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Ultrastructural permeability of the murine muscle spindle capsular perineurium to electron microscopically… Dow, Pierre Roger 1978

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ULTRASTRUCT'URAL PERMEABILITY OF THE MURINE MUSCLE SPINDLE CAPSULAR PERINEURIUM TO ELECTRON MICROSCOPICALLY DEMONSTRABLE MACROMOLECULAR TRACERS by PIERRE ROGER DOW B . S c , University of Washington, 1948 D.D.Sc., University of Washington, 1952 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Department of ANATOMY) We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA June, 1978 © Pierre Roger Dow, 1978 In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree at i h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . It i s u n d e r s t o o d that c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f ANATOMY The U n i v e r s i t y o f B r i t i s h C o l u m b i a 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date 27 June. 1978 i i ABSTRACT The muscle spindle i s invested by a capsule of continuous, multilayered perineural epithelium, creating a p e r i a x i a l space around the i n t r a f u s a l muscle fib r e s and t h e i r innervation. The u l t r a s t r u c t u r a l permeability of the muscle spindle capsule was assessed i n anterior t i b i a l i s muscles of adult mice after systemic i n j e c t i o n of the exogenous protein tracer, horseradish peroxidase (HRP). After varying time intervals ranging from 2 to 240 minutes, anesthetized animals were systemically perfused with f i x a t i v e . Muscles were then removed and processed by routine cytochemical methods i n order to demonstrate the d i s t r i b u t i o n of peroxidase a c t i v i t y . While bare c a p i l l a r i e s were never encountered within the intracapsular p e r i a x i a l space of the muscle spindle, they frequently and intimately abut against i t s outer perineural capsule. Two minutes a f t e r i n j e c t i o n of HRP, reaction product was l o c a l i z e d i n c a p i l l a r y endothelial plasmalemmal v e s i c l e s , j u s t p r i o r to i t s accumulation i n the tissue space immediately surrounding the subjacent capsular perineurium i n the equatorial region. In the polar region of the muscle spindle capsule, however, there was evidence of reaction product i n the p e r i a x i a l space at the same two minute time period. Ten minutes after administration of the tracer, a small population of perineural vesicles contained HRP. At the 12.5 minute i n t e r v a l , the sarcolemmae and T-tubules of polar i n t r a f u s a l muscle fibres were subsequently l a b e l l e d . Equatorial (sensory) regions of muscle spindles appeared to be r e l a t i v e l y less permeable to the i i i entrance of the tracer than more d i s t a l polar regions. By 15 minutes post-injection, HRP was seen traversing the perineural capsule i n equatorial zones; and by 30 minutes, perineural and Schwann c e l l phagocytosis of the exogenous protein was extensive. i v TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS iv LIST OF FIGURES v i ACKNOWLEDGEMENTS v i i INTRODUCTION 1 A. Morphology of the Muscle Spindle 1 B. The H i s t o r i c a l Background of the Understanding of the Muscle Spindle's Capsular Architecture 7 C. Scope and Aim of the Present Investigation 1 0 MATERIALS AND METHODS _ 1 2 A. General Procedures 1 2 1 . Choice of experimental animal 1 2 2 . Muscle selection 1 2 3 . Determination of muscle spindle location and d i s t r i b u t i o n i n the anterior t i b i a l i s muscle 1 2 4 . Choice of anesthetic 1 3 5 . Selection of tracer elements 1 4 6 . Fixation technique 1 6 B. Sp e c i f i c Procedures 1 6 1 . Experiments with lanthanum tracers 1 7 2. Experiments with f e r r i t i n 1 7 3 . Experiments with horseradish peroxidase 1 8 4 . Controls ' 1 9 V Table of Contents (Cont.) Page RESULTS 20 A. Control. Normal Morphology of the Capsule and i t s Blood Supply i n the Mouse Muscle Spindle 20 1. Polar region 20 2. Juxta-equatorial region 21 3. Equatorial region 21 B. Tracer Experiments 23 1. Lanthanum experiments 23 2. Horseradish peroxidase experiments 24 FIGURES 27 DISCUSSION •• 58 A. The Capsule and i t s Vasculature 58 B. Correlation of Capsular Morphology and Experimental Results 61 C. Conclusions 66 SUMMARY 67 BIBLIOGRAPHY 69 v i LIST OF FIGURES Figures Pages 1-5 Normal muscle spindle and capsule 27-31 6-8 The lanthanum experiments 33-37 9-21 The horseradish peroxidase experiments 39-58 v i i ACKNOWLEDGEMENTS To Dr. W.K. Ovalle, my supervisor and mentor during this course of study, I extend sincere thanks. His a b i l i t y to bridge the generation gap and provide e f f e c t i v e and needed guidance was greatly appreciated. The encouragement and sincere i n t e r e s t expressed by the other members of my committee, Dr. Sidney Friedman and Dr. Charles Slonecker, were a great factor i n i t s success. F i n a l l y , without the cheerful, chafing assistance and guidance of Ms. Susan Shinn, probably none of this would have come to pass. My thanks are also extended to the Department of Anatomy for providing an environment that encourages graduate study. 1 INTRODUCTION A. Morphology of the Muscle Spindle. The groups of l i t t l e muscle fibres now known as i n t r a f u s a l fibres were f i r s t discovered by Hassall i n 1851. The actual spindle i t s e l f , however, was not i d e n t i f i e d u n t i l ten years l a t e r . Kuhne gave i t the name Muskelspindeln i n describing i t s spindle shape, and this name has persisted to this day. Suggestions as to i t s function were put forth i n 1890 and 1894 (Onanoff; Kerschner). I t was not u n t i l several years l a t e r that the experiments of the English physiologist Sherrington, supported by the f u l l y - i l l u s t r a t e d observations of R u f f i n i , led to the general acceptance of the concept that muscle spindles are highly-organized sense organs. By 1930, several investigators had established the fact that t h i s complex neuromuscular sensory receptor possessed a primary and secondary afferent ending (B.H.C. Matthews). The demonstration of the existence of a motor supply came i n 1957 (Eyzaguirre; Gray) and placed t h i s neuroreceptor i n a class by i t s e l f . As P.B.C. Matthews pointed out i n 1964, the importance of mammalian muscle spindles may be judged by the number of nerve f i b r e s supplying them. In many muscles, the t o t a l number of afferent and efferent neural components greatly outnumber the ordinary alpha motor fibres supplying the extrafusal muscle. Inasmuch as the murine muscle spindle w i l l be extensively described l a t e r i n r e l a t i o n to the work done, s u f f i c e i t now to describe a normal mammalian spindle i n general terms. Located i n the perimysial connective tissue between the bundles of extrafusal muscle fib r e s i n s k e l e t a l muscle, one can fi n d at various points an encapsulated group of i n t r a f u s a l muscle fibres that stand out by the comparative smallness of t h e i r s i z e . Lying i n close proximity, nerve axon bundles, c a p i l l a r i e s and mast c e l l s are often seen. The size of the structure w i l l vary dependant on where the cross-section was taken. There are three generally-recognized p o s i t i o n a l points. Equatorial, meaning through'the widest portion at the centre; polar, that s l i c e taken close to the tapered extremities; and juxta-equatorial, being defined as that point half-way down the taper, p r i o r to the shape c h a r a c t e r i s t i c of the polar regions. Upon closer examination, i t w i l l be observed that the neural structures l y i n g close to the capsule are surrounded by the same sheath that comprises the capsule i t s e l f . The adjacent blood vascular components are usually found l y i n g on the capsular surface, or are seen as an i n t e g r a l part of the w a l l . Strangely enough, there i s no report i n the l i t e r a t u r e of c a p i l l a r i e s being found inside the capsule. The gross anatomy of the capsule can best be described by either of the two terms one constantly runs into when this receptor i s spoken of: spindle-shaped or fusiform. B a s i c a l l y these terms relate to a shape that i s c y l i n d r i c a l , wide i n the centre and tapering at both ends. This should make i t easier to understand the terms 'equatorial' and 'polar'. I t s o v e r a l l length i n humans i s about seven to ten mm (Barker, 1974). A feature of the equatorial region i s the distance that the sheath l i e s from and surrounding the centrally-located i n t r a f u s a l f i b r e s . This creates a p e r i - a x i a l space which appears to be f i l l e d with an 3 amorphous substance or f l u i d . Obviously, t h i s configuration i s absent i n the polar regions where the capsule closely encompasses the i n t r a f u s a l f i b r e s . Furthermore, i n i t s equatorial region, the capsule i s a double structure i n that i t has an outer and a separate inner portion. The l a t t e r l i e s close to the central fibres and does not extend i n polar directions as far as i t s outer counterpart. The i n t r a f u s a l muscle fib r e s l y i n g i n the centre of th i s spindle are of two d i s t i n c t l y d i f f e r e n t types. Although described at an early stage i n muscle spindle investigations, the i d e n t i f i c a t i o n of the two major forms wasn't done u n t i l recently. The nuclear bag and nuclear chain fi b r e s that we know today were i d e n t i f i e d by the work of Boyd i n 1962 and Cooper and Daniel i n 1963. The p o s s i b i l i t y of a t h i r d type was introduced by Ovalle and Smith i n 1972. The two main types are named after a description of t h e i r n u c l e i as observed i n the spindles' equatorial region. They are also d i f f e r e n t i n length. The nuclear bag fibres exhibit many rounded nuclei t i g h t l y packed i n t h e i r widest equatorial position. On each side -of this nuclear bag are the myotube regions containing muscle c e l l s with a single centrally-located nucleus. These f i b r e s have the widest diameter of the two, and are also the longest, often extending beyond the confines of the capsule i t s e l f . They appear to in s e r t into the perimysium adjacent to the extrafusal muscle f i b r e s . On the other hand, the nuclear chain f i b r e s appear exactly as named. The equatorial regions of the nuclear chain fi b r e s contain a single row of centrally-placed oval n u c l e i surrounded by a peripheral layer of myofibrils. They are shorter, often ending within the capsule, and smaller i n s i z e . One should not get the concept that a muscle spindle i s composed of a fixed number of i n t r a f u s a l f i b r e s per capsule. There i s a great v a r i a t i o n i n the number of nuclear bag to nuclear chain fibres per capsule as w e l l as the t o t a l number of fib r e s present. This relationship of quantity and type varies per muscle and species. In man, the lumbrical muscles contain spindles with one or two nuclear bag fibres and no or few nuclear chain fi b r e s (Cooper and Daniel, 1963). On the other hand, the same muscle may contain up to fourteen i n t r a f u s a l f i b r e s : three or four being nuclear bag and the rest being nuclear chain. This r a t i o of nuclear chain exceeding nuclear bag seems to be consistent. In describing the nerve supply of the muscle spindle, one has to divide i t into at least two types. As has been pointed out previously, the sophistication of this neuromuscular sensory receptor l i e s i n the fact that i t receives a motor input as w e l l as being a peripheral sensory afferent to the central nervous system. Further, the innervation which w i l l concern us mostly w i l l be that of the i n t r a f u s a l fibres within the body of the spindle. The vascular supply to the capsule i s probably involved with i t s own autonomic supply. Since the main axons to the i n t r a f u s a l fibres pierce the body of the capsule, i t i s d i f f i c u l t to assess any direct capsular innervation from these large f i b r e s . There i s some controversy as to the existence of autonomic innervation of the capsule i t s e l f . However, since Stacey i n 1967 and 1969 observed and reported Group I I I fi b r e s supplying extensive ramifications within the capsule w a l l of muscle spindles, i t i s a d e f i n i t e p o s s i b i l i t y . The largest nerve f i b r e to enter the spindle does so at the equator and i s the primary afferent to the i n t r a f u s a l muscle. Each spindle has one and apparently only one each of these Type l a axons. I t w i l l have an average diameter of 17 mm (Guyton, 1976) and a transmission velocity approaching one hundred meters per second. This i s as rapid as any sensory nerve i n the body. The f i r s t subdivision of this axon after piercing the capsule i s usually dichotomous and generally takes place within the p e r i a x i a l space p r i o r to contacting the i n t r a f u s a l f i b r e . I t s termination i s upon the equatorial portion of the nuclear bag and nuclear chain fibres i n an annulospiral configuration. Further, Landon i n 1966 demonstrated that the axon terminals l i e on the surface of the muscle fibres i n a shallow groove. I t i s interesting to note that the primary afferent impulse to the central nervous system from a muscle spindle w i l l involve a terminal contact with a l l the i n t r a f u s a l f i b r e s , expressing i t s e l f on a single axon. The secondary sensory nerve i s a Type I I f i b r e averaging 8 mm i n diameter and having a transmission v e l o c i t y of approximately t h i r t y to forty meters per second. The number of secondary endings varies from zero to f i v e . The s i t e of i n t r a f u s a l innervation by this afferent group i s l a t e r a l to the annulospiral endings of the primary nerve. In contrast to primary endings, the secondary endings are confined almost e n t i r e l y upon the nuclear chain f i b r e s . The terminal ramifications of the secondary endings are generally more dispersed than those of the big primary. Although the majority appear to have a s i m i l a r annulospiral form, t h i r t y - s i x per cent are a less regular "flower spray" type (Barker and Ip, 1960), The major difference shows up i n s i l v e r preparations which demonstrate that 6 the secondary endings appear as fi n e t e n d r i l s rather than wide strands of n e u r o f i b r i l s (M.C. Ip). The ul t r a s t r u c t u r e , however, of these endings i s very s i m i l a r to that of the primary endings. When we come to the question of motor innervation of the. mammalian muscle spindle, we get into the centre of a controversy. Barker (1948) described each i n t r a f u s a l f i b r e as being supplied with one or two motor end plates i n each pole. The fact that these were s i m i l a r to those found on extrafusal f i b r e s suggested a s i m i l a r function. At the present time there are two schools of thought on the whole problem of fusimotor innervation. Boyd (1962), maintains that the gamma-one fibres are terminating i n end plates only on the nuclear bag f i b r e s . Thinner gamma-two fi b r e s supply the terminals found on nuclear chain f i b r e s . Barker, Stacey and Adal (1970) f e e l that the gamma-one and gamma-two fibres supply both types of i n t r a f u s a l fibres and that a t h i r d Beta f i b r e supplies both the i n t r a f u s a l poles and extrafusal f i b r e s i m i l a r l y . I t might be a point i n time to explain the types of nerve fibres being described and th e i r o r i g i n . In the ventral grey horn of the spinal chord are located the motor neurone c e l l s whose axons terminate i n the somatic musculature and t h e i r spindles. The largest of these c e l l s are call e d alpha motor neurones and are located l a t e r a l l y i n the ventral grey. These are motor neurones s p e c i f i c a l l y for extrafusal s k e l e t a l muscle f i b r e innervation. A smaller c e l l also located i n the ventral grey horn i s ca l l e d a Beta neurone and, although not existent i n great numbers i n humans, are common i n lower vertebrates. These Beta c e l l s supply both the i n t r a f u s a l and extrafusal f i b r e s . Medially i n the ventral grey are found the smaller gamma motor 7 neurones. These are described as gamma-one and gamma-two, depending on size and conduction v e l o c i t y . They go only to the i n t r a f u s a l f i b r e s . These axons exhibit two types of terminal endings. One i s called a " t r a i l ending" and i s found i n the juxta-equatorial region on the nuclear chain i n t r a f u s a l f i b r e . The other terminal ending, ca l l e d a "plate ending", i s found on the same i n t r a f u s a l f i b r e , but i n the polar region' of the muscle spindle. The questionable Beta endings reported by Barker et. al. i n 1970 are located i n the polar region when observed. I t becomes rather obvious that the v a r i a t i o n i n size and location of neural motor input could have a marked influence on function. B. The H i s t o r i c a l Background of the Understanding of the Muscle Spindle's  Capsular Architecture. I t may seem strange to the casual observer that i n the extensive l i t e r a t u r e on this neuromuscular receptor, l i t t l e attention has been given to i t s capsule. However, the structure which, by it's very architecture, has given i t i t s name has only recently been under close scrutiny. Following the i n i t i a l "spindle" descriptive nomenclature given by Kuhne i n 1864, i t was not u n t i l almost a hundred years had passed for Merrillees (1960) to give the f i r s t morphological description of the capsule and i t s component c e l l u l a r structure at the electron microscope l e v e l . Sherrington (1894) had described the capsule as being composed of concentrically superposed membranous lamellae. These consisted.of fibrous tissue i n flattened bundles more or less fused together. A present day l i g h t microscopist would probably say the same. 8 Under the much higher resolution obtained i n transmission electron microscopy, Merrlllees was able to demonstrate that the capsule was made up of thin , concentric, tubular sheets of cytoplasm that belonged to c e l l s he called "capsular-sheet c e l l s " . He further i d e n t i f i e d them as fi b r o b l a s t s . In the spaces between the sheets of c e l l s he was able to i d e n t i f y extensive groups of collagen f i b r e s . In further descriptions of the c e l l u l a r cytoplasm he described very c l e a r l y the high content of vesicles and large caveolae. A marked s i m i l a r i t y between these capsular-sheet c e l l s and the so-called sheath of Henle surrounding small entering nerve trunks was also noted. In his descriptions of the concentric sheets as being composed of complete c e l l s without pores or fenestrations, he only mentions that these c e l l s are closely apposed at the edges.. H i s t o r i c a l l y t h i s i s important because i t shows the small attention paid to the junct i o n a l complexes that were to become so important a few years l a t e r . The change i n number- of lamellae, decreasing at the poles u n t i l f i n a l l y disappearing, was demonstrated l a t e r (Cooper and Daniel, 1967). At t h i s same time the presence of e l a s t i c tissue was observed at the poles i n the same laboratory. I t i s also i n t e r e s t i n g to note that a l l of these authors, including Gruner (1961), made s i m i l a r observations on the relationship of the accompanying blood vascular components of the capsule. C a p i l l a r i e s and small a r t e r i o l e s were noted between the capsular lamellae and not i n the p e r i a x i a l space alongside the i n t r a f u s a l f i b r e s . This presented a marked d i s s i m i l a r i t y to the close c a p i l l a r y relationship seen so commonly i n extrafusal muscle fib r e s . One can begin to see where the concept of the capsular sheath as a d i f f u s i o n and impermeable b a r r i e r came into existence. When the capsular sheet c e l l s which were reported as having adjoining edges i n close apposition were described as terminal bar tight junctions by Landon (1966) and zonulae adherens by Corvaja et a l . (1969), t h i s b a r r i e r concept received an extra boost. The f i n a l morphology and i d e n t i f i c a t i o n of the "capsular sheet c e l l s " was accomplished by the team of Shantha, Golarz and Bourne (1968). They were able to demonstrate that the capsule of the muscle spindle represents a continuation of the perineural epithelium from the peripheral nerve which supplies i t . This meant that the capsule was actually a continuation of the leptomeninges of the central nervous system which covers the peripheral nerve to i t s termination. Some time l a t e r , Boddingus, Rees, and Weddell (1973) demonstrated that the perineurium of healthy nerves acts as a d i f f u s i o n b a r r i e r . This would be instrumental i n maintaining a balanced endoneurial environment for the nerve f i b r e s . I t had previously been shown that the capsule of the muscle spindle, at i t s polar ends, made a ti g h t c o l l a r around the in t r a f u s a l f i b r e s which would prevent loss of the f l u i d i n the p e r i a x i a l space (Shantha et a l , , 1968). I t i s not i n the scope of this paper to enter into the discussion of the i d e n t i t y of this f l u i d ; s u f f i c e to state that i t has not been i d e n t i f i e d to date. In studying the embryology of th i s neuromuscular sensory receptor, i t was shown that the p e r i n e u r i a l epithelium of the supplying nerve fasciculus extends around the myotube i n the innervated region to form the early capsule (Milburn, 1973). At twenty days the fo e t a l rat spindle becomes bi-lamellate, containing two d i s t i n c t types of c e l l s . These are basophilic f i b r o b l a s t s and elongated vesicular c e l l s s i m i l a r to those of perineural epithelium. At one day post-partum the capsule 10 i s now multi-lamellate and has begun to extend to the polar regions. In studying the fine structure of the human muscle spindle, Kennedy and Staley (1974) found the outer capsule to consist of s i x to eight concentric layers of flattened, basement membrane-covered c e l l s . The elongated, overlapping processes were found to contain many pinocytotic v e s i c l e s , and collagen fibres were seen to l i e between the layers. These would be the end product of the fib r o b l a s t s described i n the muscle spindle's embryological development. Further, the perineurial c e l l s of entering nerves are i d e n t i c a l to and fuse with the outer capsule c e l l s . The continuing innermost layer of flattened c e l l s covering single nerves i s without pinocytotic vesicles and basement membrane. This i s i d e n t i c a l to the c e l l s of the inner capsule that surrounds most i n t r a f u s a l f i b r e s . The l a t e s t work on the fine structure of perineurial endothelium by Akert e_t a l . (1976) confirms the morphology described by previous investigators. I t was to test the permeability of this p e r i n e u r i a l epithelium i n the muscle spindle capsule that t h i s investigation was undertaken. C. Scope and Aim of the Present Investigation. Although the l i t e r a t u r e on the muscle spindle i s extensive, most of i t i s confined to work done on the i n t r a f u s a l fibres and t h e i r innervation. The capsule of the muscle spindle and i t s adjacent blood supply have a l l but been ignored. The question of how nutrients and possibly toxins can reach the encapsulated i n t r a f u s a l f i b r e s has not been answered. In the case of c l i n i c a l myopathies' this factor could be of major importance. This investigation was therefore designed to ascertain the 11 permeability characteristics of the outer arid inner capsules' perineural epithelium using the heme protein horseradish peroxidase and examining the results under transmission electron microscopy. 12 MATERIALS AND METHODS A. General Procedures. 1. Choice of experimental animal. In determining what animal would serve our needs best, the following factors were considered: a v a i l a b i l i t y , cost, muscle architecture and p o s s i b i l i t y of obtaining dystrophic variants at a l a t e r date. The mouse seemed to f i l l a l l of these requirements. Furthermore, the ready a v a i l a b i l i t y of Swiss whites at the university vivarium enabled us to s t a r t immediately. 2. Muscle selection. I t was f e l t that the use of a muscle e a s i l y accessible and possessed of a reasonable amount of muscle spindles would be i d e a l . The extensor digitorum longus (EDL) appeared at f i r s t to be the muscle of choice from the standpoint of s i z e . However, the overlying anterior t i b i a l i s proved to be much more readily accessible and of a size suitable for examination of the entire specimen i n a single cross section. The only problem presented by t h i s muscle was that one has to remember to remove the biceps femoris from i t s overlying position at the s i t e of o r i g i n of the anterior t i b i a l i s . 3. Determination of muscle spindle location and d i s t r i b u t i o n i n the anterior t i b i a l i s muscle. 13 A preliminary investigation of the anterior t i b i a l i s revealed at the light microscope level, that most of the muscle spindles were located in either the anterior or posterior position of the muscle. The central belly of the stretched muscle specimen appeared devoid of muscle spindles. Further, this muscle i s composed of two kinds of extrafusal fibres: large-diameter (white) fibres and small-diameter (red) fibres. The quantity of muscle spindles was much higher amongst the latter, than in the former. The methods of Thompson and Hunt (1966) and Dubowitz and Brooke (1973) employing nitro blue tetrazolium (NBT) for determination of succinic dehydrogenase were employed to show histochemical differences in muscle fibres. Fresh specimens of the mouse anterior t i b i a l i s were frozen in iso-pentane in liquid nitrogen. Blocks were then mounted in a cryostat (-20°C) and lOu sections were cut at -20°C. Transverse sections were then stained with either the NBT method or by conventional hematoxylin and eosin. These staining procedures made the different groups of muscle fibres and muscle spindles readily v i s i b l e at the light microscope level. I t must be noted that the mouse t i b i a has a marked f l a t lateral surface that denotes the medial aspect of the anterior t i b i a l i s muscle. The crest i s also prominent where the muscle originates. At the base of the medial posterior angle one can locate the entry of the neurovascular bundle. This i s the area where small red fibres predominate as revealed by histochemical stains. It is also markedly visible to the eye. 4. Choice of anesthetic. Several i n i t i a l attempts were made to use ether in the 14 standard b e l l j a r procedure, but i t was f e l t that depth of anesthesia as w e l l as time factors would prove not to be suitable for our purposes We then t r i e d c h l o r a l hydrate as suggested by Barnes and Etherington (1964) and found i t very d i f f i c u l t to control the dosage variance for each i n d i v i d u a l animal. In our hands i t was not successfu The f i n a l choice of sodium pentobarbital proved to work i n a predictable manner. I t was used i n a solution of 150 mg/15 ml of isotonic s a l i n e . This was injected i n t r a - p e r i t o n e a l l y at a dosage of 0.5 ml of solution per 35 gram mouse. I t requires some va r i a t i o n for longer time periods. I t i s better to give additional anesthesia, than to overload at the beginning. There does seem to be a d e f i n i t e s p e c i f i tolerance to the anesthetic registered by d i f f e r e n t i n d i v i d u a l mice. 5. Selection of tracer elements. " U l t r a s t r u c t u r a l tracers serve the important purpose of defining tissue or c e l l u l a r compartments and t h e i r interconnecting channels" (Feder, 1971). The choice of which to use, however, f a l l s upon some c r i t i c a l factors. The range i n s i z e , both dimensional as measured i n Angstroms or molecular weight, varies a great deal. The procedures for t h e i r use range from complex to rather simple. The constant problem of cost and a v a i l a b i l i t y i s forever with us. There can also ex i s t some t o x i c i t y problems as w e l l as side-reactions that far outweigh the p a r t i c l e size or other advantages. In reviewing the l i t e r a t u r e , several possible techniques presented themselves. The microperoxidase at a molecular weight of 1900 of Feder (1970, 1971) was made from cytochrome C. This material was then incubated with a DAB reaction. Due to the complexity of 15 preparation we decided i t unwise to pursue t h i s further. We also explored the p o s s i b i l i t y of cytochrome G as reported by Karnovsky and Rice (1969), but were unable to contact anybody who had used i t . At a molecular weight of 18,000, myoglobin as reported by Kendrew (1960) and Hastings and Enders (1974) looked very good. This would be a good size p a r t i c l e for demonstration of the p o s s i b i l i t y of muscle spindle capsular permeability. Myoglobin i s a protein molecule smaller than peroxidase and lacking the carbohydrate moiety. Since Hastings e_t a l . (1972) reported that when compared to horseradish peroxidase, the uptake was s i m i l a r , i t was f e l t that the l a t t e r might prove more useful, especially since the l i t e r a t u r e i s so voluminous. The use of the heme protein horseradish peroxidase as a tracer with a molecular weight of 40,000 dates back to the work of K e i l i n and Hartree (1951). I t remained, however, for Graham and Karnovsky (1966) to formulate a technique that i s followed to this day. The HRP catalyzes the oxidation of 3-3' diaminobenzidine by ^2^2' y i e l d i n g a n insoluble, brown, electron opaque reaction product. This occurs at the s i t e of the enzymatic a c t i v i t y , i . e . , the location of the peroxide molecule. The work of Karnovsky and Cotran i n 1966 had added the further benefit that i n mice the HRP does not appear to cause the degranulation of tissue mast c e l l s . This could have been an important factor since mast c e l l s are often seen i n close proximity to muscle spindles. A variable i n dosage of approximately 30% of injected HRP doesn't seem to a l t e r the quantity of reaction product seen leaving the c a p i l l a r i e s (Willaims and Wissig, 1975). The diameter of the tracer at approximately 16 5 nra was a l s o appealing. A much l a r g e r t r a c e r w i t h an approximate innervcore diameter of 55 $ and a molecular weight of 462,000 i s f e r r i t i n ( F a r r a n t , 1954). Haggis (1965) p i n p o i n t e d the molecular weight at 480,000, s t i l l a l a r g e s i z e . Farquahar (1961), W i s s i g (1964), H a r r i s o n e_t _ a l . (1965) and Bruns and Palade (1968) had a l l done impressive work demonstrating the usefulness of t h i s i r o n oxide. I t has been shown to be w e l l t o l e r a t e d by the animal, though i t i s o f t e n d i f f i c u l t to v i s u a l i z e m i c r o s c o p i c a l l y against any background g r a n u l a r i t y . However, i f the se c t i o n s are s t a i n e d w i t h a l k a l i n e bismuth s u b n i t r a t e , the f e r r i t i n i s g r e a t l y enhanced and becomes r e a d i l y v i s i b l e (Ainsworth and Karnovsky, 1972). A t r a c e r used to demonstrate f u n c t i o n s i n v i t r o i s lanthanum n i t r a t e (Revel and Karnovsky, 1967). I t was f e l t t hat p o s s i b l y t h i s might be u s e f u l i n a s i t u a t i o n where the i s o l a t e d muscle could be immersed i n the s o l u t i o n . 6. F i x a t i o n technique. For the p r e p a r a t i o n of t i s s u e s f o r e l e c t r o n microscopy, p e r f u s i o n f i x a t i o n presents the l e a s t membranous d i s t o r t i o n (Hyatt, 1970; Handenschild et_ a l . , 1972). Although formaldehyde has been used as a t i s s u e f i x a t i v e f o r a h i s t o r i c a l l y long time, Karnovsky (1969) improved on i t by the a d d i t i o n of glu t a r a l d e d y d e . The l a t t e r chemical i s e x t e n s i v e l y discussed by Hopwood (1973). However, the Karnovsky (1969) s o l u t i o n seems to be the one i n widest usage. B. S p e c i f i c Procedures. I 7 1. Experiments w i t h lanthanum t r a c e r s . This technique i s p u b l i s h e d i n d e t a i l (Revel and Karnovsky, 1967; R a v i o l a and Karnovsky, 1972) and b a s i c a l l y i n v o l v e s the use of n e u t r a l lanthanum. The a n t e r i o r t i b i a l i s m u s c l e was removed from both legs of white Swiss a n e s t h e t i z e d mice. The whole muscle was d i s s e c t e d out and immersed i n Karnovsky's (1965) formaldehyde-glutaraldehyde f i x a t i v e , c o n t a i n i n g 2% lanthanum i n a 1:1 r a t i o . I t was then washed i n 0.1 M cacodylate b u f f e r and p o s t - f i x e d at room temperature i n osmium t e t r o x i d e cacodylate c o n t a i n i n g a 2% s o l u t i o n of n e u t r a l i z e d lanthanum. A f t e r f i x a t i o n b l o c k s were dehydrated and embedded i n E p o n - A r a l d i t e , and sectioned f o r l i g h t and t r a n s m i s s i o n electron-microscopy. 2. Experiments w i t h f e r r i t i n . F o l l o w i n g sodium p e n t o b a r b i t o l i n t r a p e r i t o n e a l a n e s t h e s i a , Swiss white males of an average 45 gm weight were opened v e n t r a l l y and the i n f e r i o r vena c-ava exposed. 0.5 ml of f e r r i t i n (Sigma Chemical Co., St. L o u i s , Mo., NOF-3128) at a c o n c e n t r a t i o n of 93 mg/ml was then s l o w l y i n j e c t e d i n t o the i n f e r i o r vena cava. The animals were subsequently k i l l e d at 2, 5, 10, 15, 30, 60, 120 and 240 minute i n t e r v a l s by f i x a t i v e (Karnovsky, 1965) p e r f u s i o n i n t o the d o r s a l a o r t a . The a n t e r i o r t i b i a l i s muscle from each l e g was then removed under d r i p f i x a t i o n and f i x e d f o r 2-6 hours at room temperature. F o l l o w i n g washing i n 0.1 M cacodylate b u f f e r i t was p o s t - f i x e d i n 1% osmium t e t r o x i d e f o r one. hour, washed again i n b u f f e r and s t a i n e d i n b l o c k i n u r a n y l a c e t a t e . Normal dehydration and embedding preceeded s e c t i o n i n g f o r t h i c k and t h i n s e c t i o n examination. The g r i d s were s t a i n e d w i t h a l k a l i n e bismuth 18 subnitrate (Ainsworth, 1972) for f e r r i t i n enhancement. 3. Experiments with horseradish peroxidase. Adult Swiss white mice were anaesthetized with sodium pentobarbitol i n t r a p e r i t o n e a l l y , then injected v i a i n f e r i o r vena cava with HRP (Sigma Type II) at a concentration of 40 mg/0.5 ml of normal saline. At given time int e r v a l s ranging from two minutes to 240 minutes, they were then perfused i n the dorsal aorta with Karnovsky's f i x a t i v e (diluted 1:1 with 0.1 M sodium cacodylate buffer). The anterior t i b i a l i s muscle was then dissected out of both legs, minced and fixed for 3-6 hours at room temperature, or for 60 minutes only and then stored i n buffer overnight at 4°C. For preparation of the reaction product, the methods described by Graham and Karnovsky (1966) and Raviola and Karnovsky (1972) were employed. Tissues were rinsed i n cacodylate (0.1 M) buffer and either embedded i n 7% agar (for cross-sectioning at 200u on a Sor v a l l tissue sectioner) or processed as minced pieces. They were then rinsed for 30 minutes on 0.05 M Tris-HCl (pH 7.6), incubated for 40 minutes to one hour with mechanical rotation i n 0.05% 3-3' diaminobenzidine i n 0.05 M Tris-HCl with 0.01% H 20 2 added to the above solution. When f u l l y incubated the tissues present a. dark red-brown colour." These incubated tissues are then washed for 60 minutes i n Tris-HCl (pH 7.6), then 30 minutes i n 0.1 M sodium cacodylate buffer and post-fixed i n 1.0% OsO^ and rinsed. Tissues which were fixed at normal lengths of time (3-6 hours) were then stained en bloc with saturated aqueous uranyl acetate, washed, dehydrated and embedded. Subsequently, i t has been reported (Raviola 19 and Karnovsky, 1972) that longer f i x a t i o n times and uranyl acetate en bloc staining i n t e r f e r e with the v i s u a l i z a t i o n of the HRP. reaction product. ' The f i x a t i o n time was then cut to one hour and the uranyl acetate was eliminated from the procedure. After p o s t - f i x a t i o n , tissues were dehydrated through the usual ascending series of alcohols, i n f i l t r a t e d overnight i n Epon-Araldite 1:1 with 100% ethanol, i n f i l t r a t e d the next day i n 100% r e s i n , embedded and polymerized for 48 hours at 60°C. Thick sections were cut and stained with toluidine blue for l i g h t microscopy orientation, then the blocks were trimmed and thin sections cut on a Reichert 0MU-3 ultramicrotome. These were post-stained with Reynold's lead c i t r a t e (1963) and examined with a P h i l i p s 300 transmission electron microscope. To check on the appearance of reaction product, sections were observed before staining with lead c i t r a t e . 4. Controls. Adult Swiss white mice, following sodium pentobarbitol anaesthesia, were injected v i a the i n f e r i o r vena cava with iso t o n i c normal s a l i n e . At corresponding time i n t e r v a l s they were perfused with Karnovsky's f i x a t i v e (1965) and the anterior t i b i a l i s muscle dissected out of both legs. The minced tissue was subjected to standard f i x a t i o n and embedding techniques, 20 RESULTS Inasmuch as three tracer elements were employed, i t i s f e l t that the best projection of the results observed i s by description of each on an i n d i v i d u a l basis. A. Control. Normal Morphology of the Capsule and i t s Blood Supply i n  the Mouse Muscle Spindle. The descriptions that follow are from the three main regions of the muscle spindle's architecture (Fig. 1). Normal mice systemically injected with normal saline were used as controls. 1. Polar region. The morphology of these two most d i s t a l portions of the muscle spindle expresses i t s e l f with a c h a r a c t e r i s t i c c y l i n d r i c a l , t i g h t l y packed appearance (Fig. 1). The outer capsule i s composed of usually no more than a single layer of perineural epithelium (Fig. 2). The underlying p e r i a x i a l space i s a l l but non-existent. The i n t r a f u s a l f i b r e s , usually three or four i n number, are t i g h t l y packed against each other and t h e i r inner capsule i s missing. Lying alongside, one w i l l often see a peripheral nerve trunk and i n one instance a blood vessel was seen inside the outer capsule. When the most d i s t a l portion of the polar region i s reached, the outer capsule disappears e n t i r e l y and the compact bundle of i n t r a f u s a l f i b r e s l i e s free, surrounded by connective tissue and the larger extrafusal fibres (Fig. 2). Although 21 the i n t r a f u s a l fibres seen in this region are presumed to be of the nuclear bag and not nuclear chain type, i t i s very d i f f i c u l t to make a positive i d e n t i f i c a t i o n as such. 2. Juxta-equatorial region. Proceeding centrally from the polar regions one reaches that portion of the muscle spindle where circumferential expansion begins to take place. These portions l i e on both sides of the most central or widest part and are called juxta-equatorial (Fig. 1). Morphologically at t h i s point the most c h a r a c t e r i s t i c features are the increase i n size of the p e r i a x i a l space, the increase i n lamellae of . perineural e p i t h e l i a l c e l l s comprising the outer capsule, the increase i n number of i n t r a f u s a l fibres due to the appearance of the ends of the nuclear chain fi b r e s and the presence of an inner capsule. The blood vasculature and neural components that accompany the outer capsule become more numerous. The blood vessels here are often seen amongst the lamellae of the outer capsule. Usually, the inner capsule at t h i s stage i s i n close apposition to the i n t r a f u s a l fibres i t surrounds. Underneath the inner capsule and on the muscle fibres themselves various terminal end plates of supplying peripheral nerves can be seen. 3. Equatorial region. Prominent at this circumferentially widest part of the muscle spindle i s the marked increase i n size of the p e r i a x i a l space (Figs. 1, 3 and 4). The inner capsule s t i l l surrounds the i n t r a f u s a l fibres but at a distance from them and not closely abutting as previously noted. The muscle fibr e s themselves can be c l e a r l y seenand i d e n t i f i e d as the large nuclear bag type and the smaller nuclear chain type. Multiple-myelinated and unmyelinated nerve axons can be seen alongside the i n t r a f u s a l f i b r e s , under the inner capsule, under the outer capsule, and even i n the l a t t e r ' s lamellae as w e l l as adjacent to i t s outer surface. The outer capsule i n this central region has become d e f i n i t e l y • thicker i n that the number of perineural e p i t h e l i a l c e l l s l y i n g i n layers'over each other has increased (Fig. 5). This usually i s around three to f i v e layers i n depth at t h i s point. Heavy deposits of collagen f i b r i l s can be seen running circumferentially. Strangely enough, the inner capsule i s s t i l l e xhibiting a single c e l l layer i n thickness (Fig. 4). Many vesicles and even caveoli are present i n the outer capsule perineural epithelium but absent i n the inner capsule c e l l s . i Further, i t must be noted that the pinocytotic vesicles present i n the capsular perineurium are much more numerous i n the outer lamellae than the innermost lamellae of the outer capsule. I t would suggest that the inner capsule i s more of a b a r r i e r than i t s outer counterpart. Although the perineural c e l l s of the outer capsule exhibit a basement membrane on both sides, the inner capsule c e l l s do not. As to the junctional complexes between the perineural epithelium c e l l s , the only consistently v i s i b l e one would have to be cal l e d a maculae adherens (Fig. 5). Neither t i g h t nor gap junctions were observed i n the present study. There i s evidence that the contact between adjacent c e l l s comprising one lamella w i l l be i n the form of dovetail or overlap architecture. Some of the overlaps can be very extensive and yet reveal no apparent j u n c t i o n a l complexes (Fig. 5). The blood vascular components seen near or on the surface of 23 the outer capsule i n the equatorial region can vary greatly i n size (Fig. 3). Large a r t e r i o l e s often abut the capsular perineurium's outer surface as we l l as small c a p i l l a r i e s and veins. Within the lamellae of outer capsule perineurium c a p i l l a r i e s are often seen; however, none have been seen i n the p e r i a x i a l space within the outer capsule. Lymphatics have not been i d e n t i f i e d as such. B. Tracer Experiments. In a l l experimental animals injected with tracer or with tissues immersed i n tracer, there was no apparent change i n the normal u l t r a s t r u c t u r a l architecture of the muscle spindle. The only difference noted was the presence of tracer or reaction product within the tissues. Although lanthanum n i t r a t e forms a v i s i b l e c o l l o i d usually i n the form of lanthanum hydroxide, HRP i t s e l f i s not readily v i s i b l e . The electron dense reaction product of the enzymatic reaction of HRP with 3-3' diaminobenzidine as i t passes through the tissues i s what one actually sees i n an electron micrograph. The v i s i b i l i t y of this reaction product i s further enhanced by the. use of osmium tetroxide during post-fixation (Graham and Karnovsky, 1966b). 1. Lanthanum experiments. As had been reported by Revel and Karnovsky (1967) and Raviola and Karnovsky (1972), lanthanum c o l l o i d i s readily v i s i b l e under electron microscopy. In these experiments the anterior t i b i a l i s muscle of Swiss white mice had been immersed for s i x t y minutes i n a solution of buffered lanthanum n i t r a t e . There was extensive lanthanum precipitate abutting the thin layer of capsular perineurium (Fig. 6) 24 seen surrounding a polar spindle. A higher magnification of t h i s (Fig. 7) revealed a dense accumulation of c o l l o i d a l tracer outside the outer capsule. Smaller sized lanthanum p a r t i c l e s were also v i s i b l e i n the p e r i a x i a l space inside the spindle capsule and abutting the sarcolemma of the i n t r a f u s a l f i b r e s . Moreover, lanthanum precipitate was occasionally observed i n vesicles of the perineural epithelium of the outer capsule (Fig. 7). There was also evidence of reaction product penetrating the sarcolemma of i n t r a f u s a l fibres i n polar zones where i t was v i s i b l e i n the sarcotubular T-system i n these fibres (Fig. 8). From the foregoing, there seems to be adequate evidence to suggest that lanthanum n i t r a t e was able to penetrate the outer perineural capsule of polar muscle spindles where i t entered the p e r i a x i a l space as w e l l as the sarcotubular system of the i n t r a f u s a l f i b r e s . 2. Horseradish peroxidase (HRP) experiments. In this series of experiments, fixed quantities of HRP were injected systemically into Swiss white mice. Following intraperitoneal anesthesia, muscle spindles and t h e i r neighboring blood vessels found i n the anterior t i b i a l i s muscle were examined at d i f f e r i n g time i n t e r v a l s ranging from two minutes to two hours post-injection. Electron microscopic l o c a l i z a t i o n of HRP reaction product was c a r e f u l l y undertaken i n order to elucidate the putative routes taken by material leaving the microcirculation and entering the muscle spindle and i t s contents. I t should be noted that, i n most cases, c a p i l l a r i e s were encountered just external to, and intimately abutting, the outer perineural capsule of the muscle spindle (Fig. 9). They were not, however, seen inside the capsule i n the p e r i a x i a l space of equatorial 25 or juxta-equatorial regions. Beginning with the e a r l i e s t specimen at the two minute time i n t e r v a l , the enzymatic reaction product was readily observable i n the lumina of c a p i l l a r i e s adjacent to polar spindles (Fig. 9). In Figure 10, tracer i s evident and can be seen i n pinocytotic vesicles of c a p i l l a r y endothelium adjacent to a muscle spindle. Surprisingly enough, the polar spindle seen i n Figure 9 c l e a r l y shows HRP accumulated on the outer surface of the capsular endothelium and at the same time a gradation of smaller tracer p a r t i c l e s can be seen inside the p e r i a x i a l spindle space as w e l l . Further staining of the i n t r a f u s a l f i b r e sarcolemma i s also evident. This extensive presence of tracer was only observed at t h i s early time period i n the d i s t a l polar regions of the muscle spindles examined. At f i v e minutes and at the l a t e r seven and a half minute time period, there was v i s i b l e evidence of extensive pinocytotic a c t i v i t y i n c a p i l l a r y endothelium adjacent to a muscle spindle (Fig. 11). The capsular perineural epithelium of the muscle spindle (Fig. 12) s i m i l a r l y demonstrates this i n t r a c e l l u l a r a c t i v i t y . I t became apparent at the ten minute time i n t e r v a l that there was a marked difference i n the rate of tracer entry i n polar regions as opposed to equatorial regions (Figs. 13, 14, 15). Figure 15 shows reaction product present inside the outer capsule i n a polar region. At the same time i n t e r v a l , however, i n the equatorial region tracer was only observed i n pinocytotic vesicles of the c a p i l l a r y endothelium of vessels external to the outer capsule (Fig. 14). I t was not present i n the subcapsular p e r i a x i a l space of the equatorial region of muscle spindles observed i n this time period. These differences suggest that 26 the a c t i v i t y and movement of tracer upon an equatorial spindle at ten minutes post-injection i s about the equivalent of what i s seen i n a . polar spindle at the two minute time period. At twelve and a half minutes, the reaction product was v i s i b l e i n the T-tubular system of i n t r a f u s a l fibres i n polar regions (Figs. 16, 17). In equatorial regions, at the same time i n t e r v a l , however, the presence of HRP was only seen i n pinocytotic vesicles of the outer capsular perineurium of muscle spindles examined (Fig. 18). There i s , thus, v i s i b l e evidence that the capsule of the muscle spindle i s permeated by the heme-protein tracer horseradish peroxidase i n polar as well as i n equatorial regions. There i s , however, a markedly different time factor for the penetration of the tracer i n these two regions. In the l a t e r time zones running from t h i r t y minutes to four hours, another type of reaction to the tracer made i t s e l f apparent. Figure 19 shows the c o l l e c t i o n of reaction product i n membranous vesicles highly suggestive of lysosomal a c t i v i t y . This i s further exemplified i n the four hour time i n t e r v a l seen i n Figures 20 and 21. These l a t e r time figures strongly suggest normal c e l l u l a r function r e l a t i n g to elimination of foreign p a r t i c l e s . 27 PLATE I Figure 1: A schematic i l l u s t r a t i o n of a t y p i c a l murine muscle spindle derived from multiple transverse and longitudinal sections observed during the course of this study. Note the physical relationship of the outer (External) capsule to the inner (Internal) capsule and the i n t r a f u s a l (Nuclear bag and Nuclear chain) muscle f i b r e s . Note also how this relationship changes from central equatorial to d i s t a l polar regions. The branching microvasculature which extensively supplies the muscle spindle can be seen adjacent to the external capsule. For reasons of c l a r i t y , the innervation of the muscle spindle has been omitted i n this drawing (prepared with the assistance of Ms. Marguerite Drummond). 29 PLATE I I Figure 2: A muscle spindle i n i t s polar region i s shown i n this low power l i g h t micrograph. The tissue was embedded i n Epon-Ar a l d i t e , stained with toluidine blue and cut i n cross-section for examination. Clearly v i s i b l e are the i n t r a f u s a l fibres ( i f ) and t h e i r accompanying nerves enclosed with an outer capsule (arrowheads). Adjacent to th i s muscle spindle are blood vessles, a peripheral nerve trunk (n) and three extracapsular i n t r a f u s a l f i b r e s (arrow). A neighboring extrafusal f i b r e (EF) i s also indicated. X 1,200. Figure 3: This low power l i g h t micrograph shows the equatorial region of a muscle spindle i n cross-section for comparison with Fig. 2. The outer capsule (c) can be seen surrounding a large p e r i a x i a l space (as t e r i s k ) . The delicate inner capsule (arrows) completely invests the i n t r a f u s a l fibres ( i f ) . A blood vessel (bv) and small nerve trunk (n) are also v i s i b l e adjacent to the outer capsule. X 1,200. 31 PLATE I I I Figure 4: Low magnification electron micrograph showing a transverse section of a muscle spindle i n the equatorial region. The multi-lamellated outer capsule (c) can be seen surrounding the large p e r i a x i a l space (asterisk). The delicate inner capsule (arrowhead) surrounds the i n t r a f u s a l ( i f ) f i b r e s . Three myelinated nerve fibr e s (n) are seen inside the outer capsule. X 2,780. Figure 5: High power electron micrograph showing the multi-lamellated architecture of the normal murine muscle spindle outer capsule. An overlapping junction (heavy arrow) between two perineural e p i t h e l i a l c e l l s i s present. Numerous vesicles can be seen (fine arrows) i n the perineural e p i t h e l i a l c e l l s . Collagen f i b r i l s (c) l i e between the capsular lamellae. X 42,860. 32 33 PLATE IV Figure 6; Low magnification electron micrograph showing the polar region of a muscle spindle following immersion i n a solution of lanthanum n i t r a t e . The c o l l o i d a l tracer i s readily v i s i b l e (arrowheads) outside the capsule as w e l l as i n the p e r i a x i a l space. An i n t r a f u s a l (IF) and an extrafusal (EF) f i b r e are indicated. X 5,960. 3U 35 PLATE V Figure 7: High power electron micrograph showing the perineural epithelium of the outer capsule (OC). C o l l o i d a l lanthanum tracer i s seen l y i n g external to the outer capsule on the extreme l e f t . I t i s also v i s i b l e between the lamellae of the outer capsule and within the pinocytotic vesicles (arrows). Smaller lanthanum p a r t i c l e s abut the saroolemma (arrowheads) of an i n t r a f u s a l f i b r e (IF). X 76,400. 3 6 37 PLATE VI Figure 8; High power transverse section of an i n t r a f u s a l f i b r e (IF) i n the polar region of a muscle spindle. Lanthanum tracer i s v i s i b l e (curved arrows) i n the p e r i a x i a l space, abutting the sarcolemma of the muscle f i b r e , and i s also present i n i t s T-tubules (straight arrows). X 64,000. 38 39 PLATE VII Figure 9; The extensive presence of horseradish peroxidase reaction product i s seen i n this low magnification electron micrograph. This cross-section of a polar spindle and i t s accompanying c a p i l l a r y (C) shows extensive tracer present adjacent to i t s capsule (arrows) and within the p e r i a x i a l space (arrowheads) at two minutes post-injection. X 4,100 41 PLATE VIII Figure 10: High magnification electron micrograph showing reaction product (curved arrow) i n the c a p i l l a r y lumen (L) of a blood vessel adjacent to the outer capsule (c) at two minutes post-injection of HRP. Reaction product can also be seen (arrows) i n pinocytotic vesicles on the lumen side of the c a p i l l a r y endothelium. An i n t r a f u s a l f i b r e ( i f ) i s also indicated. X 75,600. hi 43 PLATE IX Figure 11: Transverse section of a c a p i l l a r y endothelial c e l l at i t s nuclear (N) l e v e l i s shown i n this electron micrograph. Extensive reaction product i s present i n the c a p i l l a r y lumen and pinocytotic a c t i v i t y (arrows) i s cl e a r l y v i s i b l e at f i v e minutes post-injection of HRP. X 45,100. Figure 12: High magnification electron micrograph of the perineural epithelium of the outer capsule of a muscle spindle at f i v e minutes post-injection of HRP. The tracer (curved arrows) i s v i s i b l e both outside the capsule as w e l l as i n the p e r i a x i a l space. Pinocytotic a c t i v i t y i s present (large arrow) i n the perineural capsular endothelium. X 45,500. 45 PLATE X Figure 13: Low power electron micrograph. Transverse section of an equatorial muscle spindle and an adjacent blood vessel (bv) at the ten minute post-injection time i n t e r v a l . The multi-lamellated outer capsule (c) surrounds the p e r i a x i a l space (asterisk) and i n t r a f u s a l fibres ( i f ) . A tracer-laden c a p i l l a r y (arrow) i s seen next to the outer capsule. X 3,830. Figure 14: High magnification electron micrograph of the same ca p i l l a r y as i n Fig. 13 showing reaction product (arrows) outside the endothelial c e l l w a l l . X 73,000. 4 6 47 PLATE XI Figure 15: At ten minutes post-injection of HRP, this high magnification electron micrograph shows tracer (arrow) present inside the outer capsule (C) of the muscle spindle i n i t s polar region. X 75,400. U8 49 PLATE XII Figure 16: Low magnification electron micrograph at twelve and a half minutes post-injection of HRP. This i s a transverse section of the polar region of a muscle spindle. Tracer i s v i s i b l e abutting the sarcolemma (arrows) of an i n t r a f u s a l f i b r e ( i f ) and i n i t s T-tubules (arrowheads). X 15,900. Figure 17: A high magnification electron micrograph of the same i n t r a f u s a l f i b r e seen i n Fig. 16 revealing reaction product i n i t s T-tubules (arrow). X 43,600. 50 51 PLATE XIII Figure 18: High magnification electron micrograph at twelve and a ha l f minutes post-injection of HRP. Reaction product i s v i s i b l e i n a pinocytotic v e s i c l e (arrow) of the outer capsular perineurium i n the equatorial region of a muscle spindle. X 78,000. 52 53 PLATE XIV Figure 19: This high magnification electron micrograph shows reaction product l o c a l i z e d i n a large lysosome at the periphery of an extrafusal muscle f i b r e (EF). This transverse section was taken from t h i r t y minute post-injection tissue. X 88,100. 55 PLATE XV Figure 20: At 240 minutes post-injection, this high magnification electron micrograph shows entrapped tracer (arrow) i n the muscle spindle's outer capsular perineurium. This i s highly suggestive of lysosomal a c t i v i t y . Note also the abundance of pinocytotic vesicles devoid of reaction product i n the outer capsule c e l l s . X 69,400. 56 57 PLATE XVI Figure 21: This low magnification electron micrograph of the outer muscle spindle capsule (OC) and an adjacent myelinated peripheral nerve trunk (N) i s seen at 240 minutes post-i n j e c t i o n of HRP. Here, reaction product (arrow) i s seen undergoing lysosomal a c t i v i t y i n the perineural epithelium (P) of the nerve. X 32,000. 58 58« DISCUSSION' One of the most in t e r e s t i n g and strangely least-discussed morphological aspects of the mammalian muscle spindle i s the relationship between i t s outer capsule and the blood vessels supplying i t . The presence of a capsule around the i n t r a f u s a l muscle fibres suggests the concept of a special environment within as opposed to one without. One could draw a p a r a l l e l with the relationship of a peripheral nerve and i t s surrounding perineurium. The structure of the capsule, i t s organization, and i t s permeability therefore become important factors i f the function of th i s incredible neuromuscular sensory receptor i s to be f u l l y understood. Aside from the capsule, the question of blood supply and i t s role has to relate to the l a t t e r ' s morphology. What are we seeing i n the microvasculature that could help the understanding of function? This and several other vascular-related concepts were explored. The following discussion w i l l attempt to answer some of these questions. A. The Capsule and i t s Vasculature. The fundamental question that was asked•in this project was "How do nutrients and/or toxins reach the i n t r a f u s a l f i b r e s of a muscle spindle?" The l a t t e r are l y i n g inside an inner capsule (Barker, 1974), surrounded by a f l u i d of unknown composition (Brzezinski, 1961) and a l l of this i n ensheathed by the muscle spindle's outer capsule (Smith, 1975). The problem of nutrient transport almost becomes mechanical i f one i s to further examine the accompanying blood vascular components and t h e i r physical location i n relationship to the capsule. Sherrington (1894-95) i n his early description of the muscle spindle described the p e r i a x i a l f l u i d inside the outer capsule as being lymph. This probably was due to the fact that even today i n a l i g h t micrograph one can often see pericytes i n this space that look very much l i k e lymphocytes. Further, i n pa r a f f i n sections stained with hematoxylin and eosin the presence of an amorphous gel of some sort i s c l e a r l y v i s i b l e . Since Brzezinski (1961), using histochemical techniques, demonstrated that the p e r i a x i a l f l u i d was not lymph, we are back to the basic question of what blood vascular components are responsible for oxygenation and n u t r i t i o n of the i n t r a f u s a l muscle f i b r e s . One of the ch a r a c t e r i s t i c morphological features of most mammalian muscle spindle outer capsules i s the close relationship seen between the capsular c e l l s and various elements of microcirculation. The occurrence of capillaries- l y i n g upon and amongst the capsular lamellae has been w e l l described (Cooper and Daniel, 1963; Banks and James, 1972; Landon, 1974; Ovalle, 1976). The int e r e s t i n g point to note i s that only Gruner (1961), Cazzato (1968) and Banks (1972) report having seen a c a p i l l a r y within the p e r i a x i a l space inside the outer capsule. Further, this was only reported on human tissue. In a l l of the specimens examined i n th i s study we only once saw an intracapsular c a p i l l a r y , and th i s was i n a d i s t a l polar region. We are therefore l e f t with a close examination of the capsular morphology as the probable main s i t e for nutrient transfer i n and out of a muscle spindle. Despite the fact that muscle spindles have been reported as having simple and compound forms (Boyd, 1959), l y i n g i n p a r a l l e l 60 (Thompson, 1970), and even sharing common capsules (Lund, 1978), the architecture we are concerned with i n this study i s the si n g l e , c l a s s i c a l muscle spindle as described o r i g i n a l l y by R u f f i n i i n 1898. As stated by Robertson (1957), the sheaths ( i . e . , capsule) consist of greatly flattened, closely apposed concentric c e l l s with double, membranes between thei r apposed surfaces. I t wasn't u n t i l Merrillees (1957; 1960) began to describe the "sheet c e l l s " of the capsule that the u l t r a s t r u c t u r a l morphology began to become apparent. The e p i t h e l i a l c e l l s that comprise the multi-lamellated structure of the outer capsule are not fib r o b l a s t s (Shantha and Bourne, 1968) but are d e f i n i t e l y s i m i l a r to perineural epithelium seen i n peripheral nerve trunks (Shantha, 1968; Akert et a l , , 1976). Further, Shantha et. (1968) suggested that the perineural epithelium represents simply a continuation of the pia-arachnoid from the central nervous system. When Milburn i n 1973 was able to show that the capsule was formed by an extension of perineural epithelium of the supplying nerve fasciculus covering a developing myotube, this reinforced the feeling that a muscle spindle had i t s own i n t e r n a l environment s i m i l a r to the peripheral nerve that supplied i t . Most of the l i t e r a t u r e discussing this perineural epithelium of the outer capsule s k i r t s the issue of the blood-brain b a r r i e r concept. There i s no question that on face value the existence of an i n t e r n a l environment s i m i l a r to a peripheral nerve or the CNS would appeal i n the case of as sophisticated a neuromuscular receptor as the muscle spindle. However, one s t i l l can't escape the suggestion that there must be a way i n or out of this structure. I f one explores the l i t e r a t u r e of capsular structure i n the 61 polar regions, the presence of e l a s t i n i s noted (Cooper and Daniel, 1967) and the fact that the outer capsule f i t s t i g h t l y over the extending i n t r a f u s a l muscle fibres i s described (Shantha, 3.968). I t i s i n t e r e s t i n g to note that i n the cat, which may have no relevance to the mouse used i n this work, the capsule only extends over 30-50% of the t o t a l spindle length (Swett, 1960). From this we can only deduce that portals of entry other than through the capsule or i t s vasculature have not been described. B. Correlation of Capsular Morphology and Experimental Results. When a tracer i s injected into the blood stream of an experimental animal, i t w i l l have to leave the c a p i l l a r y microvascular bed p r i o r to entry into the i n t e r s t i t i a l spaces. The c a p i l l a r y endothelium i s therefore the f i r s t b a r r i e r to be overcome (Reese and Karnovsky, 1967; Karnovsky, 1967; Clementi and Palade, 1969; Simionescu et a l . , 1972, 1973, 1975; Renkin, 1977). Since the l i t e r a t u r e c l e a r l y establishes the permeability of endothelial c e l l s to various molecular weight tracers i t was f e l t that the use of horseradish peroxidase (HRP) was w e l l j u s t i f i e d (Graham and Karnovsky, 1967). I t was further f e l t that the l o c a l i z a t i o n of a tracer i n the i n t e r s t i t i a l space surrounding a muscle spindle would demonstrate a route of entry into the p e r i a x i a l space i f one existed. In the close examination of capsular perineurium of normal mice, at the electron microscopic l e v e l we were able to i d e n t i f y many pinocytotic vesicles i n these c e l l s as w e l l as the odd caveoli. Their d i s t r i b u t i o n i n each c e l l was d e f i n i t e l y i n the same zonal arrangement described by Simionescu et a l . (1974). In the nuclear and organelle 62 regions of the perineural c e l l , the vesicles are absent, but i n the zones peripheral to the l a t t e r , they were i n great abundance. The mechanism for t r a n s - c e l l u l a r transport certainly i s present i n the capsular perineural morphology of the murine muscle spindles observed. I t i s interesting to note, however, that the c e l l s comprising the outer layer of the capsular perineurium had a much higher v e s i c l e / c e l l content than those of the innermost layer of the outer capsule. This may be related to the c e l l i t s e l f or i t may be due to the simple physical problem of having less space on the innermost ring of a concentric lamellation of c i r c l e s than on the outermost. Further, the innermost perineurial e p i t h e l i a l c e l l s appeared smaller i n size (see Fig. 5). I t would be e a s i l y possible to pursue ad nauseum the theories of permeability and existence of junctional complexes between these perineural c e l l s . Suffice i t to say that the few junctional complexes that we observed suggested a macula adherens structure. The majority of i n t e r c e l l u l a r contacts seen i n each lamella appeared to be extended overlaps or dovetails (Fig. 5). A further exploration of entry routes takes us back to the polar region. The capsular morphology has changed here to a single layer of perineural epithelium i n contrast to i t s multi-layered architecture i n the equatorial region. The inner capsular layer i s no longer present. I t i s d i f f i c u l t to say whether or not the l o o s e - f i t t i n g capsule seen i n Epon-Araldite sections of the polar region presents the true picture of capsular-intrafusal f i b r e relationship or whether this i s a r tefactual. One certainly gets the' f e e l i n g that t h i s polar area i s not a t i g h t l y sealed region. Upon examination of the tissue specimens by transmission 63 electron microscopy, the presence of the HEP i n various s i t e s i n different time periods was noted. The results observed i n the two minute time period which was our shortest one of a l l , were most surprising. In the polar region we found enzymatic reaction product a l l around the outer capsule, i n the outer capsular lamellae and i n the p e r i a x i a l space abutting the sarcolemma of the i n t r a f u s a l muscle fib r e s . Very simply stated, the tracer had been able to get i n everywhere. However, i n the equatorial region of the same time period, i t was an e n t i r e l y d ifferent picture. The presence of the HRP against the outer capsule's p e r i n e u r i a l c e l l s was s i m i l a r to that seen i n the polar region; aside from quantity, the density and p a r t i c l e size appeared the same. Penetration into the capsule or p e r i a x i a l space had not occurred i n this region. The tracer was seen i n the lumen of the capsule's blood vascular bed and i n pinocytotic vesicles leaving the c a p i l l a r y endothelium. I t had not, however, been picked up by the vesicles i n the capsular perineural endothelium. As l a t e r time periods were observed, a d e f i n i t e pattern of tracer movement r e l a t i n g to time and spindle morphology began to emerge. In the equatorial regions the movement of HRP across the outer capsule's perineural epithelium didn't reveal i t s e l f u n t i l almost ten minutes l a t e r than what was happening i n the polar regions. I t would suggest that the equatorial region has a more selective b a r r i e r action than that observed i n the polar regions. One could postulate that since the main annulospiral neural endings are found i n t h i s equatorial region, and that probably i t represents the area of highest function i n a muscle spindle, the maintenance of a special environment i s e s s e n t i a l . By the time that twelve and a half minutes tracer 64 post-injection has passed, the polar region i s observed to have reaction product present i n the T-tubu.lar system of the i n t r a f u s a l muscle f i b r e s . This would have to be regarded as penetration into the deepest part of the muscle spindle that could reasonably be expected. Penetration into the equatorial region at twelve and a half minutes was found to be no further than through the outer capsule, into the p e r i a x i a l space and abutting the sarcolemma of the i n t r a f u s a l muscle f i b r e . There was, however, another marked difference i n the two regions. We never did see i n equatorial regions the same massive quantity of reaction product that was observed i n the polar regions. I t would be reasonable to suggest that entrance of tracer i n polar regions could relate to contraction and relaxation of the spindle, causing a pumping action. This would be hard to defend i n an anaesthetized laboratory animal. The fact remains that the tracer movements observed i n the polar regions indicate routes of entry not existent i n the equatorial region. I t i s d i f f i c u l t at th i s point to assay the ba r r i e r effectiveness of the inner capsule. The fact that i t i s no longer present i n the polar region may be highly important to th i s area's permeability. The other factor may relate to the capsule's extending only over 30-50% of the muscle spindle's t o t a l length (Swett, 1960). Despite the observations of Shantha et_ al. (1968) on the ti g h t c o l l a r effect of the capsule i n the polar regions, i t may not present an e f f e c t i v e b a r r i e r to exogenous substances. This great variance i n tracer penetration has to be looked at i n another way as w e l l . The p o s s i b i l i t y of morphological differences has been explored but t h e i r relationship to time has not. There can be l i t t l e question that i f we see extensive HPJ? present i n polar regions 65 at two minutes i t has to have entered the i n t e r s t i t i a l spaces p r i o r to two minutes. Either that, or for some reason we are not seeing the reaction product i n the equatorial region at the same time period. I t has been shown that the length of f i x a t i o n time tissues are subjected to can have a tremendous effect on the v i s i b i l i t y of HRP (Rosene and Mesulam, 1978). The time difference of one hour can mean a 98% loss of enzymatic reaction product. There i s no question that when we cut our f i x a t i o n time from two hours + one hour to a t o t a l time of one hour, our v i s i b l e reaction product was much more i d e n t i f i a b l e . Notwithstanding this p o s s i b i l i t y of f i x a t i o n error, i t i s reasonable to assume that the HRP presence was constant i n our tissues. I t should be noted, however, that i n a l l our tissues the reaction product was either present i n the muscle or not; That i s to say, that there were many areas of extrafusal muscle where no tracer had entered i t s vascular bed at a l l . This was probably due to the known closure of c a p i l l a r i e s i n s k e l e t a l muscle during periods of rest or mild exercise (Martin, 1932). One could safely say that an anaesthetized mouse i s resting. There can be l i t t l e question from the observable r e s u l t s , that what happens i n the pre-two minute post-injection time i n t e r v a l i s important. Some recent work by Renkin (1978) and Simionescu et a l . (1978) indicates that multiple pathways are present i n c a p i l l a r y endothelium and also emphasize the importance of the early time periods i n the use of injected tracers. I t could be that the c a p i l l a r y endothelium which represents the f i r s t b a r r i e r , may be morphologically different i n the polar regions than i n the equatorial regions. I t certainly has been wel l demonstrated that these differences e x i s t i n different tissues (Karnovsky, 1965, 1966, 1967). The p o s s i b i l i t y of 66 morphological variance here can not be ignored. C. Conclusions. The v i s i b l e presence of HRP reaction product i n the peripheral space and T-tubular system of the i n t r a f u s a l muscle fibres demonstrates that the capsular perineurium of the muscle spindle i s permeable. I t further revealed a marked difference i n permeability i n d i s s i m i l a r morphological regions of the same muscle spindle. Entry into the polar regions was much more rapid than into the equatorial counterpart. Unfortunately, the route of entry i s obscure. There i s d e f i n i t e evidence of pinocytotic a c t i v i t y i n the equatorial capsular perineurium, but i n the polar regions there appears to be too much tracer present to correlate i t with having come through t o t a l l y by pinocytosis. The continuation of a lanthanum immersion study presently under way may give us the answer to th i s question. Further, the development of precise tracer i n j e c t i o n and tissue f i x a t i o n techniques involving the under two minute time period are c l e a r l y indicated. As i n a l l research, opening one door only reveals many more. 67 SUMMARY 1. The permeability of the perineural epithelium of the murine muscle spindle capsule to exogenous tracers, was assessed by the use of horseradish peroxidase (HRP) and lanthanum n i t r a t e . The results were evaluated by conventional transmission electron microscopic techniques. 2. The microvasculature supplying the muscle spindle, and l y i n g adjacent to i t s capsule, were found to be readily permeable to the tracers employed. 3. Extensive reaction product was found inside the polar regions of the muscle spindle capsule as early as two minutes post-injection. I t was present abutting the sarcolemma of the i n t r a f u s a l fibres and i n the p e r i a x i a l space. 4. In the polar region there was d e f i n i t e evidence of reaction product i n the i n t r a f u s a l f i b r e T-tubular system at 12.5 minutes post-injection. As compared to the polar region, the equatorial regions showed marked variances i n the early time zones. Reaction product i s v i s i b l e only i n the lumen and pinocytotic vesicles of the adjacent c a p i l l a r y endothelium. 5. In the c e n t r a l , equatorial region of the muscle spindle, the reaction product was v i s i b l e within the outer capsular lamellae 12.5 minutes a f t e r systemic i n j e c t i o n of HRP. 6. At the same 12.5 minute post-injection time i n t e r v a l , tracer was present i n the i n t r a f u s a l T-tubuiar system of the spindle's 68 polar region. 7. Although the perineural epithelium of the outer and inner capsule of the muscle spindle has revealed i t s e l f as being permeable to the 40,000 molecular weight tracer horseradish peroxidase, several questions remain unanswered: a) The polar region appears to be more permeable at an e a r l i e r time i n t e r v a l than the equatorial. One could question i f the paths of tracer entry are the same i n each region. b) From the evidence obtained i n th i s study i t i s apparent that the e a r l i e r than two minute time i n t e r v a l s merit serious attention. c) The question also arises as to whether or not a normal muscle spindle capsule exhibits the same permeability as i t s counterpart i n a mouse affected with muscular dystrophy or other myopathies. 68a ERRATA, p. 69 Boddingus, J . , R.J.W. Rees and A.G.M. Weddell. 1973. Leprosy neuropathy i n mice and men: an electron microscope st Nederlands Tydschrift voor Geneeskunde 117: 363. 69 BIBLIOGRAPHY Ainsworth, S.K. and M.J. 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