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Histological and ultrastructural study of mice with hereditary muscular dystrophy Cooper, Ann 1962

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HISTOLOGICAL AND ULTRASTRUCTURAL STUDY OF MICE WITH HEREDITARY MUSCUIAR DYSTROPHY by Ann Cooper B.S.A., University of British Columbia, 19%.. A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE DEGREE OF Master of Science in the Department of Neurological Research We accept this thesis as conforming to the required standard for THE UNIVERSITY OF BRITISH COLUMBIA Ap r i l , 1962. In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make i t freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Ann Cooper Department of Neurological Research The University of British Columbia, Vancouver 8, Canada. Date A p r i l 1962 i i ABSTRACT The history of human muscular dystrophy with reference to clinical, histo-logical and biochemical studies is reviewed. The value of the recent discovery of an experimental mouse with hereditary muscular dystrophy with clinical, histological, and biochemical similarities to human muscular dystrophy i s dis-cussed. The histology of normal muscle, with special reference to ultrastructure, is also reviewed. For this investigation mouse muscle samples for the electron microscope were fixed in Palades osmic acid solution, embedded in methacrylate or epon and stained with either lead hydroxide or phosphotungstic acid. A method for obtaining one day old muscle samples, while keeping the animals alive until the dystrophic symptoms are noted clinically, is outlined. Light microscope sections were obtained from electron microscope blocks and examined after staining with toluidine blue. Experimental results by light and electron microscope observations showed there to be no histological differences between one day old dystrophic and normal muscle. However one day old muscle showed histological differences compared to older muscle. The chief differences were the smaller size of fibers, random distribution of mitochondria and enlarged nuclei, and the presence of abundant interfibrillar sarcoplasm with a conspicuous granular components. The regular repeating pattern of the endoplasmic reticulum of adult muscle fibers was hot seen. Some "atypical" fibers showed similarities to altered fibers seen in older dystrophic mice. The mitochondria were swollen and vacuolated with few cristae and pale matrix. Endoplasmic reticular components were vacuolated and adjacent myofibrils were disorganized. Atrophy of fibers was f i r s t noted at Uj. days of age and were conspicuous at 63 days. By 63 days alterations were also noted in i i i mitochondria and endoplasmic reticulum and these changes became more promi-nent with the progress of the disease. Atrophy of the myofibrils was evident and the Z-bands were often irregular and out of register. Connective tissue also increased greatly. Several miscellaneous structures are also discussed. The histological findings are compared to those found by other workers. Several suggestions put forward by others as to the possible cause of the disease are summarized. On the basis of morphological findings i t is suggested that ribonucleo-protein synthesis in the nuclei of dystrophic muscle fibers is increased but that there may be some intermediate stage at fault which prevents the conversion of amino acids to myofilaments. iv ACKNOT^ LEDGEMSNTS This work was supported by a grant from the Muscular Dystrophy Association of Canada. The author wishes to extend to them thanks for the financial support of the project. The writer is indebted to Dr. William C. Gibson, Kinsmen Professor and Chairman of the Department of Neurological Research for directing and arranging the financial support of this project. Particular thanks are extended to Dr. W.H. Chase and Dr. J.R. Miller not only for their continued guidance and criticism throughout the course of the work but also for the invaluable scientific training. Appreciation is also extended to Mrs. K. Morris who typed the manuscript. -V-TABLE OF CONTENTS ABSTRACT i i ACKNOWLEDGEMENTS i v I. INTRODUCTION 1 I I . REVIEW OF LITERATURE 2 1. Muscular Dystrophy i n Humans 2 A. Historical • 2 B. Classification and C l i n i c a l Characteristics 3 C. Histopathology 6 D. Histo-Biochemistry 10 2. Muscular Dystrophy i n Mice 15 A* Introduction . 15 B. Classification and Characteristics • 15 C. Histopathology 16 D. Histo-Biochemistry 18 3 . Histology of Normal Muscle 19 III. METHODS AND MATERIALS 3U IV. EXPERIMENTAL OBSERVATIONS 38 1. C l i n i c a l 38 2. Light Microscope 39 3. Electron Microscope lj.2 V. DISCUSSION 50 VI. BIBLIOGRAPHY 59 VII. ILLUSTRATIONS 66 •1-I. INTRODUCTION Although muscular dystrophy was f i r s t described over 100 years ago, l i t t l e progress has been made since that time towards an understanding of the basic defects responsible for this affliction. Recent clinical, biochemical, histochemical, and histological studies have added knowledge and increased our understanding of this disease, but i t s fundamental nature is s t i l l unknown. Progress has been hindered in the past by the lack of a suitable experimental animal. The recent appearance of a mouse strain manifesting a myopathy simi-lar to that in human muscular dystrophy has provided a new approach to the study of the disease. In this study the mouse strain with hereditary muscular dystrophy has been used to study the histological changes which occur in the muscles of the affected animals. It has been found possible to study muscle biopsies from newborn mice that subsequently developed muscular dystrophy and to follow the development of the morphological changes at different ages. Both the light and the electron microscopes have been used, and the changes seen here have been compared to those described by other workers who studied human and mouse muscular dystrophy. LT. REVIEW OF LITERATURE 1« Muscular Dystrophy in Human3 A. Historical The term muscular dystrophy i s used to describe a group of diseases having characteristic microscopic changes in the muscle with wasting but without demon-strable pathological changes in the nervous system ( 1 , 69, 85, 108). This disease was confused for years with primary neuronal degeneration, as many descriptions of neuromuscular disorders had been given with muscular atrophy as the major clinical finding (85). Although Meryan, 1852, observed muscular dystrophy in several families and noted that the spinal and peripheral nerves were normal, he nevertheless s t i l l confused the condition with pro-gressive neural muscular atrophy. Duchenne, in 1885, described a simple pro-gressive muscular dystrophy of children but considered i t to be the juvenile form of progressive muscular atrophy. However, in 1886, Euleriberg and Cohnheim reported the findings at autopsy of a case of "hypertropic paralysis" and remarked on the absence of any changes in the central nervous system. Between muscle fibers fatty tissue was noted which they believed was degenerated con-tents of sarcolemmal tubes. According to Adams (l) i t was not until staining methods to demonstrate neural degeneration improved that there was a clear distinction between the primary muscular and neuronal diseases. There has been discrepancy in the literature as to who was the f i r s t person to describe the disease. As pointed out by Gowers (cited from Adams - 1 ) , isolated cases of muscular dystrophy had been noted by Bell, 1830, Partridge 18L8, Aran 1850, and Little 1853. Probably the f i r s t person who noted its familial and thus possible genetic relationship was Gowers in 1879, who -3-recognized that muscular hypertrophy and atrophy could occur in variable portions in the same family (1). Griesinger, in 1865, made surgical biopsies and noted abundant adipose tissue. Duchenne, in the same year, also noted an increase in "the muscle connective tissue. Leyden, in I876, drew attention to the morphological similarities between a l l familial primary muscular atrophies. It remained for Erb in 1881; (31) to give the f i r s t detailed study of the juvenile form of progressive muscular dystrophy and it s relation to pseudo-hypertrophy of muscles. He noted that there was no alteration in the spinal cord and peripheral nerves and that the disease was easily differentiated from the spinal form of progressive atrophy. Erb was not certain i f the changes were primarily in the muscle fibers or the connective tissue or i f both parti-cipated. He suggested that juvenile muscular atrophy, hereditary muscular atrophy and the pseudohypertrophy type could be combined and called progressive muscular dystrophy. It was not until 18°1 that he added a very detailed c l i n i -cal and histological study of many cases of progressive muscular dystrophy. B. Classification and Clinical Characteristics The classification of muscular dystrophy has been a source of dispute for many years (1, 107). Perhaps i t was due, in part, to the lack of sharp de-marcations of clinical, pathological, biochemical and genetic aspects. In spite of this dispute the various classifications are fundamentally similar. They are described according to onset of disease, areas f i r s t affected, and genetic background. The following classification has been adopted from Walton (10$, Dowden (27) and Denny-Brown (26), as the classification which appears to be the most up to date and well differentiated. It follows quite closely the classification of Batten, 1909 (cited from Hurwitz - £0). There are five groups: -li-lt Facioscapulohumeral dystrophy 2. Limb girdle; .atrophy 3. Distal myopathy ll . Ocular myopathy S>. Duchenne muscular dystrophy 1. The facioscapulohumoral dystrophy (Landouzy-Dejerine dystrophy) occurs in either sex, and at any age from childhood until late adult l i f e . It begins with atrophy of the shoulder girdle and face with the pelvic region subsequently affected. A characteristic feature is that these patients are unable to whistle and have thick gaping lips. There i s a normal l i f e expectancy. 2. The limb girdle atrophy is probably inherited by an autosomal recessive mechanism with expression in either sex, but in rare instances seems to be trans-mitted as an autosomal dominant or possibly as sex-linked recessive. This group II may be divided further into the Erb dystrophy and the Leyden-Mobius dystrophy. It is known as the Erb juvenile dystrophy i f the arm and shoulder are involved f i r s t . The patient is f i r s t noted to have difficulty in raising the arms above the shoulders. Weakness in flexion of the elbows follows in one to two years. Hurwitz separated this form into a distinct group called Juvenile or Erb type, ti The Leyden-Mobius dystrophy is characterized as beginning in the thigh and pelvic muscles. These patients are noted to have difficulty in climbing stairs due to weakness of extension of hips and knees. There is variability in severity and rate of progression of limb girdle atrophy with disability usually twenty years after onset. Thus most patients have a shortened l i f e span. 3« Distal myopathy (Steinert's Disease) is thought to be inherited as an autosomal dominant character with some sex-limitation to males. The onset is usually between the ages of 1*0 and 60. It is characteristically benign, beginning in the small muscles of the hands, in the feet and legs, and eyelids. The most common symptoms i s weakness of hand grip and difficulty in relaxing from a strong grasping effort. k* The ocular myopathy (Kelok-Nerin syndrome) or (Progressive Dystrophic Ophthalmoplegia) when inherited appears to be autosomal dominant, but in most cases there is no family history of the condition in the previous two genera -tions. This, the mildest form of the muscular dystrophies, affects only the extraocular muscles. It begins in middle l i f e with bilateral ptosis of both upper eyelids, and slowly increasing limitation of a l l eye movements. There is a general weakness in facial and neck movement in later stages. 5. Duchenne or progressive muscular dystrophy is the most severe form. It is sex-linked recessive with expression in the male but rarely in the female. It begins early in l i f e , usually in the f i r s t decade, but occasionally as late as the third decade. The child is noted to have difficulty in rising from a sitting position (the classic Gowers test earlier discussed by Erb). There is usually a waddling gait, with frequent stumbling. Atrophy begins symmetrically in the pelvic girdle musculature and later involves the shoulder girdle. By the age of fifteen the child becomes greatly disabled. The l i f e span is shortened considerably, often the child does not survive beyond late teens. Thus this dystrophy, which is the most common form, has a very malignant course (70, £9). Erb (32) in 1891, described the clinical symptoms of two cases of muscular dystrophy in children aged nine, one of whom had a younger brother with the same disease. The typical picture might be briefly summarized as follows. The patients had never been able to run properly and could not jump, and from the age of seven onwards frequently f e l l with characteristic disturbances of movement and grasping. The stomach was projected, shoulder blades spread, calves of the leg were remarkably voluminous, upper legs and arms were thin; -6-shoulders loose, gait waddling, and characteristic climbing up on the legs was observed when rising from the floor (the patient had to push on his knees to straighten up because of weakness of the leg extensors). C. Histopathology Erb (31, 32, 33) has described in elaborate detail the general structures of the muscle under the microscope from numerous patients afflicted with various forms of muscular dystrophy. He mentioned the difficulty in following up case histories from onset to end by biopsy studies and thus when only one sample was studied i t was difficult to t e l l the stage of the disease. However, i t has been established that the various clinical types of muscular dystrophies (facial, scapulohumeral, pseudohypertrophic) possess common histopathological features (31, U5). Thus, in spite of the differences in distribution, course, and onset of the muscular dystrophies the essential changes are a l l the same. Adams (1) 1953, Hassin (1*5) 191*3, Wohlfahrt and Wohlfart (111;) 1935, and others (see Adams - 1) have written reviews on the histopathology of the muscular dystro-phies from which the following summary has been compiled. Light Microscope Under the light microscope the most striking features of the dystrophic muscle are the great variation in size of individual fibers, the large amounts of areolar connective tissue, the accumulation of fat cells, and the appearance of centrally placed nuclei. It i s possible to see gradations of these changes within one muscle. Well preserved fibers with prominent striations are mixed with paler hypertrophic fibers having less distinct striations. Adjacent fibers may show disruption, thickening by connective tissue proliferation (endomysium), and finally the formation of irreversible connective tissue scars (85, 77, 50, 1*5). There is normally an i n i t i a l swelling of -the fibers followed by atrophy (1). Great irregularity in fiber diameter is noted, and when viewed in cross-section many of the fibers are rounded instead of the normal polygonal shape, with pale and dark fibers intermingled. Some fibers look homogenous and possess a distinct sarcolerama while others are granular or represented by empty vacuoles. Hypertrophic fibers with a normal internal structure are inter-mingled with atrophic broken-up f i b r i l s loosely scattered within their sarco-lemmal rings. Some of the smaller fibers are practically devoid of nuclei while in other larger fibers there is a migration of the sarcolemraal nuclei into the substance of the fiber often with the formation of chains. Several workers have noted that these nuclei, which may be more vesicular and vary in shape from the normal, are surrounded by atrophic muscle or connective tissue (1, h$s 109). The sarcolemmal nuclei are also increased an number and might occur clumped and surrounded by a halo devoid of striations, but elsewhere striations are normal (1, 27, 50, 85). (According to Hassin - h$)>the nuclei do not invade the muscle fibers but accompany them). Fat cells and connective tissue appear between the muscle fibers, increasing with atrophy until they have replaced the muscle fibers in some areas. There is no apparent pathology found in the neural tissue even in the peripheral nerves occurring within the affected muscle (1, U5). The nerve endings of the diseased area may show some atrophic changes but only in shrink-age of arborizations within the sole plates (1, 85, 109). Changes in the anterior and lateral horn cells of the spinal cord consisting either of atrophy or of diminution in the number of cells, have been described by Holmes and Gil (in Hurwitz -50). However Hurwitz (50) and Hassin (U5) believe there to be no evidence of a degeneration process in the nervous system in the majority of patients with muscular dystrophy. ••8— # In 1891 Erb discussed the histology of many patients afflicted with muscular dystrophy (33). Since this was such an outstanding piece of work, i t i s of interest to briefly summarize here some of his pathological findings and note how l i t t l e has been added to this knowledge since that time. One of the case histories cited was a patient with the juvenile form of disease. The pathological findings were as follows: The muscle fibers, when viewed in cross-section, were very altered. They were mostly hypertrophic (100 jx in diameter) with only a few atrophic (15 ^ u or under). In some areas were seen accumulations of small fibers which remained separate from one an-other. The fibers were mostly rounded in form. There were gaps and fissures, and numerous centrally located nuclei. In some areas vacuoles could be seen. The connective tissue was very distinct, very wide and greatly increased in thickness. The blood vessels were somewhat thickened with many nuclei in the walls. In longitudinal section the picture was similar. Another case history cited was a patient with pseudohypertrophy. The muscle fibers of the hypertrophic and atrophic areas were very irregular with evidence of much fiber splitting. When viewed in cross-3ection they were mostly noted to be only slightly polygonic in outline with very rounded edges. There was an increase in the size of nuclei with many being centrally located. Accumulation of fatty tissue was usually noted as rows of fat cells. The connective tissue was abundant, compact, and wavy. The nerves were normal. Electron Microscope Very l i t t l e research work has been done on the ultra-histopathology of human dystrophics. Van Breeraen (102, 103) I960 and Molbert (73) I960, apparently have been the only people who have studied muscle biopsies of human muscular dystrophy cases under the electron microscope. Van Breemen studied 56 cases of which the gastrocnemius muscle from a 6 year old patient was shown in electron micrographs. Mobert studied the gastrocnemius muscle of a 7 year old g i r l with progressive muscular dystrophy. According to van Breemen the earliest alteration noted was vacuolation of the endoplasmic reticulum,the vacuoles appearing singly or in small groups between the normal myofibrils and mitochondria. In areas where large vacuoles or groups of vacuoles appeared, the sarcoplasm was increased and many mito-chondria were lobulated. In the same muscle successive stages of degeneration could be clearly established. The endoplasmic reticulum became progressively vacuolated and later underwent disintegration. The mitochondria were lobulated and also disintegrated in a progressive manner (showing swelling, loss of density and fewer cristae) leaving debris from these organelles in the spaces between apparently non-functional myofibrils. Subsequently the myofibrils became dense, striations irregular and diffuse, and here mitochondria almost disappeared although they remained at the periphery in large numbers. There was a progressive loss of sarcoplasmic inclusions and loss of contractile bands. The structured substance which remained consisted principally of myofibrils in which the myofilaments were s t i l l apparent though swollen. According to Mobert, the ultra structure of the muscle was greatly dis-turbed and many transitional stages could be seen. The mitochondria in some fibers were normal, however in other fibers, many had lost their inner struc-ture and were irregularly distributed between the myofibrils. The Z-bands were seen only spasmodically in narrow represented regions of the cel l . The myo-filaments were often no longer distinguishable, being replaced by granular material throughout the sarcoplasm of the fiber. Often parts of the myofibrils appeared as homogenous bands with the myofilaments loosened and split. The sarcoplasm was noted to have a heavy granular structure. Isolated fibers showed a vesicular cytoplasm which was interpreted as remnants of the endoplasmic -10-retieulum. Some of the nuclei were peripherally located, with loss of nucleo-plasmic structure, others were centrally located. At the beginning of the disease the nucleoplasm was very compressed with concentrated osmiophilic sub-stance and a sinuous nuclear membrane. As the destruction progressed the nucleus became less dense. Nuclear pores were noted. The homogenous basement membrane , found next to the sarcolemma, was in contact with connective tissue f i b r i l s . Advanced degeneration of the muscle was marked by a thick disaggre-gated basement membrane and eventually the muscle fiber was surrounded by thick n connective tissues, Mobert concluded that muscular dystrophy i s a primary muscle disease with injury to the myofibrils manifested by progressive granular decomposition of the myofilaments and loss of mitochondria, D, Histo-Biochemistry Although much work has been done on the biochemical and histochemical aspects of muscular dystrophy no investigation in these areas has yet come near to revealing the basic pathological mechanism responsible for the disease. Although i t i s certain that the primary fault is in the biochemistry of the muscle, i t is uncertain whether the muscles, which have been demonstrated at present, represent primary disturbances or whether they are secondary effects. It i s at present not possible to correlate these biochemical abnormalities because our knowledge of the biochemistry of normal mechanisms is only in i t s infancy with many aspects s t i l l unsolved. Degeneration and necrosis of muscle tissue from any cause will release enzymes and other components into the blood stream and urine where they may be detected. Of the many enzymes which have been detected in increased amounts in the blood and urine of patients suffering from muscular dystrophy, none appears to be specific for the disease. A diagnostic test for muscular dystrophy based on such studies is not yet available although there are some promising leads. -11-In reviewing the literature i t was found difficult to adhere to a classi-fication of enzymes and metabolites which would be meaningful. The following discussion will outline the literature under the headings (1) glycolytic enzyme, (2) muscle electrolytes, (3) miscellaneous enzymes and metabolites. (1) Glycolytic enzymes Glycolysis i s the breakdown of carbohydrates to pyruvic and lactic acid. This series of reactions i s an important mechanism for the production of energy in the body. The following enzymes have been noted altered in dystrophic patients - aldolase, lactic dehydrogenase, phosphatase, phosphorylase Ma" and "b", phosphoglucorautase, phosphohexose, isomerase, and phosphylatLon of fruc-tose. Because of the progressive disappearance of muscle tissue in the course of muscular dystrophy i t i s possible that some of the c onstituents of muscle breakdown pass not only into the blood but also into the urine (93). Aldolase, the enzyme which degrades fructose-l,6-diphosphate to dihydroxyacetone phos-phate and glyceraldehyde-3-phosphate, is greatly increased in the serum but decreased in the muscle of dystrophics. The highest levels in the serum are noted in the earliest stages of the disease and might reflect a rapid breakdown of relatively large amounts of muscle tissue (96, 99, 109). Serum levels of this enzyme may be used as a diagnostic test for muscular dystrophy. Lactic  dehydrogenase, which converts lactic acid to pyruvic acid in the presence of reduced DPN, has been reported to be increased in the serum of dystrophic patients and may also reflect rapid breakdown of muscle (96, 99, 109). Phosphatase, the enzyme present in many tissues, has been reported to be in-creased ih serum of dystrophics and may also reflect rapid breakdown of muscle. This enzyme i s thought to play an important part in the destruction of muscle fibers as i t is found in high concentration in the connective tissue (6). Acetylphosphatase activity is also increased in connective tissue and muscle -12-but the significance of this increase is obscure (6). Phosphorylase "a" and "b^ which converts glycogen to glucose-l-phosphate, i s decreased in muscle of dystrophics (91, 92, 97). Phosphoglucomutase, which converts glucose-1-phosphate to glucose- 6-phosphate, is decreased in dystrophic muscle (91, 92, 97). Phosphohexosisomerase, which converts glueose-6-phosphate to fructose-6-phosphate, i s increased in human dystrophic serum (92). Phosphosylation of  fructose, i s enhanced in muscular dystrophy while the subsequent metabolism of the carbohydrate is arrested due to deficient oxidation of pyruvic acid (19). (2) Muscle electrolytes The cations that exhibit the highest asymmetry in their intra-and extra-cellular distribution are potassium and sodium. Potassium is found in high coneentration within the cells and in low concentration in the plasma while sodium is low in cells but high in plasma. Interference with the normal meta-bolism of the cell brings about a loss of intracellular potassium and a gain of sodium until these and other ions reach equilibrium concentrations with the external medium. According to the present hypothesis ionic accumulation itself needs no metabolic energy, however the maintenance of the fixed charged system depends on a continual expenditure of energy (Adenosine Triphosphate - ATP) for repair, maintenance and performance of biological functions (60). In muscular dystrophy there is a notable increase in the sodium and decrease in the potassium levels per gram muscle (16, 109). A decrease in the serum level of sodium (and magnesium) and increase in the serum of potassium has also been noted in some patients with muscular dystrophy (65). These changes might suggest a partial replacement of intracellular by extracellular space, and is indeed seen histologically as an increase in the connective tissues between muscle fibers. -13. (3) Miscellaneous enzymes and metabolism Creatine metabolism. Creatine functions as a source of high energy phos-phate. The transfer of this high energy phosphate from creatine phosphate to Adenosine Diphosphate (ADP) results in the formation of ATP and requires the enzyme creatine-kinase (1U±). In the urine of dystrophics there is an increase in the creatine and decrease in the creatinine which probably reflects degener-ation, of muscle ( 1 , 27). There is an increased plasma concentration of crea-tinine and a normal concentration of creatine ( 1 , 27). The serum phosphokinase is higher especially in young dystrophic patients (29, 98), but muscle creatine phosphokinase is decreased i n young dystrophics whereas the adult muscle does not show such enzyme alterations (105). The creatine phosphate and total creatine has been reported to be lower in the dystrophic muscle (27). Amino acid metabolism. Amino acids form the building blocks of proteins. The amino acids threonine, valine, leucine, arginine and taurene are found in higher concentrations in the urine of dystrophics and these levels are parti-cularly high in young patients (U9). Blahd (lit) noted a qualitative increase in the urinary excretion of methionine or valine, isoleucine or leucine, methionine sulforide or sarcosine, methyl histidine, cysteic acid and unidentified sub-stances. An increased number of amino acids were found in the urine of normal siblings and the maternal parents of muscular dystrophic patients (possibility of an inhibited metabolic abnormality). Nucleotides. AMP, ADP, ATP act as carriers or transport agents for phos-phase groups, a function intimately bound up with bio-energetics. They are important in the storage and utilization of energy. In dystrophic muscle there is a lowered activity of ATP (89). ATP'ase activity i s decreased in the muscle according to Vignos (10£). Nucleotidases are apparently non specific enzymes splitting the nucleotides - U l -into phosphoric acid and nucleosides. By histochemical techniques connective tissues give a very strong reaction for 5-nucleotidase and i t , like phosphatase, is thought to participate in muscle fiber destruction and replacement by dense connective tissue. The connective tissues contain enzymes which dephosphorylate a number of metabolically important phosphate esters (6, 8, 15, 17)• Ribose. Minot et al (72)have found excretion of ribose in the urine of dystrophic patients and have suggested the use of ribosuria as a diagnostic test in humans dystrophics. However, Mathews and Smith (67) have failed to demon-strate ribose in urine of dystrophics. A test for ribosuria was carried out in 89 patients with muscular dystrophy by Walton (106) and since only 12 were positive he concluded that this test had no value in diagnoses. Cholinesterase. Acetylcholine is concerned in the chemical mediation of transmission of nervous impulses. The physiological function of acetylcholine esterase is thought to be the disruption of the substrate thus allowing for a single rather than continuous nervous discharges. A slight decrease of cholin-esterase has been found in human dystrophic muscle ( 6 ) . Glutamic oxaloacetic transaminase is increased in the serum of dystrophics (99) . Succinic dehydroginase activity is decreased which suggests a lowered aerobic metabolism ( 6 ) . Deoxyribonucleic Acid (DNA) and Ribonucleic Acid (RMA). There is a notable increase of DNA (60$) and RNA (l|C$) per gram of dystrophic muscle. This in-crease might have been because the muscle volume but not the nuclear volume is decreased (115)« -15-2. Muscular Dystrophy in Mice A« Introduction Little progress has been made towards an understanding of the underlying mechanisms responsible for muscular dystrophy although much data has accumulated over the past century. The primary abnormality of the dystrophic muscle remains obscure. In the past, one of the factors responsible for the present status was the lack of suitable material to study a l l phases of the disease as i t was not possible to use humans for experimental work (90). However, the recent discovery of a mouse strain at Jackson Laboratory, Bar Harbor, Maine, with a hereditary primary muscular disease has offered a new approach to the problem by providing an excellent experimental animal. These dystrophic mice have stimu-lated a great deal of research which might possibly shed light on the problem of human dystrophy. There appears to be many similarities - genetic, clinical, histopathological, and histochemical which could link the mouse dystrophy with the human muscular dystrophics (108). B. Clas sif ication In the original work by Michelson et al (69) the dystrophic mouse mutation was found to be caused by a single autosomal recessive factor and this has been confirmed recently by Loosli (61). By breeding this strain with 13 different populations there has shown, a genetic modification of the penetration of the dystrophy. The f u l l recessive expression of 25$ i s obscured by the phenomena of incomplete penetration, thus only about 19% of the original 129 dystrophic mouse strain become dystrophic. Michelson et al (69) noted that the affected mice did not show any clinical symptoms until about 11+ days of age. The f i r s t overt sign of the disease was paralysis of the hind limbs. The affected mice were usually smaller in size and -16-lighter in weight than normal l i t t e r mates. They were noted to be frequently tensed and convulsively nodded the head up and down. They developed a d ecided -waddling gait followed by paralysis of the hind limbs. The mice had a shortened l i f e span, generally not living beyond two months. However, longevity could be greatly increased by improved management such as powdered food, easy access to water in dishes, and separate pens (71). Thus the clinical symptoms, early on-set, waddling gait, disease commencing in hind limbs and shortened l i f e span, are comparable with juvenile human muscular dystrophy. C. Histopathology Light Microscope The general pathology of the muscle, as studied by Ross et al (90) and West (112) resembles that of the human dystrophics. There is great variability in fiber size observed conspicuously at a l l ages (69, 90, 112). This variabil-ity in diameter, seen in cross-section, is noted to be more extreme in older animals. The fibers tend to be round (90) rather than polygonal in shape. West (112) noted that they were rounded at two weeks, polygonal at k weeks, rounded again in severely affected animals. Michelson et al noted a splitting of the fibers to give daughter fibers which are a l l contained within a single endo-mysial tube. The sarcolemmal nuclei have been noted to migrate into the muscle fiber with the frequent occurrence of chains. There is also an increase in the size and the number of the sarcolemmal nuclei. However West (113) noted that in regions of increased nuclear numbers there are no mitotic figures to account for the increase. There is a notable increase in the connective tissue especially in small caliber fibers which West noted as early as 2 weeks of age. This increase in collagen and elastin content has been confirmed by histochemical techniques (22). Michelson et al noted rows of fat cells between the fibers, and this has been confirmed by biochemical techniques where a marked lipid and -17-and cholesterol rise was noted in the dystrophic muscle (22, 9h)» Contrary to human dystrophy there was no i n i t i a l swelling of the muscle fibers preceding atrophy (112). Electron Microscope The ultrastructure of muscle fibers of dystrophic and l i t t e r mate controls between three weeks and three months of age has been studied by Ross et a l ( 9 0 ) . The abnormalities noted under the light microscope have been confirmed by use of the electron microscope and in addition alterations in the mitochondria and endoplasmic reticulum were noted which will be summarized below. Ross et a l observed that there was a loss of myofibrils which was at f i r s t confined to localized sites and then progressed to involve the entire fiber. Often normal fibers were seen next to cells that had undergone notable degener-ation but as the disease progressed the number of normal fibers diminished. There was a distinct loss of myofilaments in the regions occupied by nuclei, however, below and above the level of the nuclei the myofibrils appeared normal except for increased amounts of sarcoplasm. The most frequent phenomenon, in regions devoid of nuclei, was the gradual loss of filaments which frequently occurred in a manner so that the myofibrils lost continuity leaving isolated elements within the c e l l . This breakdown appeared to occur as a result of frag-mentation and dissolution of myofilaments. The breakdown did not appear to originate in any specific site within a sarcomere. The nuclei, some centrally located, appeared normal even in the most advanced stages of the disease. The sarcolemma showed no definitive alteration of fine structure. The mitochondria normally possessed a very dense matrix which almost ob-scured the intimal structure. Within the degenerated cell, the mitochondria possessed less dense matrix and were larger than the normal. (Note that this is contrary to van Breemen's observations of human dystrophics - see above). -18-The inner structure suggested that the increased size might have represented swelling rather than actual growth. The mitochondria appeared to be more long lived than the f i b r i l s and could be seen in areas almost devoid of contractile elements. The endoplasmic reticulum appeared distended and swollen with obvious ballooning, however occasionally few short segments of this membraneous system showed a normal morphology. In areas with very pronounced loss of myofibrils the endoplasmic reticulum appeared as large isolated membrane bound vesicles. D. Histo-Biochemistry Many of the biochemical abnormalities found in human dystrophics have also been described in dystrophic mice. There is an increase of aldolase (21, 96, 112), lactic dehydrogenase and phosphatase in the serum (96, 109), while a decrease of aldolase, phosphatase "a" and "b" and phosphoglucomutase occur in the muscle of dystrophic mice (l|6). In fact, overall glycogenolysis is dimin-ished. Sodium i s increased and potassium decreased (k)* There is an increased urinary excretion of creatine and decreased excre-tion of creatinine (27), however, Perkoff (86) noted the creatine excretion unchanged with a decreased creatinine excretion. An increased plasma concen-tration of creatinine but a normal concentration of creatine has been reported (27). Muscle creatine i s decreased which might be the result of an approxi-mately two-fold increase in total l i p i d (55)• There i s a decreased amount of myogen and myosin but an increased myoalbumin by electrophoresis of muscle proteins of dystrophic mice (23). McGaughey (6k) found no differences in the urinary excretion of amino acids. Excretion of <=C -ketoglutarate in the urine per gram of body weight is increased (6k) in the dystrophic mouse. A l l dehydro-genases which require TPN as a co-factor are increased in muscle of dystrophic .19 mice, whereas a l l dehydrogenases which required DPN as a co-factor are decreased in the affected muscle. Muscle glucose-6-phosphate dehydrogenase activity i s increased 300$ and isocitric dehydrogenase activity i s also i n -creased (63). There is a lowered activity of ATP in the diseased muscle and a higher activity of ADP, AMP, DPN, GTP which suggests that these may have contributed through secondary pathways, to make up for the deficiency of ATP (115). Ketogenesis - The formation of acetoacetate, -hydroxybutyric acid and acetone are difficult to demonstrate as they are rapidly metabolized into other substances. There is a definite accumulation of acetoacetate in dystro-phic mouse muscle which might be the result of a lack of an enzyme to metabolize i t further, as normal muscle i s known to oxidize acetoacetate to carbon di-oxide, water and energy (38). Urea excretion in the urine i s increased in dystrophic mice (61*). Glutanic oxaloacetic transaminase is increased in the serum of the diseased animal (96). 3. Histology of Normal Muscle Since the pathological findings of muscular dystrophy point to a primary degeneration of the skeletal muscle with no signs of nervous disorder, i t is essential that before attempting to study the dystrophic muscle the normal architecture of the muscle be understood. There are doubtless s t i l l many un-recognized differences to be noted in abnormal muscle, but not until the time when normal cell has been fully understood will a l l these differences be apparent. Muscle constitutes almost half of man's total body mass. The muscle is made up of muscle fibers which are supported by connective tissue. There are three connective tissue sheaths - the epimysium which surrounds the whole muscle, the perimysium which surrounds muscle fiber bundlesj these two sheaths contain nerve fibers and blood and lymphatic vessels, while the third sheath, 20-the endomysium, which surrounds the individual fibers, contains capillaries, nerve filaments, fibroblasts, histiocytes and mast cells (1). Recent work on the ultrastructure of muscle has been studied by Draper and Hodge (28), Huxley, A.F. (51), Huxley, H.E. (53, 5U), Ham (U3), Bennett (9, 11). and Bennett and Porter (8). Each muscle fiber is enveloped in a sarcolemma which was f i r s t described by Schwann in 1839 (from Bennett - 11), The sarcolemma, difficult to see under the light microscope, is seen under the electron microscope to be composed of two dense membranes, an outer and an o inner, each about U.0 A thick and separated by a less dense interlamina about o 20 A thick (9, 11, h3)» According to van Breemen (101) the sarcolemma is 8 p. thick with reticular fibers embedded in i t . It separates the contents of the fiber from the connective tissue fibers without. It is responsible for main-taining the difference in concentration of sodium and potassium ions between the outside and inside of the fiber (11). According to many workers^ the inner surface of the sarcolemma has adherent strands of endoplasmic reticulum (9, 101). Van Breemen (101) noted indentations which seemed to continue into folds that had apparently connections to Z-and M-bands (9, 101). The membrane appears to be elastic and have remarkable electrical properties which conduct action potentials down the fiber (9, Ul)« Fiber The contractile part of the skeletal muscle c e l l is composed of fibers. There are large numbers of fibers arranged in numerous bundles which compose an individual muscle which is surrounded by the endomysial sheath. Each of these bundles, enclosed in a perimysium, i s composed of individual fibers. Each fiber, enclosed in i t s endomysium is composed of many myofibrils, which are made up of many, minute, long myofilaments. 21-The muscle fibers, arranged in bundles, vary in thickness but l i e parallel within a given bundle. Each muscle fiber i s actually a large multi-nucleated ce l l ( 1 , 28) . The average fiber diameter i s 1*0 - 50 jx and anything thicker than 80//may be considered hypertrophic by many pathologists ( 1 ) . However, according to Huxley (5U) and Guyton (i|l) the average diameter i s 10 -100p.. The length of the fibers range from several mm to over 30 mm according to Adams et a l or from several mm to 1*0 - 100 mm according to Guyton. As a rule the fibers do not branch or anastomose. Each muscle fiber bundle is composed of numerous myofibrils usually k -20jx in width (8, %k) which are the most conspicuous element of the cell ( 1 , 8 ) . They are orientated in a longitudinal direction and run the f u l l length of the fiber parallel to one another. The myofibril is about 0,5 - 1 . 0 i n width and remarkably uniform in length. Some of the superficial ones may be deflected slightly by the sarcolemmal nuclei, and at the end of the fiber they are usually more tightly packed together. In extremely tapered fibers those myofibrils which are situated peripherally may not reach the end of the fiber(l). The cross-striations within the myofibrils arise from the repeated variations in density of the protein along the myofibril (5U). The repeating unit, called the sarcomere, is about 2 - 3 jx in length. With the light microscope i t is possible to see transverse bands which are regularly spaced within the sarco-mere. The A-band, also known as an isotropic or "Qrt band, i s about 1.5 jx in length. It is darker than the "I" band, also known as the isotropic or " J w band, which is 0.8 jx in length (Huxley - 5k)• The very dense Z-band is in the center of the I-band. The M-band, narrower and lighter than the Z-band, b i -sects the center of the A-band and thus the sarcomere. The H-band, a very narrow line within the M-band, is usually not seen with the light microscope. One sarcomere i s measured from Z to Z-band. Normally a l l the Z-bands of the 22-muscle fiber are in perfect alignment with one another when viewed in longi-tudinal section ( 1 ) . n Kolliker in 1888 (cited from Bennett - 9) reported that the myofibrils were composed of finer elements of submicroscopic dimensions which he termed "faserchen". He postulated that these might contain the contractile protein myosin. The electron microscope has revealed that such fine structures, now known as myofilaments exist. They are about 100 in diameter according to Bennett (9) and Huxley (53) or 150 A in diameter according to Bennett (8). According to Huxley, who worked on frogs 1 muscles, there are two kinds of f i l a -ments present which interdigitate with each other, and this observation is supported by van Breemen (101) who worked on human gastrocnemius muscle. The filaments differ in diameter, length, position and composition. The fine o myofilaments, actin, about 50 A in diameter and 2 jx in length are attached to the Z-band and extend from i t ; t o near the middle of the A-band,but not in to the H-band area. The coarse filaments, believed to be composed of myosin, are o 100 A in diameter and 1.5 p long and extend the length of the A-band (5U)« Hodge (U7) however, challenges Huxley's hypotheses, and believes that the thin bands are found to traverse a l l bands of the sarcomere in continuous array with thickenings or additional material present in the A-band. The myosin i s located in the A-band and the actin in the I-band areas. Spiro (97) noted the same number and type of filaments in a l l parts of the sarcomere,and therefore, assumed that there is a continuity of the filaments from Z-band through I and A up to the H^ when several of them associated to form thicker filaments. Hall, et al (1*2) in 19U6 described a series of nodosites along the f i l a -ments which Draper and Hodge (28) in 191*9 discussed as recurring every 250 -o U50 A along the filaments in a l l bands. Hoffraan-Berling and Kausche (U8) in -23-1950 discussed the fine ultrastructure of the cross bridges in frog muscle ti and noted that the "interfibrilaren Brucker" (cross -bridges) looked like beads between the myofibrils bridging one myofilament with the next. They could not t e l l i f the cross-bridges went the length of the sarcomere but noted o them in the A-band occurring every 220 - 250 A along the sarcomere. Bennett's (U) work on avian muscle reported periodicity at less than 200 A. Hodge o (U7), working on toad and rabbit, considered the bridges to be 250 - 1*00 A apart along the axis, the magnitude of spacing depending on the state of con-traction of the myofibril. He does not support Huxley's view as to their probable composition, as actin and myosin are known to be orientated longi-tudinally. He supposes that the cross-bridges might be composed of tropomyo-sin. Huxley in 1957 (5U) described the intricate system of the cross-bridges. They project out from the thick filaments at fairly regular intervals of 60 -o 70 A and each bridge is 60 degrees around the axis of the filament with res-pect to the adjacent bridge. Thus the bridges form a helical pattern which o repeats every 6 bridges or about every ljOO A along the filament. Work with X-ray defraction studies has shown a similar pattern. Huxley suggests that each bridge is part of a single myosin molecule as the number of myosin molecules is close to the number of bridges in a unit volume. Nucleus Striated muscle fibers are multinucleated. The nuclei are typically ovoid, 1 - 3 u in width by 5 - 12 u in length, and normally peripherally placed except during fetal l i f e and in the course of muscle disease when they are also seen centrally ( 1 , 101). They have dense irregularly outlined bodies, called nucleoli, usually 1 - 3 in number, with no enclosing membrane (1 , 30, 101). These sarcolemmal nuclei tend to be more numerous at the ends of the cell than in the central portion (7h). The nucleus is bounded by a double membrane (101). The membrane is noted to be interrupted at intervals by pores in most of tissue of animals so far studied ( 2 , 5 , 101, 110). The actual diameter of o o spaces between pores vary 280 A (5) - 500 A (2) between author and animal and tissue studied which may be due in part to plane of sectioning and techniques used. Watson (110) calculated the pore's diameter to be 500 A in the rat, o o o and the spacings between pores to be 1500 A (800 - 900 A - 2, 1300 A - 5 ) , thus 5 - 15$ of the nuclear surface is exposed. The pores are formed in the double nuclear envelope by continuities between the nucleoplasm and the cytoplasm. The inner and outer nuclear membranes are continuous with one another and en-close the perinuclear space. Watson believes there to be a close relation between the pores and the endoplasmic reticulum but Beams et al (5) deny this. The chromatin of the nucleus is generally located peripherally in dense aggre-gates. The sarcolemmal nuclei are more numerous in the fetus and the newborn than in the adult animal ( l ) . They are usually fairly regular in shape but folds may be seen according to the state of contraction of the myofibrils (contracted muscle have folded nuclear membrane)(101). Mitochondria The existence of mitochondria has been known for a long time in muscle II fibers. Kolliker in 1875 (9) described mitochondria in muscle and called them interstitial granules and spoke of a relation between these bodies and regular metabolism of muscle. It was not until recent work with the electron micro-scope, by such workers as Palade (76) and Edwards et a l (30) that the structure of this organelle has been elucidated. It is now recognized that the mito-chondrium has a double membrane, although Palade (78) in 1952, originally described only one membrane, 7 - 8 jx thick. The outer membrane i s regular -25-in outline while the inner one is convoluted to form cristae. When seen in longitudinal section these cristae normally l i e parallel to each other at more or less regular intervals although occasional branching or splitting may occur. According to Freeman (36), on the basis of observations on the human erythrocyte, the inner membrane is composed of a double membrane, the two outer . o o dark membranes each 15 - 17 A , and the inner one 20 - 23 A to give an overall 0 o of 50 - 60 A. The crista itself is about 175 - 190 A acrossj according to o Watson (111) the crista is 150 - 200 A. The matrix appears to be of homo-genous density which is greater than that of the cytoplasmic ground substance (76). According to Edwards et al (30) who have studied insect, amphibian, avian, and mammalian mitochondria, there are three types of mitochondria: the small round ones, long narrow ones and larger rounder ones. The small round ones O.U p. in diameter, are found in intimate relationship with the endoplasmic reticulum at the level of the Z-band (in mouse) and are usually in pairs. The long narrow type, fewer in number, are arranged between the f i b r i l s in scattered chains of 5 - 10. These are equal to,or longer than,a sarcomere in length. The large rounder mitochondria, 3 - h u in diameter, are noted in rows ( 1 - 3 ) separating the adjacent f i b r i l s . They are generally tightly packed together and the cristae do not appear parallel. The mitochondria, according to Edwards et al^are in close contact with but not attached to the endoplasmic reticulum. Edwards et al and van Breemen (101) noted them to be more numerous near the nuclei, just beneath the sarcolemma in sarcoplasmic pockets, and more in the "belly" than in the extremity of the muscle. He also occasionally noted some "ghosts" which he considered might be metabolically exhausted mitochondria. When mitochondria are closely packed, apparent connections or openings from one mitochondrium to another may be seen. Van Breemen (101) noted evidence of -26-lateral branching of mitochondria at the Z-band and irregular distribution and orientation of the cristae tubules. Mitochondria, as biochemical machines, have been elaboratorely discussed by Green (39, I 4 O ) , UBC lecture) and men-tioned by other workers . (76). It has become quite apparent in recent years that these organelles, located almost everywhere in the body, carry out very important oxidative phosphorylation reactions. They are able to oxidize pyruvic acid to C 0 2 and H20 with energy released via the TCA cycle which links these oxidative reactions involving molecular oxygen to synthesize ATP. "The weight of evidence supports the view of the raitochondrium as a highly precise organized mosaic of a strictly determined number of enzymes and co-enzymes arranged in a repeating and invariant pattern" (UO). Green has noted that the greater the number of cristae per unit area the greater the oxidation rate of the mitochondria thus the oxidative capacity i s proportional to the quantity of mitochondria. Endoplasmic Reticulum (ErgastopLasm, sarcoplasmic reticulum) According to Bennett (10), Dobie in 181*9, i s believed to have been the f i r s t person to describe what is now called the endoplasmic reticulum. Mellend 1885, (68) gave an account of the intercellular network in striped muscle in various animals including the rat. Retzius 1881, 1890 and Cajal 1888 (cited from Bennett - 9) gave accounts of the endoplasmic reticulum by gold impreg-nation techniques. But i t was not until 1902, when Veratti (10ii) studied the "endoplasmic apparatus" in many animals including the mouse, that a very de-tailed and concise account was given. This system was virtually forgotten until i1?s rediscovery by such workers as Porter and Palade (87), working with the electron microscope, who verified the earlier works. Veratti noted that the endoplasmic reticulum was not limited to the surface of the muscle but also -27-occupied the inner area. His work showed that i t consisted of anastomosing filaments that are arranged longitudinally and parallel to the myofibrils and are crossed at regular intervals by a series of transverse reticula. He also noted that i n newborn mice, the apparatus i s irregular and appeared at random in a l l directions; the longitudinal filaments united with irregular transverse lines. However, in the adult animal the direction and arrangement of filaments are in a regular pattern. Porter and Palade (87) and Palade (79), noted that the endoplasmic reticulum is composed of tubular elements with a homogenous matrix and a limiting membrane. It i s mostly bound by a smooth membrane, as those noted within the f i b r i l s , but outside the sarcomere or directly inside the sarcolemma, i t might have granules attached to i t s surface. Retzius in 1881, (cited from Bennett - 11) noted that the endoplasmic reticulum was in close association with the mitochondria. Melland and Veratti also believed that the inner part of the sarcolemma was attached to the endoplasmic reticulum and this has been supported further in recent years by other workers (10, 30). Others (8, 10, 30, 101) have verified Veratti's work that the endoplasmic reticulum attaches directly to the Z-band in the sarcomere. It has a repeat pattern in each sarcomere forming "lace-like sleeves around and among the myofibrils" (10li), Van Breemen also noted the endoplasmic reticulum attached to the M-band. Edwards, who studied various phyla from insects to mammals, also believed the endoplasmic reticulum to be connected to the membrane surrounding the nucleus? thereby showing the possible relation between nucleus, contractile material and sarcoplasm. Porter and Palade who worked on Arabystoraa larva denied this re-lation* The probable function of the endoplasmic reticulum had been suggested by Retzius in 1881 (cited from Bennett - 10, 11), who considered that i t might transmit excitatory impulses from the sarcolemma to the myofibrils deep within -28-the muscle fibers. According to Porter and Palade (81), Edwards et al (30) , and Peachey and Porter (83) , a wave of depolarization of the sarcolemma (the nerve impulse which probably was the result of the release of acetylcholine from the motor end plate of the peripheral nerve fiber) might be picked up or transmitted to the endoplasmic reticulum and then spread rapidly as an action potential along i t s limiting membrane to a l l parts of the sarcomere ( f i b r i l ) . Presumably (lateral) conduction would be to the threads opposite the Z-or I-bands. Since the conduction is believed to be saltatory, from one sarcomere to the next, the only morphological system for such conduction to the centers of symmetry i s the endoplasmic reticulum. Excitation might be by the release of an inhibitor or removal of magnesium ions through the membrane of the reti -culum into the fluid phase. According to Bennett (9) the Z-band would be the preferential site in which the excitatory signal might enter the myofibrils and activate the myofilaments. Porter and Palade also suggest that the endoplasmic reticulum might serve to channel products of metabolism to a l l parts of the c e l l . It seems, however, that the sarcoplasm would be a more efficient medium of diffusion of nutrients and removal of wastes. Bennett (8) suggests that endoplasmic reticulum might participate in exchange of materials between myofibril and sarcoplasm, a hypo-thesis also mentioned by Cajal (cited from Bennett - 9, Peachey and Porter -83). The endoplasmic reticulum may act as an intracellular impulse conductor as i t i s a continuous membrane which repeats itself longitudinally and trans-versely across the whole diameter of the cel l . It also has a close association with the external membrane of the cell and with the myofibrils where contraction is presumably initiated. With the change in the diffusion distance, the time distance-relation in the muscle is brought into proper order. - 2 9 -Glycogen Since glycogen is known to represent a major energy resevoir in muscle as well as other tissues, i t s distribution i s of particular interest. It has been demonstrated to exist in muscle by light microscopic histoehemical tech-niques such as the diastase-PAS test. It was Palade (80) in 1955 who discussed in some detail small particulate components in the cytoplasm, one of which was believed to be RNA' and the other glycogen. Other workers such as Bergman (12, 13), Adams (1) , Revel et al (88) , and Macini (66) have discussed glycogen in muscle. It i s described as forming spherical particles with a diameter of o o 150 - 300 A according to Palade, or 150 - 1*00 A according to Revel et a l or 0 o up to 500 A aggregates which are in turn made up of 75 - 150 A particles according to Bergman (13). Macini.. worked on human and rat muscle with freeze-dry histoehemical techniques and noted by the light microscope that glycogen appeared as small granules situated regularly in the isotopic bands on both sides of the I-line, at the periphery of muscle fibers surrounding the nuclei and near the interstitial blood capillaries. With Watson's (110) lead hydroxide stain the larger electron dense glyco-gen granules have been well differentiated from the smaller ?less electron dense^ RNA granules. Bergman (12, 13) worked oh the histochemistry of glycogen in frog muscle and has described glycogen deposits between the myofibrils (inter-f i b r i l l a r space) and amongst the myofibrils (interfibrillar space) verifying the earlier reports of Arnold and Goldstein, Fawcett and Selby (cited from Bergman - 13). Glycogen is more concentrated in the interfibrillar spaces adjacent to the Z and I-bands but is less concentrated in the intrafibrillar o spaces of the A, Z and I-bands. It is usually encountered as 150 - 500 A aggregates. Although Adams (1) believed there to be no direct relation between -30-the contractile process and glycogen, the localization in the Z and I-bands indicate that these areas may be associated with an enzymatic system capable of regenerating high energy ATP (12). In a l l species tested (rat, guinea pig, rabbit, etc.) the concentration of muscle glycogen at birth is usually at least twice the corresponding adult level, falling below the adult level within 1 - 3 days of birth (93). Sarcoplasm The sarcoplasm is defined as undifferentiated protoplasm of the muscle fiber which f i l l s the spaces between the contractile elements (1, i;3). It i s believed, by some, to be a sticky substance which causes adherence of the myo-fi b r i l s (1) and is believed to constitute about $0% of the muscle fiber (ill). Retzius (cited from Bennett - 8) noted three components of the sarcoplasm, besides the myofibrils; - the endoplasmic reticulum, mitochondria, and serous fluid of the matrix. The specific proteins of the sarcoplasm have proved difficult to localize in the muscle fiber (1), but the sarcoplasm i s believed to take part in the nutrition of myofibrils since i t completely surrounds them. It is known to contain fat droplets, glycogen, glycogen and RNA granules, mito-chondria and Golgi apparatus (7U). According to Bennett (8) who worked on domestic fowl the sarcoplasm can be divided into four divisions; (1) The paranuclear sarcoplasm located in the region of the nucleus (2) The subsarcolemmal sarcoplasm located in the region beneath the sarcolemma (3) The interfibrillar sarcoplasm located between individual myofibrils (U) The intercolumnar sarcoplasm located between groups of myofibrils. In longitudinal section, the myofibrils are noted to be closely packed in -31-in the muscle fiber and only small amounts of sarcoplasm can be seen between the myofibrils and around the sarcolemma of nuclei (1, 101). Even in accumu-lations next to the nuclei the actual amount of sarcoplasmic matrix is small since mitochondria are massed in these spaces (101). Contraction Contraction i s known to occur in the myofibrils. There have been numerous attempts to measure the sarcomere and their constituents during the resting state and stretched state and in the process of contraction but no consistent results have been obtained (1). According to Adams (1), Meerkel, in 1872, wrote that in the contracting muscle fiber the A-bands diffused through the whole sarcomere so that the fiber acquired a homogenous appearance. Contraction proceeded, the anisotropic substance appeared at each end of the sarcomere against the Z disc. Later workers showed that these observations were inaccu-rate. According to Bennett (9), Kolliker, in 1888, was the f i r s t person to clearly formulate the modern concepts of organization of the contracted mater-i a l in striated muscle tissue. He also postulated that these filaments might contain the contractile protein myosin. According to Huxley and Bergman (12) there is an electrically polar-ized membrane around each fiber. If the membrane is temporarily depolarized5 the muscle fiber contracts and i t is by this means that the activity of the muscle is controlled by the nervous system. The sequence of events might then go as follows: The nerve is stimulated and the impulse passes down the nerve fiber to the motor end plate, there i t i s thought to release acetyl choline which passes across from the pre- to post-synaptic membrane (the muscle mem-brane or sarcolemma), and causes a depolarization of the area. This depolari-zation might be picked up or transmitted, i f the muscle is activated enough, -32-via the endoplasmic reticulum (83) to within the muscle fiber, perhaps to attach on to the Z-bands of the sarcomere ($2). A.F. Huxley ($2) has noted that, by depolarization with micropipettes in areas of the sarcomere, the influence of the membrane depolarization i s conveyed to the interior of fibers^ by spreading along some structure in the I-band which he suggests may be the Z-band. If the wave of depolarization is great enough a twitch results (5h). If the excitation of the contractile apparatus is the result of the liberation of ATP, as suggested by Szent Gyorgyi, perhaps the relative potential drop at the Z-band i s sufficient to trigger ATP to release high energy phosphate and thus results in muscular contraction (12). Muscle derives its energy from the breakdown of ATP->ADP with the release of large amounts of energy. The resynthesis of ATP from ADP requires energy and this is supplied by phosphocreatine which in turn has obtained the high energy phosphate from the breakdown of carbohydrate. The above hypothesis^ regarding the mechanism of contraction,may or may not be entirely correct. What is known for certain is that when contraction occurs the muscle fiber changes during the process. With the advent of the election microscope,two main concepts have arisen as to how the myofilaments are associated or dis-associated with one another. One was put forward by H.E. Huxley (53, 5U), the other by Hodge (ij7). Huxley does not believe that contraction involves the shortening of the coarse or the fine filaments,but that i t i s the result of the fine filaments sliding further into the interstices between the coarse f i l a -ments, pulling the discs to which they were attached with them, until they meet in the middle. Thus the A-band remains constant in length during contraction and relaxation. However the I-band changes in accordance with the length of the muscle. -33-According to Hodge,only a single set of filaments extends continuously through a l l levels of the sarcomere. During contraction the globular actin (i-band) is shortened by interaction with fibrous myosin (A-band) so that as contraction proceeds those parts of the myofilaments in the I-bands are pro-gressively incorporated into the A-band until the I-band has disappeared. In strong contraction A-band material migrates to the Z-bands to form contraction bands. It i s , however, not certain whether this migration involves myosin or another A-band component. -3k-III. METHODS AND MATERIALS The strain 129 mice used in this study were obtained from a colony main-tained at the University of British Columbia since 1958• The original animals for this colony were obtained from the Roscoe B. Jackson Memorial Laboratory. A total of 20 dystrophic and 15 normal animals aged one to 225 days were studied under the light microscope. One or two blocks from each animal were examined. 22 dystrophic and 13 normal animals between the ages of one and 225 days (in-cluding a 260 day old dystrophic and a two year old normal) were studied in the electron microscope. An average of 10 blocks were cut from each age group. LIGHT MICROSCOPE > ELECTRON MICROSCOPE Age in Number of Number of Age in Number of Number of days Normals Dystrophics days Normals Dystrophics 1 9 10 1 k 10 1U 1 1 Hi 1 2 21 1 2 21 1 2 28 1 1 27 1 1 63 1 2 35 1 1 158 1 63 1 2 165 1 158 1 196 1 165 1 205 1 1 205 1 1 225 1 225 1 1 260 1 2 years 1 -35-Dystrophics and controls were always prepared at the same time to insure that fixing and embedding techniques were similar, and that any differences noted were not a result of technical difficulties. Except for the one day old mice, the animals were killed by dislocation of the neck. In order to keep the muscle cool, the hind leg was removed and placed on a cork covered with ice and paraffin. By aid of a dissecting microscope, a muscle fiber from the t i b i -alis anterior, having a maximum diameter of 2 mm and length approximately 1 cm, was raised with jeweller's forceps. Tiny wooden slivers were placed under both ends so that a l l sides of the fiber would be exposed to the fixative. This operation took less than one minute. The muscle was then immersed in a Palade's solution (78) at 0°C for one hour and l e f t to stand at room temperature for 15 minutes. After removal from the fixative, the leg was rinsed briefly in several washings of distilled water in a flask, and then dehydrated through 50 -70 - 80 - 95% alcohols with several changes of 5 minutes in each concentration (modification of Lane - 56) . While in 9$% alcohol the muscle fiber was separa-ted from the leg and cut into small pieces with a sharp razor blade. Care was taken not to squeeze the fiber. In the earlier stages of the investigation the tissues were then embedded in a 15 - 85$ mixture of methyl-butyl metha-crylate according to Lane (56). In the later stages, the tissue blocks were embedded in Epon according to the method of Luft (62), using a hard mixture. Obtaining one day old mouse muscle presented some problems. Since i t is not possible to detect those animals with dystrophy at this age, i t was necess-ary to obtain a muscle sample and to keep the young alive and healthy until the age when the disease could be detected by physical signs. The newborn mice were separated from the mother just before operation and placed in a petri dish with a f i l t e r paper on the bottom. A 60 watt lamp about 10 inches above the dish served as a source of heat. The mice were identified by clipping toe combinations on both hind legs. An anaesthetic may be used but better results were obtained without i t . Using a pair of sharp scissors, the right hind leg was amputated just above the lower femur and immediately placed on a cork covered with ice and paraffin. The wound was plugged with a 3 mm cube of gel foam. The mouse was held 3 inches from a 60 watt bulb for half a minute, and then placed in another petri dish with f i l t e r paper on the bottom and a light bulb as a source of heat. This whole operation was completed in one minute. The muscle fiber from the amputated limb was then prepared as described above for the older animals. If the tibialis anterior muscle was used, an appropriate sample included the whole muscle. The mice were periodically examined and after about one hour were returned to the mother if the wounds were dry. A source of heat was kept near and a wad of cotton wool placed in the next pan to cover the young and keep the mother with them (Figure 1 ) . The sections were cut with a Servall microtome about 60 m u thick, (grey color) for high magnification observations or about 90 - 150 m u thick,(gold color) for low power observations according to Peachey (81). The grids were coated with colloidion according to the method of Lane (56) or with formvar according to Pease (810 or were carbon shadowed (81+). Two stains were used. A 1% phosphotungstic acid in 95$ alcohol was used to stain myofilaments. The block was stained 1-2 hours in 95% alcohol and then washed in several changes of 9% alcohol, or a section mounted on a form-var covered grid was stained by immersion for 15 minutes and then washed in distilled water. Because of the poor contrast with Epon embedded tissues, lead hydroxide stain was used to increase general contrast (110). In addition, this stain was excellent for demonstrating glycogen. Lead hydroxide was stored in syringes according to the method of Peachey (81) and the tissues were stained in a nitrogen atmosphere apparatus according to Parson's method (80 A). The -37-tissues were stained 5 - 2 0 minutes. The specimens were examined in a Siemens electron microscope. Kodak contrast process orthofilm was used, and developed in Kodak D-ll for high contrast. Kodak Kodabromide F3 or F5 was used for printing. Sections for study by light microscope were cut from Epon and methacry-late embedded blocks at 1.5 - 2.0 u. They were stained with toluidine blue for 1 hour according to Trump (100). - 3 8 -IV. EXPERIMENTAL OBSERVATIOHS 1. Clinical The clinical signs of the 129 mouse strain affected with progressive mus-cular dystrophy were described by Michelson et al (69) in their original report of the occurrence of the mutation. Similar observations have been noted here and are summarized as follows. The f i r s t signs of the disease in mice, dragging of the hind legs and atrophy of musculature of pelvic girdle region, were noted at Ii; days of age (Figures 3 , U). However in numerous animals the signs were noted at 10 - 11 days, and in a couple at 9 days of age. In the latter the affected hind limbs were f i r s t observed after excercising. To detect them at a younger age would be very difficult by this sign alone, although affected ani-mals were noted by about one to two weeks of age to be usually lighter in weight and smaller in size than non-affected mice. There was no i n i t i a l swelling of the muscle. In the case of the animals who had a limb removed at birth, i t was im-possible to detect good signs until about 18 - 21 days of age, since the loss of a hind limb made normal movement difficult. The f i r s t signs of limb dragg-ing were often followed by secondary defects such as poor coat conditions, eye defects and later by a humped back. In some of the affected animals in good physical condition (shiny coat, no eye defects, larger size and delayed waddling gait), the progress of the disease took a somewhat slower course. These animals usually lived beyond six months, generally to 7 or 8 months of age. The oldest one was 260 days (Figure 6 ) . One of the female dystrophics, in excellent con-dition, was bred and delivered young. On the other hand i t was noted that i f an animal was runtish, perhaps with a secondary eye defect, poor coat, and decided waddling gait before the end of the f i r s t month, i t rarely lived beyond the third month. Hence i t was often possible to foretell how long the animal -39-would live when nmscle samples from older animals were needed. As already-mentioned, longevity was greatly increased by improvements on management (71). The head bobbing trait appeared at about 11+ days, the same time as the hind leg symptoms (Figure 2). To our present knowledge the exact significance of this sign was unknown, but i t appeared to be associated with handling and general disturbance. As the disease progressed, the animals dragged the hind limbs until they were unable to return them to the normal position; finally the legs became very stiff and remained in an extended position and any move-ment was done with the fore legs (Figure 5 ) . The back became very hunched, coat very poor with notable mangy areas, caused in part by the inability to clean themselves and free their coats of mites. There was a heavy infection of mites identified as rayobia muculi (95).-The weight of the animal, after reaching a peak at three months, decreased gradually to a minimum of 13 - 15 grams. The fore limbs remained in good con-dition to the very end as the animals were able to hold food and drag them-selves around. From a clinical point of view the peripheral nerves in the hind legs appeared to be normal because i t was noted that, even in the very old dystro-phics, hind leg twitching occurred particularly with fore leg scratching. Thus the signs pointed to a primary atrophy of the muscles. . 2. Light Microscope No histological differences were observed between the muscles of one day old dystrophics (Figure 7) and one day old normals (Figure 8). In both biopsies, interstitial and muscle nuclei were increased in size (up to 16 n in length by 7.0 p. in width) and number when compared to older muscles. The nuclei were distributed haphazardly within the fibers, instead of being loca-lized in the periphery. Often nuclei were seen in chains within the fibers. -ko-They were spherical to ellipsoid in shape rather than elongated as they are in older muscles. Approximately $0% of the oval nuclei and a l l of the spheri-cal ones were pale with prominent nucleoli. Rarely very large round nuclei with a pale matrix and prominent nucleoli were seen. Mitochondria could not be identified at this age. The fibers were notably shorter and narrower than those observed in animals Hi days of age and older. Irregularity in fiber size made accurate measurements difficult; however, they rarely measured greater than 0.3 mm in length by 0.6 u in diameter, compared to the adult forms which measured about 3 - 6 ju in diameter and an immeasurable length. Stria tions were evident but often not in register with adjacent myofibrils. There was a suggestion that the normal fibers were more uniform in size, but this may have been owing to difficulty i n orientation of some of the tissue blocks. By lit days of age normal muscle had the characteristic "clear cut" appear-ance of adult muscle (Figure 9 ) . Although the fibers had not reached their maximum size they were of comparatively uniform diameter and length. The nuclei were sparse and peripherally located. The typical nuclei of one day old muscle had already been replaced by the more elongate adult forms identified by their dark matrix and undistinguishable nucleoli. Connective tissue was sparse. The f i r s t obvious changes in the dystrophic muscle were noted at lk days of age (Figure 10). More nuclei were present than in the normal sibling but less than noted at birth. Many were very prominent because of their similarity to the spherical and ellipsoid nuclei, with pale matrix and prominent nucleoli of the one day old muscle. Centrally located nuclei, in chain formation, were present but to a lesser degree than noted at birth. Some fibers were notably shorter and narrower than in the normal sibling. Interstitial nuclei and connective tissue were easily seen between the atrophic fibers. - l l l -By 21 days of age the normal muscle had assumed the appearance of the mature "clean cut" fibers with scattered peripheral nuclei and scanty connec-tive tissue (Figure 11). This was in direct contrast to the 21 day old dys-trophic muscle fiber, where atrophy was obvious and connective tissue prominent (Figure 12). There were many peripheral and interstitial nuclei usually of an elongatedform, however a few were swollen with spherical shapes and distinct nucleoli, typical of one day old muscle. Some of the fibers lacked uniformity in cross striations because many of the Z-bands were not in register. Long dark streaks between myofibrils were conspicuous throughout some fibers in normal and dystrophic muscles of 21 days of age and older. These streaks could be identified under the electron microscope as mitochondria. The histological changes seen in dystrophic animals from the age of 30 days until death were similar among the age groups selected for the study. The appearance of muscle from 165 day old dystrophics wil l be described. The fibers were noted to vary greatly in length and diameter. Many of4 the fibers were notably atrophic, while some of the larger ones showed signs of hyper-trophy. Some of the fibers lacked regularity in cross striation, while others had a homogenous internal structure and appeared to be remnants of sarcolemmal tubules. The nuclei were conspicuous by their increased numbers and enlarged size. They were oval, with either a pale matrix and prominent nucleoli, or with a dark matrix and indiscernible nucleoli. Nuclei were abundant in the peripheral and interstitial regions and sporadic in the interfibrillar regions. Some nuclei were seen in chain formation between the fibers and appeared to belong to large endometrial tubes. The typical nuclei of one day old muscle were s t i l l evident in 63 and 165 day old dystrophic fibers. Connective tissue was clustered by the atrophic fibers and the remnants of fibers. -2*2-This picture was in direct contrast to the muscle from a 158 day old normal animal where fibers were very uniform in length and diameter with ob-scure peripheral nuclei and "the lack of connective tissue (Figure Hi). Cross striations were parallel adding to the uniformity of the fiber. Rarely a few centrally located nuclei were also present in most of the normal muscles studied, 3 . Electron Microscope The ultrastructure of one day old mouse muscle exhibited notable differ-ences when compared to muscle of normal animals lh days of age and older. Since the structure of one day old muscle does not appear to have been described in detail elsewhere, i t is f e l t that a description is merited even though no qualitative ultrastructural differences have yet been detected between normal one day old muscle and one day old muscle removed from animals which subse-quently became dystrophic. Some of the fibers showed distinct similarity to the altered fibers seen in older dystrophic mice. However not enough specimens have been studied between birth and the f i r s t appearance of atrophic fibers (at lli days of age) to trace their development. The nuclei of newborn muscle were very conspicuous under low magnification because of their large numbers, random distribution and variety in size and shape (Figure 15). Some were torpedo shaped with irregular indentations and tapered ends. Others were elongated with smooth membranes resembling the nuclei of adult muscle. The nuclei were noticeably larger than in the adults, measu-ring 3 - 6 u in width by 8 - 16 u in length while the average adult ones were 1 - 3 p in width by 5 - 12 u in length. The nucleoli were large and composed of very dense granules. Similar dense material was abundant along the peri-phery of the nuclei. The nuclear double membrane was interrupted by pores but no continuities between the nuclear membrane and the endoplasmic reticulum -U3-could be seen. No mitotic figures were noted. The fibers varied in length and diameter (Figures 15, 19). The average contained 10 - 15 myofibrils in longitudinal section, although occasionally much larger fibers were seen. The fine structure of the sarcomeres resembled that of adult muscle (Figure 32), although i t was noted that only about $0% of the Z-bands were in register (Figure 16). The length of the sarcomere in one day old muscle varied from 1.0 - 1.8 n, the average being closer to 1.8 u. In the adult fibers the sarcomere varied from 2 - 2.U u, the I-band being 1,0 u in length and the A-band being l . U u in length. The A-band was composed of coarse and fine filaments. The former had hazy granules attached to them at n o regular intervals of about 280 A, giving a cross-banded structure. Between each coarse filament appeared a finer, less dense filament, which normally stopped at the A,H-band junction but which occasionally was seen to continue through A and I-bands to insert onto the Z-band. Occasionally i t was impossible to trace the fine filaments through the I-band when they were not parallel and left the plane of section. The Z-band was dense and the fine filaments of the I-band merged into i t on either side. On leaving the Z-band the fine filaments appeared to be coarser and denser for a short distance. Many of the smaller myofibrils were separated from each other by large amounts of sarcoplasm (Figures 15, 16). On the other hand, the larger fibers were composed of tightly packed myofibrils with sparse interfibrillar sarcoplasm. Small mitochondria, components of endoplasmic reticulum and small dense granules formed the chief structures wiihin the sarcoplasm. Mitochondria were distribu-ted irregularly between the f i b r i l s (Figure 15). They were not localized near the Z-band as seen in adult normal muscle. Under low power they appeared as dark bodies, while under high power they showed the typical triple-membraned structure with internal cristae and opaque matrix. At the periphery of the -hh-fibers the mitochondria were occasionally bilobular or showed other irregular shapes. The endoplasmic reticulum appeared as tubules or vacuoles without any regular structure within each sarcomere. The regular repeating structure of the endoplasmic reticulum in adult muscle was not seen. A conspicuous feature of one day old muscle was the presence of a large number of granules between the f i b r i l s , at the periphery of, the fibers and at fiber ends (Figure 17). They were concentrated in the vicinity of mitochondria, endoplasmic reticulum and nuclei. A smaller number of granules were also seen between individual myo-fi b r i l s in the I-band and M-bands (illustrated in 63 day old muscle - Figure 23). Two types of granules could be distinguished and these were intermixed (Figures . o 17, 27). Large granules, about 220 A in diameter appeared to be composed of smaller, tightly packed particles about UO A in diameter. These granules re-acted very strongly with lead hydroxide stain. These have been tentatively identified as glycogen since they were similar in appearance and were in the same region of the muscle as the diastase sensitive granules described by Bergman (13). Experiments with diastase digestion, as outlined by Bergman were not successful with methacrylate embedded muscles of either dystrophic or normal . o mice. The second type of granule was about 150 A in diameter. They could be easily overlooked because they stained poorly with lead hydroxide. They were found in clusters between the f i b r i l s , but in the periphery they were also seen attached to one another showing a spiral arrangement, or adhered at regular in-tervals to the outer membranes of the endoplasmic reticulum. These granules were identified as ribonucleoprotein as described by Palade (80) . The Golgi apparatus was seen occasionally near the pole of peripheral and centrally located nuclei (Figure 17). It consisted of single membraned tubules and flattened sacs with a homogenous pale matrix. Its morphology was comparable to the endoplasmic reticulum, but there were no attached granules. Connective tissue elements, fibroblasts and collagen fibers, were identified between muscle fibers, but were not increased in amount in one day old muscle. The above description refers to muscle fibers which had a fine structure most nearly resembling normal adult muscle. The chief differences were the smaller size of fiber, the random distribution of mitochondria and enlarged nuc-l e i , and the presence of abundant interfibrillar sarcoplasm with a conspicuous granular component. Quite frequently these muscle fibers lay adjacent to fibers which showed variations in structure (Figure 19). These latter "atypical" fibers showed many features identified in later dystrophic age groups as being typical of the atrophic fibers. These features were present, both in normal one day old mice and in muscle biopsies of mice that subsequently displayed the clinical features of muscular dystrophy, A conspicuous variation in the structure was the presence of swollen vacuo-lated mitochondria, having a pale matrix and few cristae (Figure 20). They were present uniformly throughout the fiber between the myofibrils. They varied greatly in size from 0;$ p. to 2.0 u. Large vacuoles, measuring up to 1.5 u were seen also, between myofibrils and appeared to represent swollen portions of the endoplasmic reticulum. The vacuoles contained a homogenous material of very low density. The adjacent myofibrils were disorganized with irregular Z-bands and many absent myofilaments. Milder degrees of the same changes in mitochondria and endoplasmic reticulum were often seen in other fibers. At Hi days of age the muscle fine structure resembled very closely that of adult mice. Unfortunately a representative number of animals, between 1 and Hi days, has not been examined to permit a description of this maturation pro-cess, however, i t is probably significant that compact fibers resembling adult -1*6-muscle were seen in the one day old group. Atrophic muscle fibers in the dystrophic mice were f i r s t noted at li * days of age and were conspicuous at 63 days, but further work is necessary to deter-mine the time of onset and nature of the structural differences which become visible as the dystrophic process progresses. Evidence of fiber structural changes at Ik days has been obtained but needs confirmation. By 63 days abnormal atrophic fibers were seen next to normal-appearing ones. There was an overall decrease in the number of myofilaments per myofibril re-sulting in f i b r i l s sometimes less than a third of the normal diameter. This appeared to be a more or less specific feature for dystrophy and rarely was seen in muscle from normal animals (Figure 21). Often incomplete f i b r i l splitting was present for a distance of 5 - 10 sarcomeres. The f i b r i l s usually split and re-united at the Z-bands. These areas were usually surrounded by endoplasmic reticulum, glycogen granules and sometimes mitochondria. F i b r i l splitting was seen rarely in normal muscle but, even when present, i t was localized to a small region of the fiber and did not disrupt the "clean cut" architecture. Atrophic fibers presented a disorganized appearance. Changes, which became more prominent with the progress of the disease, also appeared in the mitochondria and endoplasmic reticulum. Slightly swollen mito-chondria and endoplasmic reticular components, which often formed small vesicles between many normal appearing tubules, could be seen in the aptrophic fibers at 63 days. At 165 days of age the mitochondria were notably swollen (not apparent in Figure 2li) and varied in size (up to 2.1* u in length) and distribution. They were often between myofibrils adjacent to the I-bands. They contained fewer cristae and had a pale matrix. The normal tubular structure of the endoplasmic reticulum was often replaced by large and small pale vacuoles. By 205 and 225 days of age the mitochondria were greatly swollen and vacuolated (Figures 25, 26, 27, 31). They often measured over 3«6 w in length by 1.0 u in width, and extended over the length of a sarcomere, seeming to push the myofibrils out of alignment. The normal endoplasmic reticular, elements (identified in Figure 30) were rarely seen, being replaced by large empty vacuoles. Sarcoplasm was often abundant between the atrophic myofibrils containing the altered mitochondria, components of endoplasmic reticulum, and glycogen and RNA granules. Such areas resembled closely the atypical fibers of one day old muscle. Compare these alterations to the normal 205 fiber in Figures 28 and 30. Normal muscle also contained fibers with pale mitochondria of various sizes but they differed from the altered mitochondria of dystrophic animals (Figure 29). They were large but not swollen and, although they occasionally extended the length of a sarcomere, they did not unduly disturb the adjacent myofibrils and were generally localized within a small area of the fiber. They normally had an orderly system of cristae, but occasionally part of the internal struc-ture was diffuse, obliterating the clear outline of the tubules. This diffuse matrix, however, was noted in mitochondria of normal appearing fibers of dys-trophic animals. The endoplasmic reticulum of these fibers was not vacuolated, but exhibited an elaborate lattice-work of regularly repeating longitudinal and transverse tubules, unlike the vacuolated endoplasmic reticulum of dystrophic fibers. Some nuclei of older dystrophic fibers were also similar to newborn nuclei (Figure 25). They were frequently increased in numbers and size, sometimes dis-playing irregular indentations and enlarged dense nucleoli. They were occa-sionally centrally located within the muscle fiber, either singly or in short chains. Connective tissue, consisting of fine collagen strands, increased greatly -1*8-with "the progress of the disease. It was most abundant in the vicinity of the atrophic fibers especially at fiber ends. It also crowded between adjacent small fibers. This accumulation was in direct contrast to the normal muscles where only small amounts of connective tissue were seen in the interstitial spaces. In the 225 day old altered fibers (Figure 26) two additional elements were noted. The f i r s t structure resembled altered mitochondria in size, general outline and location, however differed by having only a single membrane and no cristae. The general outline of the diffuse matrix resembled the remnants of former cristae. This structure might be interpreted as degenerated mitochondria but confirmation would be necessary in other dystrophic muscle. The other stru-cture was oval to circular in shape and 0.3 - 0.6 u in diameter with a very dense, homogenous matrix and single membrane. They were located in the inter-f i b r i l l a r spaces near mitochondria. A very abnormal Golgi apparatus was seen in an adult dystrophic specimen of 260 days of age (Figure 18) . In contrast to the one day old normal Golgi apparatus, this one was swollen and similar in density to the nearly vacuolated constituents of the endoplasmic reticulum. Miscellaneous structures Multivesicular bodies have been seen in a number of altered fibers of dystrophic animals (Figures 3U, 35) . Their presence has not yet been demon-strated in normal mouse muscle. However, i t is probable that they are present but to a much less degree than in dystrophic muscle. They may be easily over-looked because of their small size (0.2 - 0.1* u in diameter) and their low density. The pale matrix contained six or more small, circular, single nembraned vesi-cles. They were frequently seen in the periphery of the fiber next to -2+9-interfibrillar mitochondria, and externally, in interstitial blood vessels. Another circular body, about 0,5 u in diameter, was seen once i n the vicinity of multivesicular bodies of an altered fiber. It varied from the former by having a small, clear, circular area and seveial tiny vesicles in a dense, granu-lar matrix. This body, like the multivesicular body, had a single membrane, "Myelin figures" were noted in several dystrophic muscles. They appeared as dark membranes wound in a helix, like a watch spring, and were located in the interstitial space and between the f i b r i l s . Their size resembled small mito-chondria, except for the large "myelin figures" observed in the 260 day old dystrophic muscle (Figure 35)• These were surrounded by a clear matrix with a double membrane and the"myelin figures" often displayed a lamellar structure. They were located in abnormal fibers and greatly distorted the adjacent f i b r i l s . Large vesicles with the appearance of "fried eggs" have been noted in normal fibers of both normal and dystrophic animals (Figure 29). They were seldom observed and could be easily overlooked unless fibers were examined under high magnification. They were the size of small mitochondria, about 0.5 n in diameter, and were located between interfibrillar ones at the Z-bands and be-tween peripherally clustered ones. They had a single, irregular membrane and a pale matrix except for a dense, sparsely granular, central area and a sparsely granular peripheral area. -50-V. DISCUSSION In general, the histological observations of dystrophic mouse muscles are the same as those noted by previous workers and closely parallel the pathological characteristics of the Duchenne type of human muscular dystrophy. In one day old mice no notable histological differences were noted between normal and dystrophic muscle. However these muscles are histologically diff-erent from U* day old and older muscle. The variety in fiber size at birth suggests that maturation progresses at variable rates. The length of the sarco-meres is shorter than in adult f i b r i l s . This may be because the f i b r i l s are in a more contracted state during fixation or i t may be possible that growth occurs within the sarcomere. The prominent, large, spherical or slightly ellipsoid nuclei with promi-nent nucleoli are easily differentiated from the more elongate adult forms under the light microscope. Lash et a l (57-),studying the regeneration of skeletal mouse muscle, noted that the nuclei became enlarged and spherical or ellipsoid in shape with prominent nucleoli several days after transection of muscle and these nuclear characteristics preceded the appearance of structural proteins. It may be postulated, therefore, that these nuclear changes are related to the synthesis of nucleoproteins necessary for the production of structural protein. The electron micrographs of one day old muscle reveal additional histo-logical differences compared to older muscle. The nuclei show conspicuous in-foldings of the nuclear membrane, which, according to Lash,reflects either high functional activity or is the result of compression. An elevated level of functional activity appears the likely condition here. The very dark, electron dense, peripheral areas of the nuclei are most evident at birth, but are also present in older dystrophics. It would be necessary to use the Feulgen nucleic acid stain to compare the nucleic acid content of typical enlarged one day old -51-nuclei with the more elongated adult forms. According to Adams ( 1 ) , the nuclei of human muscle are a l l peripherally located by the end of the fourth fetal month; however, in one day old muscle, many are found to be s t i l l centrally located, often in chain formation. It is suggested here that some of the interfibrillar nuclei, especially in dystrophic muscle, never migrate peripherally, which i s contrary to Dowden's (27) sugges-tion that peripheral nuclei migrate centrally during muscle degeneration. According to Lash, neither the significance, nor the mode of relocation of the muscle nuclei during differentiation i s known. Before any conclusion may be made a critical analysis of more young dystrophic animals would be necessary to determine i f a l l the nuclei migrate peripherally before atrophic fibers appear. Since no mitotic figures are noted, probably a l l the nuclei have appeared by birth. If nuclei do not degenerate notably during the l i f e of the dystrophic animal, this may account for the appearance of greater numbers per muscle volume in the young animals. According to Brachet (18), i t has been established that nucleoli are well developed and rich in RNA in a l l cells which are the site of intensive protein synthesis. A number of enlarged nuclei with the characteristic prominent nuc-leoli and dense granular peripheral areas are present at lU , 21, 65, and 165 days of age in dystrophic muscles as observed under the light microscope. It may be assumed that these nuclei are actively producing proteins. Increased amounts of sarcoplasm between the f i b r i l s , at one day of age, probably facilitates the access of nutrients to within the ce l l and the removal of wastes. Glycogen and RNA granules are most abundant at this age and may also reflect active synthesis of proteins for contractile elements. Mitochondria are numerous in the periphery and at fiber ends but are irregularly and spora-tically distributed between the f i b r i l s . This suggests that they may be -52-produced peripherally and migrate inwards, as new f i b r i l s are developing to finally reside at -the I-bands. Thus actively developing fibers may be con-sidered to be ones which contain large amounts of sarcoplasm, many large nuclei, and abundant glycogen and RNA granules within a small caliber fiber. Electron micrographs have shown "atypical" fibers to be present at birth and, although insufficient studies have been made to correlate these findings with the structure of older atrophic dystrophic muscle, the morphological simi-larities are suggestive of a relationship between the two. The mitochondria are swollen and the endoplasmic reticulum vacuolated. These alterations are very similar to those of the older atrophic fibers of dystrophic mice, but a better understanding of the normal histology of muscle at birth and prenatally is necessary before any conclusions can be drawn. The ultrastructural alterations noted here and confirmed by others (73, 90, 102, 103) include atrophy of f i b r i l s and alteration of mitochondria and ii endoplasmic reticular components. Ross et al (90) and Mobert (73) noted a gradual loss of the filaments of the myofibrils which occurred at random sites along the myofibril in such a manner that the myofibril lost its continuity and produced isolated contractile elements within the cell . Ross presented evidence to suggest that the breakdown of the myofibrils resulted from fragmentation and dissolution of the myofilaments. A loss of myofilaments was also noted in this experiment, but atrophy involved the whole fiber rather than isolated areas. Only in one 260 day old specimen were there noted areas of actual degeneration or "breaking down" of the contractile elements which Ross noted at 90 days and which, in this experiment, does not appear to be typical of the disease. The delayed degeneration of the fi b r i l s in this experiment may be the result of healthier animals through improved management. Mobert also noted, in human dystrophics, that in regions of fiber degeneration the Z-bands were seen only -53-sporatically and the myofibrils, no longer distinguishable, were replaced by granular material. Such advanced signs of degeneration are not noted in this ti experiment. From the micrograph present in Mobert's article i t i s very d i f f i -cult to distinguish the ultrastructures of the c e l l . However this may be the result of reproduction difficulties, it Mobert, van Breemen and Ross noted an alteration in the mitochondria of dystrophic humans and mice. In van Breemen's study in human dystrophics, mito-chondria were described as being smaller and denser than in normal muscle. This is i n contrast to Ross who noted that the mitochondria in degenerating cells were larger with irregular shapes and possessed a less dense matrix than seen in normal cells. Ross mentioned that these differences between altered mitochondria were due to species variation or to technical difficulties. The characteristic appearance of mitochondria noted by Ross were also noted in this study. In addition, the mitochondria are often missing between the myofibrils at the I-band region or are located in other areas, mainly at the A-band. Their irregu-larity greatly disturbs the regular architecture as found in normal fibers, n Mobert also noted a loss and irregular distribution in the mitochondria. Ross suggested that the enlarged mitochondria and endoplasmic reticulum may be due to imbibition of fluid. Van Breemen noted that the vacuolated mitochondria sub-sequently underwent disintegration. Van Breemen (103) considered the endoplasmic reticulum to be the f i r s t effected system and the f i r s t to disappear from the cell during degeneration. He noted vacuolation of the endoplasmic reticulum which progressively altered and finally disintegrated. Ross and Mobert also noted a swelling and vacuolation of the reticulum in cells afflicted with dystrophy, but did not mention in what sequence these alterations occurred. Vacuolation was also noted in this inves-tigation and became more apparent with the progress of the disease, -5U-simultaneously with the alteration of the mitochondria and the general dis-organization of the fiber. Although greatly altered in old fibers, vacuolation is s t i l l present. According to Ross there is a distinct loss of f i b r i l s , increased amounts of sarcoplasm, and the occurrence of dense mitochondria in the region occupied by centrally located nuclei in the dystrophic mice. An absence of fi b r i l s was noted in the interfibrillar nuclear region in both dystrophic and normal muscles and appears to be a normal occurrence. As even peripheral nuclei exhibit this phenomenum, no structural elements, which Ross suggested might represent rem-nants of the Z-bands of pre-existing myofibrils, were noted in these central areas. The mitochondria with dense indiscernible matrices may be the result of tech-nical difficulties rather than a dystrophic characteristic. Van Breemen (103) noted a vacuolated Golgi apparatus in dystrophic human muscle and considered i t to be abnormal, Vacuolation has been observed in an abnormal fiber of a 260 day old dystrophic animal. Baker (3) has,discussed the presence of "myelin figures" in tissues which he considered to be due to the unmasking of hydrolytic lipids from lipoprotein complexes to reform themselves into laminated bodies. These myelin figures have been observed in only three animals - a l l dystrophic - 35, 205 and 260 days of age. However since the myelin figures are easily overlooked i t i s presently impossible and uhlikely to conclude that they are confined to dystrophic animals. Multivesicular bodies, noted in dystrophic animals, can easily be missed because of their small size and random distribution. Additional investigations are necessary to determine their location in normal muscle. It is believed that 1hey are related to lyosomes and play a role in pinocytosis (75). They are known to have acid phosphatase activity and are present in a variety of tissues. De Duve (2l|) and Novikoff (75) have also described bodies in liver which are -55-characterized by a single membrane, the presence of many electron opaque gran-ules and small internal cavity. They consider them to be lyosomes. Similar bodies have been observed in this experiment. Lyosomes contain a number of different enzymes such as acid phosphatase, acid ribonuclease and deoxyribo-nuclease and are thought to play a role, in autolysis, intracellular digestion, phagocytosis and pinocytosis (75). However their presence in mouse muscle has not yet been demonstrated. Muscular dystrophy is considered by a l l to be hereditary; however l i t t l e i s known of the way in which a genetic disturbance can produce atrophy and degeneration of muscle fiber at a certain period of l i f e (1) . Adams (1) suggests that i t may be that, through an inborm metabolic fault, certain skele-tal muscles cannot be sustained in a state of health. There have been numerous attempts to discover the fundamental biochemical abnormality. Some workers (70) have found a diminished creatine tolerance in progressive muscular dystrophy and offer this as evidence of a primary disorder of creatine metabolism, but significance of altered creatine and creatinine metabolism has not been ascertained and probably only reflects muscle breakdown (1 , 27). Although i t i s agreed that altered serum enzyme activities , parti-cularly an increase of aldolase, are of considerable value in the diagnosis of muscular dystrophy, normal findings do not exclude the diagnosis. These find-ings appear to be a secondary result of leakage from diseased muscle cells (99) . According to other workers (6 , 16) the increase of phosphatase activity in the connective tissue of muscles i n some types of dystrophy suggest that the phos-phatases play an important part in the destruction of muscle fibers by dephos-phorylating ATP and other high energy phosphates. They suggest that museular dystrophy may be due, fundamentally, to an aberration of the connective tissue. Van Breemen, who considered the endoplasmic reticulum to be the f i r s t -56-effected system, also related this endoplasmic reticular alteration with the histoehemical data of Beckett and Bourne (7) who noted the accumulation of hydrolytic enzymes situated between myofibrils in dystrophic muscle, in parti-cular 5-nucleotidase, and who suggested that these enzymes could cause func-tional failure of muscle through the excessive breakdown of high energy bonds. However van Breemen did not suggest why these enzymes did not also effect mito-chondria at the same time. According to Peachey and Porter (83) the endoplasmic reticulum may play a very important role in the transmission of excitatory impulses intercellularly. If this hypothesis, which is also suggested by Ross, is correct, a defect in this system may prevent conduction to a l l parts of the cell and result in fiber disorganization. This defect may be seen histologically by the presence of vacuolated endoplasmic reticulum and a disorganized arrangement of the fiber especially evident at the individual Z-bands. This vacuolation may be caused by an osmotic gradient towards the endoplasmic reticulum. Muscle atrophy is evident by lh days under the light and electron micro-scopes. Replacement of myofibrils is believed to be in a continuously active state in normal muscle (25) . Dreyfus et al (29 A) have estimated by r a d i o — active work on myosin (which is very similar to that of total myofibril proteins) that the l i f e duration of normal mice myofibrils i s 20 days. Experimental evidence has shown that regeneration does not occur in dystrophic fibers ( 1 ) , therefore i t may be suggested that the primary defect i s in muscle synthesis. This abnormality manifests itself early in l i f e , but does not effect all the -fibers since degrees of atrophy are evident. Although this abnormality is not apparent under the microscope until lU days after birth, i t may already be present at birth, but i s not associated with any morphological changes. Enlarged nuclei with characteristic prominent nucleoli are present in dystrophic fibers. -57-Although this suggests active protein synthesis, i t may be that either the proteins are not converted into contractile elements, or that the contractile elements are being broken down more rapidly than in normal muscle with resulting atrophy of muscle and connective tissue accumulation. Since atrophy is evident there may be some faulty mechanism within the fiber which prevents normal growth. Mitochondria are considered "powerhouses" for energy production. Certain enzymes concerned in oxidative phosphorylation such as cytochrome oxidase and succinic dehydrogenase occur only in the mito-chondria (20) . According to Green (39), mitochondria with greater numbers of cristae per unit volume are capable of carrying out more rapid oxidative reactions. The swollen mitochondria with few cristae present in abnormal fibers may be in-capable of carrying out even sufficient metabolic reactions for the maintenance of muscle. The inadequate release of high energy phosphate bonds within the cell may retard the synthesis of proteins for the repair and maintenance of muscle fibers thus resulting in atrophy. Mitochondria are capable of oxidizing fatty acids to carbon dioxide and water or to acetyl CoA which is used by the TCA cycle. An accumulation of lipids and acetoacetate in dystrophic muscle (38) may indicate that these mitochondria are incapable of oxidizing many of the fatty acids. According to Gould et al (38) accumulation of acetoacetate in dystrophic mice is due to the lack of an enzyme rather than a heat stable cofactor which may indicate the lack of an enzyme in mitochondria. In conclusion, muscular dystrophy appears to be due to a primary defect in the mechanism which produces and maintains the contractile elements. Since dystrophic muscle appears to have lost i ts ability to regenerate, the mechanism responsible for protein synthesis has probably been impaired. Since the nucleoli appear to be actively producing ribonucleo-protein there may be some intermediate stage in the synthesis to contractile elements which is at fault. -58-It i s suggested that the mitochondria, which play such an important role in metabolism, may be faulty and unable to produce enough high energy bonds (ATP) necessary for the energy supply. Fat infiltration, connective tissue prolifera-tion,raay just be secondary factors to the primary atrophy of the muscle fiber. Endoplasmic reticular alteration may be caused by a separate external metabolic upset, but more likely i t is the result of a general change in intracellular environment. -59-VI BIBLIOGRAPHY 1* Adams, R.D., Denny-Brown, D., and Pearson, CM., Diseases of Muscle, New York, Paul B. Hoeber, Inc., 1951*. 2. Bahr, G.F., and Beerman, ¥., Exp. Cell Res., 6 : 519, 1951*. 3 . Baker, J.R., J. Histochem. & Cytol., 6 : 303, 1958. 1*. Baker, N., Blahd, W.H., and Hart, P., Am. J. Physiol., 193: 530, 1958. 5* Beams, H.W., Tahmisian, T.N., Anderson, E., and Devine, R.L., Proc. Soc. Exp. Biol. & Med., 91: 1*73, 1956. 6 . Beckett, E.B., and Bourne, G.H., Science, 126: 357, 1957. 7. Beckett, E.B., and Bourne, G.H., J. Neuropath. & Exp. Neurol., 11: 199, 1958. 8. Bennett, H.S., and Porter, K.R., Am. J. Anat., 93: 61, 1953. 9. Bennett, H.S., Am. J. Physic. Med., 31*: 1*6, 1955. 10. Bennett, H.S., J. Biophysic. & Biochem. Cytol., 2:(Suppl.) 171, 1956. 11. 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Watson, M.L., and Siekevitz, P., J. Biophysic. & Biochem. Cytol., 2: 639, 1956. -65-112. Watson, M.L., J. Biophysic. & Biochem. Cytol., h: 727, 1958. 113. West, W.T., and Murphy, E.D., The Anatomical Record., 137: 279, I960. 111*. Wohlfahrt, S., and Wohlfart, G., Acta Med. Scand., Suppl. 63, 1, 1935. 115. Zymaris, M.C., Saifer, A., and Yolk, B.W., Nature, 118: 323, I960. - 6 6 -VII . ILLUSTRATIONS Figure 1 Apparatus for the preparation of one day old mouse tissue. Figure 2 19 day old dystrophic mouse illustrating the characteristic bobbing of the head. Figure 3 Hi day old female dystrophic mouse with unilateral dragging of the hind leg. Figure It Hi day old dystrophic and normal l i t t e r mate control. Note the larger normal sibling. Figure g 97 day old dystrophic mouse with slightly hunched back, general poor con-dition and dragging of the hind limbs. Figure 6 193 day old male dystrophic mouse with permanently distended legs and secondary eye defect in right eye. Figure 7 Light microscope picture of a one day old dystrophic muscle. The nuclei are large, some measuring 16 u in length by 7 u in width. Many have pale matrix and prominent nucleoli. The fibers are small and the cross stria-tions evident. The dark streaks are folds in the section. The arrow points to a very large round nucleus. Methacrylate embedded, Magnification X 500. - 6 7 -Figure 8 Light microscope of a one day old normal muscle. The histological fea-tures are comparable to Figure 7. The nuclei are prominent and the fibers numerous and small. The arrow points to a string of centrally located nuclei. Methacrylate embedded, Magnification X 500. Figure 9 This lit day old normal muscle has matured greatly from that of one day old muscle in Figure 8. The nuclei are small and sporatic. The fibers are quite regular in size and although they have not reached their adult dimensions, atrophic fibers are not present. The connective tissue is sparse. Methacrylate embedded, Magnification X 500. Figure 10 This Ik day old dystrophic muscle illustrates the characteristic features of dystrophic muscle. The muscle looks disorganized and many of the fibers are atrophic. The nuclei are more numerous than in Figure 9 . Many, similar to one day old nuclei, are large with pale matrix and prom-inent nucleoli. Connective tissue and interstitial nuclei are abundant. Methacrylate embedded, Magnification X 500. Figure 11 Normal 21 day old muscle. The fibers are almost adult size and of regu-lar appearance. The Z-bands are in register. Long dark streaks between the myofibrils of some fibers pointed out by arrows (see also Figure 12) , have been identified under the electron microscope as mitochondria. The nuclei are elongated and scarce. Epon embedded, Magnification X 500. Figure 12 Fiber atrophy is obvious in this 21 day old dystrophic. Small fibers are dispersed among larger normal looking fibers. The nuclei are more numerous -68-than in Figure 11. The connective tissue i s also more abundant. Epon embedded, Magnification X 1+00. Figure 13 The fibers in this longitudinal section of a 165 day old dystrophic muscle, vary greatly in length and diameter. Many are atrophic while some larger ones show signs of hypertrophy. The nuclei are numerous with some centrally located. Connective tissue (CT) is abundant and appears more conspicuous at fiber ends. At the upper right hand corner, indicated by arrow, are numerous dark circles which may be fat droplets. Two capi-llaries, cut in cross-section are seen in the middle area of the picture. Epon embedded, Magnification X 1+00. Figure 11+ The fibers in this 158 day old normal muscle are very uniform in length and diameter. The peripheral nuclei are obscure and the connective tissue is not apparent. Cross-striations are parallel. Epon embedded, Magnification X 500. Figure 15 A low power electron micrograph of a longitudinal section of a one day normal muscle. Note that the central and peripheral nuclei (N) assume various sizes and shapes. The central nuclei are seen in a chain formation. The nucleoli are dense and prominent. The sarcoplasm (S) is very abun-dant between the nuclei and between areas of some f i b r i l s and contain copious amounts of mitochondria and small granules. The length of the sarcomere varies from 1 - 1.8 u. The fibers are narrow and the Z-bands are not always in register. The mitochondria are irregular in distribu-tion and only a few are located in the I-band regions. Epon embedded, FbCOH)^ stained, Magnification X 7,000. - 6 9 -Figure 16 Section through several sarcomeres of a one day old normal muscle. Thick and thin filaments are seen in the A-band. Most of the thin filaments stop at the A-H band junction, howeTOr several are seen traversing the H-band to insert onto the Z-band. The I, A, Z, H-bands and M-line are identified. Cross-bridges are evident in the A-band. The myofibrils are separated by abundant sarcoplasm (S). The FTA stain obscures details of sarcoplasm components. Methacrylate embedded, PTA stained, Magnification X 35,700. Figure 17 An electron micrograph of a one day old mouse muscle. The Golgi apparatus (GA), located just below and to the right of the nucleus (N) consists of fine tubules and vacuoles with single membranes and a homogenous pale matrix. Dark sarcoplasmic granules, probably glycogen (G) are present between the f i b r i l s and in the peripheral sarcoplasm. Paler, smaller granules are dispersed among the darker granules and are also attached to vacuoles and tubules. They are thought to represent ribonucleo-protein. Almost bisecting the micrograph in half, from the top to the bottom, is seen the sarcolemma (SL) which shows numerous pinocytotic vesicles, in-dicated by arrow, on its inner aspect and a continuous basement membrane (B) on i t s outer aspect. Epon embedded, Pb(0H)2 stained, Magnification X 1*5,000. Figure 18 An electron micrograph of a 260 day old dystrophic muscle. The vacuolated Golgi apparatus (GA) is seen below the nucleus. Contrast this with the normal one day old apparatus in Figure 17. Methacrylate embedded, not stained, Magnification X 51,000. -70-Figure 19 A low power electron micrograph of a longitudinal section of one day old normal muscle. It illustrates the two types of fibers, the typical and the atypical. The atypical fiber is seen in the central portion of the picture. It contains large swollen appearing mitochondria (Mit) with a pale matrix. The sarcoplasm is abundant. The typical fiber lying above contains less conspicuous mitochondria with a dark matrix and more compact cristae. The f i b r i l s are more compact with sparse sar-coplasm. The black marks on the micrograph are artifact (lead, carbo-nate precipitate). Methacrylate embedded, Fb(0H)2 stained, Magnification X 7,000. Figure 20 One day old muscle biopsy from an animal which subsequently became dys-trophic. The mitochondria (Mit),.are located at irregular intervals be-tween the myofibrils. They vary notably in size and shape and in many internal empty vacuoles are visible. Small vacuoles (V) are located in the areas normally occupied by endoplasmic reticulum. They are irregular in distribution between the f i b r i l s . Most of the Z-bands (Z) are out of register with one another. The sarcoplasm contains small dense granules abundant in the areas occupied by mitochondria and vacuoles. Note the similarity of this micrograph with that of the older dystrophic fibers in Figure 31. An arrow points to a dark body considered to be a fat droplet. Methacrylate embedded, Pb(0H)2 stained, Magnification X 15,000. Figure 21 This micrograph, although not clear enough to show detailed structures, illustrates the large f i b r i l s of normal, 63 day old muscle. The mito-chondria are mostly restricted to the I-band areas. The longitudinal -71-endoplasmic reticular tubules are seen between the f i b r i l s and are not vacuolated. Since the f i b r i l s are tightly packed together the sarco-plasm i s very scarce. Z-lines are in register. Epon embedded, FbCCK^ stained, Magnification X 15,000. Figure 22 A longitudinal section from an atrophic fiber of a 63 day old dystrophic muscle. The myofibrils are reduced in volume. This micrograph should be compared to the normal fiber in Figure 20. Some of the Z-bands are irregular and not in register. The mitochondria vary in size and shape and give the fiber a variable appearance. Some of the I-band areas are devoid of mitochondria while adjacent areas contain enlarged mitochondria over 2.0 p. in length. The endoplasmic reticulum is not vacuolated in this micrography however in adjacent areas vacuoles are evident. Small dense granules (G) are abundant between the fi b r i l s and within the in-dividual f i b r i l s . One large pale structure located to the left of the interstitial nucleus (N) may represent a mitochrondria. Epon embedded, Fb(0H)2 stained, Magnification X 15,000. Figure 23 A longitudinal section from a 63 day old dystrophic muscle of an almost normal appearing fiber. It illustrates the localization of small dense granules, probably glycogen, in the fibers. The granules (G) are most abundant in the interfibrillar spaces (adjacent to the I-bands) in close association with the endoplasmic reticulum and mitochondria. Intra-f i b r i l l a r l y i t is located in the I-band and to a less degree in the H-band. The granules are not seen in the A-band or the Z-band. Small myo-fib r i l s are seen in the left and right hand side of the micrograph. The large, normal sized f i b r i l s are in the central region. The mitochondria and endoplasmic reticulum of the f i b r i l appear normal. The Z-bands are regular and in register with one another. Epon embedded, FbCOHOg stained, Magnification X 15,000. Figure 2h This electron micrograph illustrates the f i b r i l atrophy of a 165 day old dystrophic muscle. The f i b r i l s in the upper area near the periphery show extreme signs of atrophy. Many of them lack mitochondria at the I-band areas. The mitochondria lack the uniformity of size and position which is noted in Figure 21. Many are very long. The arrow points to some of the transverse endoplasmic reticular elements at the I-A-band junction which show swelling. Sarcoplasm is increased in a patchy manner. Part of an interfibrillar nucleus,which was seen to be in a chain formation, is seen at the lower left hand corner. A few of the Z-bands are irregu-lar. In an adjacent area many of the Z-bands are out of register with one another. Epon embedded, FbCOlOg stained, Magnification X 12,500. Figure 25 An electron micrograph of a 225 day old dystrophic muscle. At the lef t hand side of the micrograph are seen portions of three nuclei (K) which are in the central region of the fiber. They contain greatly enlarged nucleoli. The nuclei are irregular in shape and closely resemble one day old nuclei. Compare them to Figure 15. The myofibrils show variation in size. Many are atrophic. Longitudinal splitting, with increased amounts of sarcoplasm, separates them. The mitochondria vary in distribution, size and shape. The sarcoplasm contains many dense granules (glycogen) concentrated in the I-band areas and between nuclei, Methacrylate embedded, Pb(OH) stained, Magnification X 8,000. ~7> Figure 26 This micrograph of a 225 day old dystrophic muscle illustrates an altered fiber with swollen mitochondria which vary in shape and size. Several dark bodies, indicated by arrows, are located in close association with the mitochondria. Pale bodies with homogenous contents are noted near the bottom and top right hand area of the micrograph. They are also in close association with mitochondria. The Z-bands are irregular and out of register with one another (Z). The endoplasmic reticulum components appear slightly vacuolated. Methacrylate embedded, FbCOIiOg stained, Magnification X 13,500. Figure 27 An electron micrograph of a longitudinal section of muscle from a 225 day old animal illustrating the ultrastrueture of mitochondria. The triple membrane i s seen with the infolding of the inner membrane to form many cristae. The cristae contain an opaque homogenous substance. The arrows point to some of the cristae which appear swollen. Vacuoles (V) are noted between the mitochondria which may represent altered endoplasmic reticular components. Small granules are dispersed between the mitochondria and in the I-band areas. The densely staining granules are composed of smaller tightly packed particles. These are probably glycogen granules (G). The pale, poorly staining granules are smaller and may be ribonucleo-protein. Methacrylate embedded, Pb(0H)2 stained, Magnification X 52,000. Figure 28 A survey picture of longitudinally sectioned fiber from a normal 205 day old mouse. It illustrates the regular repeating structure of the mito-chondria and endoplasmic reticular components. The endoplasmic reticular elements (E) are located in the A-band level and oriented longitudinally -7k-to the long axis of the myofibrils. The mitochondria (Mit) are located at the I-band level of the myofibril. Note uniformity in size and that they do not disrupt the architecture of the contractile elements. Epon embedded, Fb(OH)2 stained, Magnification X 22,500, Figure 29 A longitudinal section of a 20J> day old normal fiber. The mitochondria are very conspicuous because of their large size. Compare them to the small typical mitochondria in Figure 28 and to the altered mitochondria in Figure 31• This fiber may be mistaken for abnormal but on closer examination the endoplasmic reticular elements are seen to be normal and not swollen. The mitochondria, although large, do not appear swollen, lack internal vacuoles and possess a f a i r l y orderly arrangement of cristae. The f i b r i l s are narrower than normal. The arrows point to large vesi-cles, between the mitochondria, with irregular single membranes and a pale dense mass in the center of a clear matrix. Epon embedded, Fb-COHjg stained, Magnification X 22,000. Figure 30 An electron micrograph of a longitudinally sectioned muscle from a 205 day old normal animal. The sarcomere at the right hand side of the pic-ture illustrates the complicated anastomosing channels of the endoplasmic reticulum (E). The other sarcomeres reveal longitudinal components at the level of the A-band which are orientated parallel to the longitudinal axis of the myofibrils. The coarse (C) and fine filaments (F) of the A-band are evident. The fine filament i s seen to terminate at the A-H-band junction but in several instances is seen to extend from one Z-band to the next without interruption. The fine filaments on leaving the Z-band are coarser for a short distance. The cross-striation in the A-band is -75-evident, Epon embedded, Pb(OH)2 stained, Magnification X 31,250. Figure 31 A peripheral region of an altered fiber from a 205 day old dystrophic muscle. The mitochondria are large and swollen (Mit). The cristae are irregular in distribution and in some areas are absent. Compare these mitochondria to those illustrated in a normal fiber in Figure 30. The components of the endoplasmic reticulum are mostly vacuolated (V), how-ever several normal longitudinal tubules can be seen. The f i b r i l s are reduced considerably in diameter. The peripheral nucleus appears normal (N). Epon embedded, Fb(0H)2 stained, Magnification X 28,000. Figure 32 A longitudinal section of a slightly altered fiber from a 205 day old dystrophic muscle. Compare the following structures with those in Figure 31 of a very altered fiber from the same muscle. The mitochondria are not strikingly altered in this fiber. The longitudinal endoplasmic reticular tubules (E) are seen between the myofibrils. Fragments of tubules, as small vesicles of the transverse system (E), are located at the A, I-band boundaries and at the Z-band. The transverse and longitudinal components appear almost normal but evidence of swelling i s obvious (V). The matrix is opaque in many of the tubules and vesicles but is clear in the en-larged vacuoles. The A, I, Z-bands of the sarcomere are indicated. The cross beading in the A-band is obvious. The myofibrils are not compact and the sarcoplasm, although not abundant, is more obvious than in normal fibers. Compare with Figure 28. Some small dense granules are in the sarcoplasm. Epon embedded, Fb(OH) stained, Magnification X 22,500. -76-Figure 33 A very altered region from a 260 day old dystrophic muscle fiber. The mitochondria are very irregular in shape and size. The dark matrix is not typical of most altered mitochondria (Mit) and was not seen in other dystrophic muscles. Large empty appearing vacuoles (V) often in close association with the mitochondria, seem to be the altered components of the endoplasmic reticulum. Large sarcoplasmic spaces (S) between the myo-fi b r i l s are devoid of contractile elements and small dense granules. In some areas (at arrow) the myofilaments appear to be broken off. The Z-bands are irregular (Z). Methacrylate embedded, PbtOHOg stained, Magnification X 1*0,000'.. Figure 3U An area including two multivesicular bodies (MV) and one granular body are indicated by the arrows. They are situated in the sarcoplasm (S) peri-pherally and interfibrillarly. They a l l are invested by a single membrane. The two multivesicular bodies have numerous tiny internal vesicles about the size of those noted in pinocytosis. The granular body contains granules smaller and less dense than those considered to be glycogen granules. It also contains a clear area in the matrix. The granular body is seen close to agranular membranes which resembles a Golgi apparatus. The mitochondria (Mit) are pale, swollen and have sparse cristae. The endoplasmic ret i -culum (V) is swollen and the sarcoplasm is abundant. The myofibrils appear atrophic. A thin section taken from a 196 day old dystrophic muscle. Methacrylate embedded, Fb(OH)2 stained, Magnification X 1*9,500. Figure 35 An electron micrograph of a 260 day old dystrophic muscle. Large "myelin figures" (MF) are seen between the myofibrils of an altered fiber. The -77-central area contains dark lamellar structure while the outer area is clear and bound by an irregular double membrane. A multivesicular body (MV) is seen in the lower lef t hand corner of the micrograph. The Z-bands are very irregular (Z). The endoplasmic reticulum appears as clear vacuoles (V) of irregular sizes. The sarcoplasm i s abundant in the region of the myelin figures. Methacrylate embedded, Pb(OH)2, Magnification X U8,000. ABBREVIATIONS A-band Basement membrane Coarse filament Connective tissue Endoplasmic reticulum Fine filament Fat droplet Glycogen Golgi apparatus H-band I-band M-band Mitochondria Myelin Figure Multivesicular body Nucleus Pinocytotic vesicle Sarcoplasm Sarcolemma Vacuoles Z-band -79-FIGURE 2 -80-FIGURE k FIGURE 5 FIGURE 6 FIGURE 7 FIGURE 8 FIGURE 12 FIGURE 11; FIGUBE 16 FIGURE a FIGURE 25 FIGURE 277 FIGURE 29 -93-FIGURE 31 FIGURE 33 -95-FIGURE 35 

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