer segments. Later, the swollen outer segments became separated from the inner segments. The isolated outer segments were then found among the apical m i c r o v i l l i of the pigment epithelium and gradually broke down into smaller sizes. Kuwabara and Corn (1968) thought that the membranous changes were a type of c e l l u l a r reaction of the photo- receptors to extreme bleaching circumstances. Continuous exposure to the l i g h t appeared to overbalance the bleaching and recovery mechanisms of the photoreceptor c e l l s . Since bleaching of the visual pigments causes a loss of some vitamin A (Wald, 1955), prolonged illumination would cause excessive loss of this prosthetic group from rhodopsin. Therefore, rhodopsin cannot be generated to restore the 148 structural integrity of the lamellar discs and consequently the la t t e r break down in a similar manner to that observed i n vitamin A deficiency. Again the stabilizing effect of vitamin A aldehyde on the structure of the outer segment discs i s implicated. Recently, Shear et a l (1973) have shown that retinas of albino rats exposed to continuous low intensity of fluorescent l i g h t (18 foot- candles) for periods of 6 to 18 hours display fine structural changes i n both the r e t i n a l e p i t h e l i a l c e l l s and the outer segments of the photoreceptor c e l l s . Adjacent e p i t h e l i a l c e l l s were separated by wide gaps and suffered retraction of the apical processes. The lamellar discs of the photoreceptor outer segments became progres- sively more tubular. These changes were reversible i n an environment of c y c l i c l i g h t (14 hours of ligh t and 10 hours of darkness). In the present study, the vitamin A deficient animals and their controls were kept under lighting conditions of a maximum of 10 foot-candles during the 12 hours of l i g h t per day. Since the vitamin A deficient animals and their controls were always k i l l e d after less than 1 1/2 hours of l i g h t adaptation at an intensity of 25 foot-candles, i t seems unlikely that the structural changes observed i n this study i n the retinas of the vitamin A deficient animals were influenced by the lighting conditions. Also the control animals in this study,which were kept under identical lighting conditions did not show significant morphological changes with aging i n either photoreceptor or r e t i n a l e p i t h e l i a l c e l l s . G) Normal Loss of Photoreceptors In control specimens of the present study, a very small number of photoreceptor cells, consisting of prominent nuclei surrounded by a narrow rim of cytoplasm -were observed lying outside the outer limiting membrane between the retinal epithelium and the neural retina. Photo- receptor nuclei were also observed p a r t i a l l y embedded i n the retinal epithelium. These findings suggest that under the lighting conditions of the present study some photoreceptor nuclei normally make their way towards the r e t i n a l epithelium where they are eventually phago- cytosed and degraded. Thus, throughout l i f e i n the rat, there may be a continual loss of photoreceptor c e l l s . Apart from the work on l i g h t damage, visual c e l l loss of this sort, to my knowledge, has not been described i n the retina of any vertebrate studied to date. The significance of this process, which may be a normal aging change, i s s t i l l obscure. H) Glycogen F i l l e d Mitochondria In this study, after 4-5 months of vitamin A deprivation degenerating inner segments of an unusual type were occasionally observed. The cytoplasm of these inner segments became very dense and contained no v i s i b l e subcellular structures except mitochondria (Fig. 41, 42). The mitodnondria were greatly enlarged by an accumulation of glycogen particles within their outer membranes. Degenerating inner segments of this type were also observed i n the control animals. Glycogen f i l l e d mitochondria have been reported to be present i n rod ellipsoids of toads (Ishikawa and Yamada, 1969), rats (Ishikawa and Pei, 1965), in the f l i g h t muscles of aging blow f l i e s (Sacktor and Shimada, 1972) and" i n the heart muscles of aging drosophila (Sohol, 1970). The presence of intramitochondrial glycogen i s believed to be related to aging since mitochondria of this type have never been reported to exist i n retinas of the newborn or young rats (Ishikawa and Pei, 1965). However, they were easily detected i n retinas of rats 1 year or older (Ishikawa and Pei, 1965). The present results confirm this finding since riutochcndria with enclosed glycogen were not found i n rats under 5 months of age. This type of degenerating inner segment with dense cytoplasm and glycogen f i l l e d mitochondria may be due to aging and not vitamin A deficiency since, inner segments of this type were found i n both the older vitamin A deficient and control animals. 151 VI SUI-2-iARY V7eanling rats, on a vitamin A free diet, supplemented with \dtamin A acid, gained weight more slowly than litter-mates fed a normal diet. The plasma vitamin A content of the experimental animals declined rapidly after they had been on the vitamin A free d i e t f o r 3 weeks. At t h i s time, the h i s t o l o g i c a l deterioration of the photoreceptors commenced. * . Light microscopic study shaved that i n the vitamin A deficient - rats the photoreceptor outer segments f i r s t degenerated, then the photoreceptor inner segments, the synaptic processes and the photoreceptor nuclei. In the. degenerating retinas of the vitamin A deficient ranimals, discarded photoreceptor nuclei were often observed displaced s c l e r a l l y lying clcse to the r e t i n a l epithelium. This phenomenon also occurred i n retinas of older control animals. Electron microscopic study showed that the lamellar discs i n the d i s t a l ends of the photoreceptor outer segments were the f i r s t to break down into vesicles and tubules after the rats were on the vitamin A free diet for 1 month. By 2.5 months of \dtarrin A deficiency, the d i s t a l ends of the photoreceptor inner segments became swollen and t h e i r polysome and mitochondrial content had greatly decreased. Later, the normally elongated and c y l i n d r i c a l inner segments retracted 152 towards the photoreceptor n u c l e i and became s h o r t and barrel-shaped. 7. By 6 months o f v i t a m i n A d e f i c i e n c y , the photoreceptor o u t e r segments had l o s t t h e i r normal, r e g u l a r o r i e n t a t i o n . Few o f them appeared i n t a c t . 8. By 6 months, the photoreceptor i n n e r segments were shortened t o d i f f e r e n t degrees. I n the cytoplasm o f the i n n e r segments, polysomes c o u l d be found o n l y i n the p r o x i m a l p o r t i o n , next t o t h e photoreceptor n u c l e i . The few mitochondria t h a t remained were o f t e n s h o r t e r than normal and the G o l g i complexes present showed s i g n s o f d i s i n t e g r a t i o n . 9. A l s o a t t h i s 6 month stage, the photoreceptor s y n a p t i c t e r m i n a l s were s e v e r e l y a f f e c t e d i n the animals on the s p e c i a l d i e t . Fewer s y n a p t i c v e s i c l e s were present, o f t e n l y i n g immediately adjacent t o s y n a p t i c s i t e s . The s y n a p t i c ribbons p e r s i s t e d b u t l a r g e gaps appeared i n t h e plasma membranes o f the s y n a p t i c processes. W i t h i n the s y n a p t i c processes, d i s p l a c e d m i t o c h o n d r i a l membranes and l o s s o f m i t o c h o n d r i a l c r i s t a e were apparent. 10. By 9 months o f v i t a m i n A d e f i c i e n c y , the photoreceptor o u t e r segments had completely disappeared except f o r a few s p o r a d i c c l u s t e r s o f s a c c u l e s . 11. By 9 months, a l l the photoreceptor i n n e r segments had r e t r a c t e d c o n s i d e r a b l y . The shortened i n n e r segments s t i l l c o ntained some polysomes and rough endoplasmic r e t i c u l u m , b u t the m i t o c h o n d r i a had e n t i r e l y disappeared. 1 5 5 12. By 9 months, the nuclear envelopes showed evidence of d i s - integration although the photoreceptor nuclear chromatin appeared unchanged. 13. Also at this 9 month stage, the synaptic processes of the degenerat- ing photoreceptors were very short and contained only a few synaptic vesicles. Synaptic ribbons could s t i l l be identified i n some synaptic processes. 14. By 11 months of vitamin A deficiency, only one irregular row of photoreceptors remained. The remaining neural retina adhered closely to the retinal epithelium. 15. At this 11 month stage, the degenerating photoreceptors consisted only of a nucleus surrounded by a narrow rim of cytoplasm containing very few organelles. The photoreceptors were surrounded by several layers of membranes probably of g l i a l origin. Synapses of the photoreceptors with nerve c e l l s of the inner nuclear layer and the concomitant synaptic ribbons had disappeared. 16. By 11 months of vitamin A deficiency the normal c e l l attachment between photoreceptors and Muller c e l l s i n the region of the outer limiting membrane had disappeared. The outer limiting membrane was now formed by c e l l junctions between adjacent Muller c e l l s . At this stage, the terminal processes of the Muller c e l l s above the outer limiting membrane were seen to bend l a t e r a l l y , possibly contributing to the membrane layers surrounding the photoreceptor remnants. ±54 17. Fadioautographic studies of HJ-metliionina incorporation into the retinas of control and vitamin A deficient animals revealed that both control and degenerating photoreceptors were capable of protein synthesis. It i s postulated that this protein might be qualitatively different i n the vitamin A deficient animals than the protein synthesized by normal photoreceptors. 18. From the detailed studies of the structural degeneration of the photoreceptors i n vitamin A deficiency, i t i s suggested that i n vision, vitamin A, besides i n i t i a t i n g visual excitation, also functions i n rraintaining the structural integrity of disc and c e l l membranes. I t i s also suggested that vitamin A probably acts at the gene level since i t s absence caused the photoreceptors to dedifferentiate along a "retrograde" pathway. 19. In the retinal epithelium, an increase of lysosomes close to the inner surface was noted by 4-5 months of vitamin A deficiency. The lysosomes were acid phosphatase positive. The increase of lysosomes i n the retinal epithelium persisted as long as degeneration of the photoreceptors continued. Only a few lysosomes were observed in the inner epithelium of control animals. 20. The apical processes of the retinal epithelium proliferated s l i g h t l y by 2.5 months of vitamin A deficiency and became very numerous and prominent by 11 months of vitamin A deficiency. 21. In the vitamin A deficient animals, the changes observed i n the retinal epithelia as a result of photoreceptor breakdown strongly supported a cause and effect relationship. The morphology of photoreceptors and retinal e p i t h e l i a l c e l l s studied i n control rats from ages 1.5 to 12 months remained consistently unchanged, except for the appearance of glycogen f i l l e d mitochondria i n the photoreceptor inner segments of older animals. V I I O r i g i n a l C o n t r i b u t i o n s I n t h i s i n v e s t i g a t i o n a d e t a i l e d study has been made o f t h e m o r p h o l o g i c a l changes which o c c u r i n t h e o u t e r r e t i n a ( p r i m a r i l y r e t i n a l e p i t h e l i a l and p h o t o r e c e p t o r c e l l s ) as a r e s u l t o f d i e t a r y v i t a m i n A d e f i c i e n c y . Only a few p r i o r s t u d i e s o f t h i s phenomenon have been made (Tansley, 1933, 1936; Johnson, 1939, 1943; Dewing and Wald, 1958, 1960; Dowling and Gibbons, 1961; Dowling, 1966) and t h e s e a r e d e f i c i e n t i n the f o l l o w i n g r e s p e c t s . 1. The l i g h t i n g c o n d i t i o n s have n o t been c a r e f u l l y c o n t r o l l e d . (Tansley, 1933, 1936; Johnson, 1939, 1943; Dowling and Wald, 1958, 1960; Dowling and Gibbons, 1961; Dowling 1966). I t has now been e s t a b l i s h e d t h a t a l o n g p e r i o d o f exposure t o l i g h t by i t s e l f can produce p h o t o r e c e p t o r damage ( I v o e l l , 1966; N c e l l e t a l . , 1966; Kuwabara and Gorn, 1968; G r i g n o l o , 1969; Weale, 1969; Tso, 1973; L a w w i l l , 1973; Shear e t a l . , 1973). 2. The m o r p h o l o g i c a l changes examined p r e v i o u s l y have c e n t e r e d on t h e p h o t o r e c e p t o r o u t e r segments o n l y . B e f o r e t h e p r e s e n t s t u d y l i t t l e was known o f t h e s e q u e n t i a l changes produced by v i t a m i n A d e f i c i e n c y i n th e p h o t o r e c e p t o r i n n e r segments, s y n a p t i c processes and n u c l e i . P r i o r t o t h i s study i t was assumed t h a t t h e pigment e p i t h e l i u m was u n a f f e c t e d i n v i t a m i n A d e f i c i e n c y (Dowling and Gibbons, 1961). 3. The p r i o r s t u d i e s have f a i l e d t o t a k e i n t o account t h e f a c t t h a t t h e s t r u c t u r e o f t h e o u t e r r e t i n a may undergo s t r u c t u r a l changes i n normal animals d u r i n g a g i n g . T h i s p o s s i b i l i t y has been c a r e f u l l y examined i n the p r e s e n t r e p o r t . I n summary, the pre s e n t r e p o r t r e p r e s e n t s a much more d e t a i l e d and c a r e f u l l y c o n t r o l l e d study o t t h i s phenomenon than has been attempted p r e v i o u s l y . From t h i s s t a n d p o i n t i t can be s t a t e d t h a t a l l the f i n d i n g s 157 reported i n the thesis possess at least some degree of originality. The following findings reported i n the thesis have not, to my ' knowledge, been reported elsewhere under any experimental conditions. In the normal retina of the rat: 1. The fine structure of cone photoreceptor c e l l s . 2. The presence of photoreceptor nuclei displaced outside the external limiting membrane. 3. The complete absence of aging changes i n the photoreceptor and pigment e p i t h e l i a l c e l l s from 1.5 to 12 months of age except for the appearance of glycogen f i l l e d mitochondria i n the photoreceptor inner segment of older animals. In the vitamin A deficient rat: 1. A l l of the changes noted i n sequential fashion i n the r e t i n a l pigment epithelium including the accumulation of lysosomes and the alterations i n the morphology of the inner (apical) e p i t h e l i a l border. 2. The ultrastructural changes noted sequentially i n the photo- receptor inner segments, synaptic processes and nuclei. 3. The alterations noted i n the outer processes of the Muller c e l l s . The proliferation of g l i a l membranes around the degenerating photoreceptor nuclei. In terms of i t s significance this thesis represents another step along the road pioneered by Wald (1935a, 1936) and Dowling and Wald (1958, 1960) leading to a f u l l understanding of the role played by vitamin A i n vision and the etiology of night blindness. It has become clear from the present work that, i n the retina the effects of vitamin A deficiency are 158 more complex than had p r e v i o u s l y been thought. V i t a m i n A d e f i c i e n c y leads t o a d e s t r u c t i o n c f the photoreceptors i n a s e q u e n t i a l manner, beginning w i t h the outer segments, then a f f e c t i n g t he i n n e r segments and s y n a p t i c processes and f i n a l l y the photoreceptor n u c l e i . The changes i n the pigment e p i t h e l i u m appear t o be secondary t o , and induced by, the presence o f photoreceptor fragments a t the i n n e r e p i t h e l i a l border. I n t h i s case, the pigment e p i t h e l i a l c e l l s a c t l i k e t r u e macrophages, rerro\dng the photoreceptor d e b r i s . I f p resent t h e o r y i s c o r r e c t , and v i t a m i n A i s normally present i n q u a n t i t y i n the photoreceptor o u t e r segments, then i t i s easy t o see why the o u t e r segments degenerate i n v i t a m i n A d e f i c i e n c y . I t i s much more d i f f i c u l t , however, t o understand why subsequently the r e s t o f t h e photoreceptor c e l l ; d i e s p a r t i c u l a r l y when one remembers t h a t t h i s p a r t of the c e l l i s not thought t o c o n t a i n more than minute amounts o f v i t a m i n A under normal circumstances (2-5oore, 1964) . The present r e s u l t s do not p r o v i d e a d e f i n i t i v e answer t o t h i s q u e s t i o n . 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