Cardiomyopathic hamsters (UM-X7.1 strain) have demonstrable myopathy involving both skeletal and cardiac muscle. In addition, they have multiple ocular abnormalities. In this study, extraocular muscles were examined by light and electron microscopy. Changes observed within affected muscle fibers were variable and included coagulation necrosis, lysis of rnyofibrils, mitochondria1 changes, and infiltration by phagocytic cells. Regenerative changes included duplication of myoblast nuclei, proliferation of sarcoplasmic reticulum, and myofibrillogenesis. The lesions are presumably myogenic in origin. The cardiomyopathic hamster may be useful as an animal model for certain types of ocular myopathy in man. MUSCLE & NERVE
2:288-294
1979
MORPHOLOGIC CHANGES IN EXTRAOCULAR MUSCLES OF THE DYSTROPHIC HAMSTER D. H. PERCY, DVM, J. H. THAKAR, PhD, and K. P. STRICKLAND, PhD
Hereditary polymyopathy has been described in a variety of strains of Syrian hamster including the BIO 14.6 arid UM-X7.1 ~trains.~,”’*~ Light- and electron-microscopic findings have been reported in the B I O 14.6 strain,1’2’5and light-microscopic studies have been performed on cardiac arid skcleta1 muscle of the UM-X7.1 strain.8 The disease is transmitted as an autosomal reccssive gcnc, and lesions of striated muscle are common in mature animals.2~’Death is usually due to congestive heart failure.‘ We have described multiple ocular defects in these animals16 in addition to the cardiomyopathy and muscular dystrophy associated with the UM-X7.1 strain. This report describes the lightmicroscopic and ultrastructural changes observed in the extraocular muscles of the dystrophic UMX7.1 strain.
From the Departments of Microbiology and Immunology (Dr. Percy) and Biochemistry (Drs. Thakar and Strickland), the University of Western Ontario, London, Ontario, Canada. Acknowledgments: This work was supported in part by National Institutes of Health grant EY01833-01 and Medical Research Council of Canada grant MT-617 Address reprint requests to Dr Percy at the Health Sciences Centre, The University of Western Ontario, London, Ontario, Canada N6A 5C1. Received for publication August 18, 1978, revised manuscript accepted for publication February 1 , 1979. 0148-639X10204i0288 $OO.OO/O 0 7979 Houghton Mifflin Professional Publishers
288
Extraocular Muscle of Dystrophic Hamstei
MATERIALS AND METHODS
Hamsters of the UM-X7.1 (dystrophic) strain were obtained from Dr. G. Jasmin, University of Montreal, P.Q., Canada. A total of 12 dystrophic animals, from 1.5 to 7 months old, were studied by light and electron rnic:rosc:opy. Eight nonaffected Syrian hamsters, from 1.5 to 7 months old, were uscd for control extraocular muscle studies. Animals were anesthetized with sodium pentobarbitone, thon perfused via the left ventricle with isotonic saline followed by 3% gliitaraldehyde. After fixation, rectus muscles were collected under a dissecting microscope and portions wcre embedded in paraffin for light-microscopic examination. Other tissues collected for light microscopy included myocardium, quadriceps muscle, and eye. The tissues for electron microscopy were cut into small pieces, dehydrated, embedded i n F,pon o r Spurr’s medium, and uscd for preparat.ion of semithin and thin sections, Grids were examined in a Zeiss EM 9s electron microscope (Oberkochen, West Germany), and microscopic findings were compared with control tissues. RESULTS
When viewed by light microscopy, the majority of fibers in control animals of all ages were well differentiatcd with distinct cross striations. Occasionally, in younger animals, individual miisrle fibers with one or two centrally loControl Hamsters.
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catcd nuclei were seen. Occasionally, there were myoblasts with rounded nuclei and abundant mitochondria in a subsarcolemmal location. Myoblasts were observed in all controls, but they were niost numerous i n animals examined at 1.5 and 2 months of age. When the tissues were examined by electron microscopy, fibers interpreted to be both “twitch” and “slow” were observed (figs. 1 and 2). Cells interpreted to be myoblasts and those that appeared to be sarcoblasts were associated with areas of active myogenesis (figs. 1, 2, and 3). Mitochondria were abundant, both tcithin myoblastic cells and in the sarcoplasm of well-diff’ereritiated fibers (figs. 2 and 3). In the dystrophic hamsters. muscular lesions were detected by light microscopy at the following rate: heart--83%; quadriceps muscles-92%; extraocular muscles-varying 1.1-om 63%’ (dorsal and ventral rectus muscles) to 100% (medial rectus muscle). I n this study, lesions \\-ere first observed in the myocardium at 2 months of age, but changes were present in skeletal muscle beginning at 1.5 months. Lesions were similar to those previously I n the extraocular rnuscles, the changes observed varied from acute degeneration of muscle fibers to regenerative arid reparative changes. For example, in some specimens, loss of cross striations in affected muscle fibers, clumping of sarcoplasmic contents, and concurrent cellular infiltration (fig. 4) \\‘ere seen. In other areas of segmental degcneration, there icas sivelling of the muscle fibers but little evidence of’ cellular infiltration. Duplication of myoblast nuclei and centrally located nuclei were frequcntly observed. I n some instances, degenerate muscle fibers were present in close proximity to hist.ologically normal structures (fig. 4). Dystrophic Hamsters.
Electron Microscopy.
Degenerative and regenera-
active areas. These included both macrophages arid granulocytic: cells (fig. 6). In some lesions interpreted to be of recent occurrence, there rcas coagulation necrosis of muscle fibers, with contraction and loss of Z lines. Fragmentation and swelling of mitochondria, and the concurrent deposition of electron-dense degcnerate material, were sometimes observed (figs. 7 and 8). Areas of degeneration n.ithin muscle fibers varied in size, intensity, and stage of‘ development. Some lesions were focal in nature and were composed prirnarily of fragmented muscle fibers, with deposition of electron-dense material and disruption of’ the normal architecture. Other lesions of the extraocular muscles were segmental in nature, Tvith complete absence of myofibrils and striations, presence of centrally located sarcoplasink nuclei. and clear demarcation between degenerate parts of muscle fibers and adjacent. intact parts (figs. 9 and 10). Reac:tive satellite cells and myoblasts w.2.ere a striking feature in damaged arcas (figs. 6 and I O), and indicated the regenerative potential of aflected muscle. I n one section, a mitotic myoblast was observed (lig. 8). Mitochondria, sarcoplasmic reticulum, and free ribosomes were frequently present in abundance in the reactive sarcoplasm (fig. 10). Iri advanced lesions of extraocular muscle, changes varied fi-om myogenesis to fibrosis. Occasionally, within al’fected muscle fibers uridergoing regeneration, there were identifiable bundles of myofibrils and Z bands, dilated sarcoplasmic reticulum, and numerous mitochondria of varying shapes and sizes (figs. $1 and 10). In some lesions, there was evidence of scarring, with collapse of sarcolemnial sheaths, hagmentation of muscle fibers, and presence of excessive amounts of collagen fibers, with distortion arid separation of individual muscle fibers.
tivc: changes varied and included nuclear abnor-
malities, fragmentation and coagulation necrosis and lysis 01’ myofihrils. alteration in appearance arid numbers of mitochondria, swelling of the sarcnplasmic reticulum, inflammatory cell infiltration, and cdlagenous tissue proliferation. Duplication of satellite cells and myoblasts was a common linding in affected muscle fibers. Reactive nuclei Tvere in close apposition, frequently centrally located, arid often accompanied by minimal sarcoplasmic change (fig. 5); this change was not observed in control anirnals. Infiltrating inflammatory cells w r e observed both within degenerating muscle fibers arid in perivascular regions adjacent t o re-
Extraocular Muscle of Dystrophic Hamster
DISCUSSION
The morphology of extraocular muscles has been studied in a variety of animals;7*’“however, f e ~ data are available on the fine structures of extraocular muscle in the hamster. Both the singly innervated “twitch” libers and the multiply innel-vated “ s l o ~ v ”fibers tvere identified in our study. Morphologic criteria used to identify slow fibers have included irregular, poorly defined myofibrils; zigzag Z lines; and an irregular, poorly defined T system.7The criteria uscd to categorize extraocular niuscle fibers vary. For example, Peachey divided
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Figure 1 . Extraocular muscle from control hamster examined at age 1.5 months. Note that the nucleus (n) of the myoblast has irregular borders and numerous sarcosornes and mitochondria in the sarcoplasm. The well-differentiated myofibrils (lower right) have wide, but zigzag, Z lines and a poorly defined T system, criteria characteristic of slow fibers. Bar = 1.0 pm.
Figure 2. Extraocular muscle from control hamster, age 2 months. Within developing fiber is the nucleus (n) of a sarcoblast. Mitochondria (rn) are abundant in this cell, and in adjacent well-differentiated fibers. Note zigzag alignment of Z bands of slow fibers (arrowheads). Bar = 2.5 pm.
Figure 3 . Extraocular muscle from control hamster at age 5 months. Note the variations in shape and size of the mitochondria (m). Distinct Z bands (z)are present in the developing sarcomeres, but the A bands arc not evident at this stage of development. Bar = 1 .O pm.
290
Extraocular Muscle of Dystrophic Hamster
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F/gure 4 Extraocular muscle (lateral rectus) from dystrophfc hamster (#4023) exammed at age 1 5 months Sarcoplasm of degenerate muscle hber contams centrally located nucler and granular material Note the clearly debneated cross striations / n adjacent muscle f/bers Toluidine blue, bar = 25 p.m
Figure 5. Medial rectus muscle from dystrophic hamster (#4025) at age 1.5 months. Several sarcoblast nuclei appear to be aligned within the sarcoplasm, but no changes are detectable in adjacent muscle fibers. Bar = 2.5 pm.
Figure 6 Medlal rectus muscle of dystrophic hamster (#4022) at age 2 months Area of acute degeneration with mfdtratmg granulocytic cells (P) and reactwe satellite cell (S) assmated w/th les/ons Some d/lat/onof sarcoplasmic retculum IS seen Bar = 2 5 p.m
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Figure 7. Ventral rectus muscle in dystrophic hamster (#3981j age 5 months. Focus of acute degeneration contains electron-dense ceiluiar debris (center) and homogeneous giobuies (arrow), which are probabiy lysosomes Bar = 2.5 pn.
Figure 8 Medlai rectus from dystrophic hamster (#3982) exammed at age 5 months Mitotic ceii IS probably a diwdmg rnyobiast Note myobiast nucleus (upper right), segmentai fragmentatmn of muscle f/ber at lower /eft, and electron-dense celiular debris (arrow) Bar = 2 5 p m
292
Extraocular Muscle of Dystrophic Hamster
the fibers into five types using criteria such as diameter, location, shape of fibrils, characteristics of' the Z and M lines, and type of inriervation.'" Of interest in our study of normal animals as the degree of myogenesis evident in hamsters of all ages. Reactive inyoblasts seen in the extraoc:ular muscles of control hamsters. when present, were morphologically similar to those observed in thc extraocular muscles of dystrophic animals. Ho"cver, the concurrent degenerative changes arid inflammatory cell infiltrates frequeritly seen i n reactive areas in dystrophic hamsters were not present in controls. 'l'he histologic changes observed in our studies of the extraocular muscles of the UM-X7.1 hamster are similar in many respects to the ultrastructural changes described in skeletal muscles of' the BIO 14.6 strain.' Changes observed in the latter strain iriclude distention of the sarcotubular system, deposition of lipid droplets within muscle fibers, etlcrna, dissolution of myofilaments, collapse of muscle fibers, and rriineraliation of sarc-oplasmic contents. In Chulfield's study,' most of the ultrastructural changes depicted were collected from hamsters after swimming exercise, which markedly increased the intensity of t h e lesions. Acute, degenerative niuscular changes appeared to 1~ t h c predominant finding. which lvas probably related primarily to Caulfield's experimental design. Important ultrastructural findings observed in our studies included those interpreted to be regenerative arid those thought to be reparative changes. R e z r ~ i k 'has ~ described the typical morphologic changes that occur in regenerative niuscle, including proliferation of satellite cells and rriyoblasts. I n our study, the duplication of satellite cells, the formation of niyoblasts, and the presencc of increased numbei-s of mitochondria and sai-coplasrnic reticulum within the sarcoplasm of reactive muscle fibers I\ ere considered t o be evidence of the regenerative capacity of al'fected hamsters. Neither the degenerative nor the regenerative changes tvere characteristic of a particular agc group. For example, while fibrosis secondary to dystrophic muscle change was observed in one animal at five months, acute sarcoplasniic changes and rnyogenesis were observed in the extraocular m i d e s of animals u p t o seven months of age. These findings indicate that age is not a limiting factor in the evolution (and possible resolution) of muscle lesions. Ultrasrructural studies have bccri performed on extraocular muscles of certain strains of dystrophic mice. Changes described in the Re-129
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Figure 9. Medial rectus muscle from dystrophic hamster (#4025) examined at age 1.5 months. Within the sarcoplasm of degenerate muscle fibers are numerous mitochondria (m), cytoplasmx degradation products (arrow), and vacuolated areas Interpreted to be degenerating mitochondria. Other vacuolated areas are probably dilated sarcoplasmic reticulum. Proliferating myofibrils, with idenbfiable Z bands (z),are abundant in some segments. Bar = 2.5 p n
Figure 10 Medial rectus muscle from dystrophic hamster (#4022), age 2 months Dupkcation of myoblast nuclei, with numerous mitochondria and prominent sarcoplasmic rebculum IS seen (arrows) Note /denbhable satelhte cell (S) Bar = 2 5 pni
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dy/dy strain include centrally located nuclei, sarcoplasmic vacuolation, mitochondria1 swelling, and lipid accumulation." Ultrastructural SI udies of' extraocular muscles of the C57 B1/6Jdy2j mouse emphasized changes observed at the neuromuscular j ~ n c t i o n .Similar ~ , ~ ~ changes were not observed in our preliminary studies, and the muscular lesions present in the UM-X7.1 hamster are presumably of myogenic origin; howrever, a more detailed ultrastructural study of the motor endplates is required. Recently, the acid phosphatase levels in dystrophic hamster muscle have been studied as an indicator of the concurrent sarcoplasmic degeneration,2 and elevated creatirie kinase is associated with the disease.6 Kuwabara and Lessell'" described the changes in extraocular muscle of two fatal cascs of human myotonic dystrophy. Abnormalities similar to those observed in our dystrophic-hamster tissue included centrally located nuclei, proliferation and irregular alignment of myofilaments, presence of large and irregular mitochondria, and irregular distribution of sarcoplasmic reticulum. They noted
the similarities between the abnormal fibers and the appearance of developing skeletal muscle cells. They suggested that the pathogenesis of the myopathy in myotonic dystrophy may be related to abnormal production and maintenance of myofibrils. We also have been impressed with the morphologic similarities between regenerating muscle fibers in dystrophic hamsters and normal myogenesis in control animals. However, structures such as degenerating organelles, mineralized debris, and infiltrating phagocytic cells were features seen only in the dystrophic animals. Cases of ocular myopathy associated with progressive muscular dystrophy have been observed in Croft et aP reviewed the recorded cases of ocular myopathy, or progressive external ophthalmoplegia, which also appears to be a hereditary disease. The changes described include mitorhondrial abnormalities in affected muscle fibers and other cells, cardiornyopathy, and pigmentary retinal degeneration. Similarly, in the UM-X7.1 dystrophic hamster, concurrent ocular lesions and cardiomyopathy are features oP the disease.
REFERENCES
1. Caulfield JB: Electron microscopic observations on the dystrophic- hamstcr muscle, Ann Y \i Acad Sci 138:151-159, 1966. 2. Cristie KN, Stoward PI: A cvtochernical study of acid phosphatase in dystrophic-hamster muscle. J Ultrktruct R k 58: 219-234, 1977. 3. Croft PR, Cuttin JC, Jewesbury EGO, Blackwood W, Mair WGP: Ocular myopathy (progressive external ophthalmoplegia) with neuropathic complications. Acta Neurol Scand 55:169-197, 1977. 4. Davidowitz J , Pachter- BR, Phillips G , Breinin GM: Structural alterations of the Junctional region in extraocular muscle of dystrophic mice. I. Modifications of sole-plate nuclei. Am J Pathol 82: 10 1 - 110, 1976. 5. Gertz EW: Cardiomyopathic Syrian hamster: a possible model of human disease. Prog Exp Tumor Res 16:242-260, 1972. 6. Hadlow WJ: Myopathies in animals. Pearson CM, Mostofi FK (Editors): The Striated Muscle. Williams & Wilkins, 1973, pp 364-409. 7. Hess A: T h e structure of vertebrate slow and twitch muscle fibers. Invest Ophthalmol 6217-228, 1967. 8. Jasmin G , Bajusz E: Polymyopathie et cardiomyopathie hkrtditaire chez le hamster d e Syrie. Inhibition stlective des lesions d u myocarde. Ann Anat Pathol (Paris) 18:49-66, 1973.
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9. Kiloh LG, Nevin S: Progressive dystrophy of the external ocular muscles. Bruin 74:115-143, 1951. 10. Kuwabara T, Lessell S: Electron microscopic study of extraocular musc-les in inyotonic dystrophy. Am J Ophthalmol 82:303-309, 1976. 11. Nicolaissen B, Brodal A: Chronic progressive external ophthalmoplegia. Report of a rase with histopathologic examination of external eye muscle and skeletal muscle. Arch Ophthalmol 6 1:202-2 10, 1959. 12. Pachter BR, Davidowitz J, Breinin GM: A phase-electron microscopic study of extraocular muscle dystrophy in the mouse. Invest Ophthalmol 11:715-722, 1972. 13. Pachter BR, Davidowitz J , Breinin GM: Structural alterations of the junctional region in extraocular muscle of dystrophic mice. 11. Hypertrophy of the neuromusrular~junction apparatus. Am JPathol 82: 1 1 1- 118, 1976. 14. Peachey L: T h e structure of the extraocular muscle fibers of mammals. I n Bach-Y-Rita P, Collins CC (Editors): The Control OfEye Mnoements. New York, Academic Press, 1971, p p 47-66. 15. Reznik M: Current concepts of skeletal muscle rcgeneration. I n Pearson CM, Mostofi FK (Editors): The Striated iMuscb. Baltimore, Williams & Wilkins, 1973, p p 185-225. 16. Thakar JH, Percy DH, Strickland KP: Ocular ahnormalitics in the myopathic hamster (UM-X7.1). Invest Ophthalmol 16:1047-1052, 1977.
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JuliAug 1979
2:288-294
1979
MORPHOLOGIC CHANGES IN EXTRAOCULAR MUSCLES OF THE DYSTROPHIC HAMSTER D. H. PERCY, DVM, J. H. THAKAR, PhD, and K. P. STRICKLAND, PhD
Hereditary polymyopathy has been described in a variety of strains of Syrian hamster including the BIO 14.6 arid UM-X7.1 ~trains.~,”’*~ Light- and electron-microscopic findings have been reported in the B I O 14.6 strain,1’2’5and light-microscopic studies have been performed on cardiac arid skcleta1 muscle of the UM-X7.1 strain.8 The disease is transmitted as an autosomal reccssive gcnc, and lesions of striated muscle are common in mature animals.2~’Death is usually due to congestive heart failure.‘ We have described multiple ocular defects in these animals16 in addition to the cardiomyopathy and muscular dystrophy associated with the UM-X7.1 strain. This report describes the lightmicroscopic and ultrastructural changes observed in the extraocular muscles of the dystrophic UMX7.1 strain.
From the Departments of Microbiology and Immunology (Dr. Percy) and Biochemistry (Drs. Thakar and Strickland), the University of Western Ontario, London, Ontario, Canada. Acknowledgments: This work was supported in part by National Institutes of Health grant EY01833-01 and Medical Research Council of Canada grant MT-617 Address reprint requests to Dr Percy at the Health Sciences Centre, The University of Western Ontario, London, Ontario, Canada N6A 5C1. Received for publication August 18, 1978, revised manuscript accepted for publication February 1 , 1979. 0148-639X10204i0288 $OO.OO/O 0 7979 Houghton Mifflin Professional Publishers
288
Extraocular Muscle of Dystrophic Hamstei
MATERIALS AND METHODS
Hamsters of the UM-X7.1 (dystrophic) strain were obtained from Dr. G. Jasmin, University of Montreal, P.Q., Canada. A total of 12 dystrophic animals, from 1.5 to 7 months old, were studied by light and electron rnic:rosc:opy. Eight nonaffected Syrian hamsters, from 1.5 to 7 months old, were uscd for control extraocular muscle studies. Animals were anesthetized with sodium pentobarbitone, thon perfused via the left ventricle with isotonic saline followed by 3% gliitaraldehyde. After fixation, rectus muscles were collected under a dissecting microscope and portions wcre embedded in paraffin for light-microscopic examination. Other tissues collected for light microscopy included myocardium, quadriceps muscle, and eye. The tissues for electron microscopy were cut into small pieces, dehydrated, embedded i n F,pon o r Spurr’s medium, and uscd for preparat.ion of semithin and thin sections, Grids were examined in a Zeiss EM 9s electron microscope (Oberkochen, West Germany), and microscopic findings were compared with control tissues. RESULTS
When viewed by light microscopy, the majority of fibers in control animals of all ages were well differentiatcd with distinct cross striations. Occasionally, in younger animals, individual miisrle fibers with one or two centrally loControl Hamsters.
MUSCLE & NERVE
JuliAug 1979
catcd nuclei were seen. Occasionally, there were myoblasts with rounded nuclei and abundant mitochondria in a subsarcolemmal location. Myoblasts were observed in all controls, but they were niost numerous i n animals examined at 1.5 and 2 months of age. When the tissues were examined by electron microscopy, fibers interpreted to be both “twitch” and “slow” were observed (figs. 1 and 2). Cells interpreted to be myoblasts and those that appeared to be sarcoblasts were associated with areas of active myogenesis (figs. 1, 2, and 3). Mitochondria were abundant, both tcithin myoblastic cells and in the sarcoplasm of well-diff’ereritiated fibers (figs. 2 and 3). In the dystrophic hamsters. muscular lesions were detected by light microscopy at the following rate: heart--83%; quadriceps muscles-92%; extraocular muscles-varying 1.1-om 63%’ (dorsal and ventral rectus muscles) to 100% (medial rectus muscle). I n this study, lesions \\-ere first observed in the myocardium at 2 months of age, but changes were present in skeletal muscle beginning at 1.5 months. Lesions were similar to those previously I n the extraocular rnuscles, the changes observed varied from acute degeneration of muscle fibers to regenerative arid reparative changes. For example, in some specimens, loss of cross striations in affected muscle fibers, clumping of sarcoplasmic contents, and concurrent cellular infiltration (fig. 4) \\‘ere seen. In other areas of segmental degcneration, there icas sivelling of the muscle fibers but little evidence of’ cellular infiltration. Duplication of myoblast nuclei and centrally located nuclei were frequcntly observed. I n some instances, degenerate muscle fibers were present in close proximity to hist.ologically normal structures (fig. 4). Dystrophic Hamsters.
Electron Microscopy.
Degenerative and regenera-
active areas. These included both macrophages arid granulocytic: cells (fig. 6). In some lesions interpreted to be of recent occurrence, there rcas coagulation necrosis of muscle fibers, with contraction and loss of Z lines. Fragmentation and swelling of mitochondria, and the concurrent deposition of electron-dense degcnerate material, were sometimes observed (figs. 7 and 8). Areas of degeneration n.ithin muscle fibers varied in size, intensity, and stage of‘ development. Some lesions were focal in nature and were composed prirnarily of fragmented muscle fibers, with deposition of electron-dense material and disruption of’ the normal architecture. Other lesions of the extraocular muscles were segmental in nature, Tvith complete absence of myofibrils and striations, presence of centrally located sarcoplasink nuclei. and clear demarcation between degenerate parts of muscle fibers and adjacent. intact parts (figs. 9 and 10). Reac:tive satellite cells and myoblasts w.2.ere a striking feature in damaged arcas (figs. 6 and I O), and indicated the regenerative potential of aflected muscle. I n one section, a mitotic myoblast was observed (lig. 8). Mitochondria, sarcoplasmic reticulum, and free ribosomes were frequently present in abundance in the reactive sarcoplasm (fig. 10). Iri advanced lesions of extraocular muscle, changes varied fi-om myogenesis to fibrosis. Occasionally, within al’fected muscle fibers uridergoing regeneration, there were identifiable bundles of myofibrils and Z bands, dilated sarcoplasmic reticulum, and numerous mitochondria of varying shapes and sizes (figs. $1 and 10). In some lesions, there was evidence of scarring, with collapse of sarcolemnial sheaths, hagmentation of muscle fibers, and presence of excessive amounts of collagen fibers, with distortion arid separation of individual muscle fibers.
tivc: changes varied and included nuclear abnor-
malities, fragmentation and coagulation necrosis and lysis 01’ myofihrils. alteration in appearance arid numbers of mitochondria, swelling of the sarcnplasmic reticulum, inflammatory cell infiltration, and cdlagenous tissue proliferation. Duplication of satellite cells and myoblasts was a common linding in affected muscle fibers. Reactive nuclei Tvere in close apposition, frequently centrally located, arid often accompanied by minimal sarcoplasmic change (fig. 5); this change was not observed in control anirnals. Infiltrating inflammatory cells w r e observed both within degenerating muscle fibers arid in perivascular regions adjacent t o re-
Extraocular Muscle of Dystrophic Hamster
DISCUSSION
The morphology of extraocular muscles has been studied in a variety of animals;7*’“however, f e ~ data are available on the fine structures of extraocular muscle in the hamster. Both the singly innervated “twitch” libers and the multiply innel-vated “ s l o ~ v ”fibers tvere identified in our study. Morphologic criteria used to identify slow fibers have included irregular, poorly defined myofibrils; zigzag Z lines; and an irregular, poorly defined T system.7The criteria uscd to categorize extraocular niuscle fibers vary. For example, Peachey divided
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Figure 1 . Extraocular muscle from control hamster examined at age 1.5 months. Note that the nucleus (n) of the myoblast has irregular borders and numerous sarcosornes and mitochondria in the sarcoplasm. The well-differentiated myofibrils (lower right) have wide, but zigzag, Z lines and a poorly defined T system, criteria characteristic of slow fibers. Bar = 1.0 pm.
Figure 2. Extraocular muscle from control hamster, age 2 months. Within developing fiber is the nucleus (n) of a sarcoblast. Mitochondria (rn) are abundant in this cell, and in adjacent well-differentiated fibers. Note zigzag alignment of Z bands of slow fibers (arrowheads). Bar = 2.5 pm.
Figure 3 . Extraocular muscle from control hamster at age 5 months. Note the variations in shape and size of the mitochondria (m). Distinct Z bands (z)are present in the developing sarcomeres, but the A bands arc not evident at this stage of development. Bar = 1 .O pm.
290
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F/gure 4 Extraocular muscle (lateral rectus) from dystrophfc hamster (#4023) exammed at age 1 5 months Sarcoplasm of degenerate muscle hber contams centrally located nucler and granular material Note the clearly debneated cross striations / n adjacent muscle f/bers Toluidine blue, bar = 25 p.m
Figure 5. Medial rectus muscle from dystrophic hamster (#4025) at age 1.5 months. Several sarcoblast nuclei appear to be aligned within the sarcoplasm, but no changes are detectable in adjacent muscle fibers. Bar = 2.5 pm.
Figure 6 Medlal rectus muscle of dystrophic hamster (#4022) at age 2 months Area of acute degeneration with mfdtratmg granulocytic cells (P) and reactwe satellite cell (S) assmated w/th les/ons Some d/lat/onof sarcoplasmic retculum IS seen Bar = 2 5 p.m
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Figure 7. Ventral rectus muscle in dystrophic hamster (#3981j age 5 months. Focus of acute degeneration contains electron-dense ceiluiar debris (center) and homogeneous giobuies (arrow), which are probabiy lysosomes Bar = 2.5 pn.
Figure 8 Medlai rectus from dystrophic hamster (#3982) exammed at age 5 months Mitotic ceii IS probably a diwdmg rnyobiast Note myobiast nucleus (upper right), segmentai fragmentatmn of muscle f/ber at lower /eft, and electron-dense celiular debris (arrow) Bar = 2 5 p m
292
Extraocular Muscle of Dystrophic Hamster
the fibers into five types using criteria such as diameter, location, shape of fibrils, characteristics of' the Z and M lines, and type of inriervation.'" Of interest in our study of normal animals as the degree of myogenesis evident in hamsters of all ages. Reactive inyoblasts seen in the extraoc:ular muscles of control hamsters. when present, were morphologically similar to those observed in thc extraocular muscles of dystrophic animals. Ho"cver, the concurrent degenerative changes arid inflammatory cell infiltrates frequeritly seen i n reactive areas in dystrophic hamsters were not present in controls. 'l'he histologic changes observed in our studies of the extraocular muscles of the UM-X7.1 hamster are similar in many respects to the ultrastructural changes described in skeletal muscles of' the BIO 14.6 strain.' Changes observed in the latter strain iriclude distention of the sarcotubular system, deposition of lipid droplets within muscle fibers, etlcrna, dissolution of myofilaments, collapse of muscle fibers, and rriineraliation of sarc-oplasmic contents. In Chulfield's study,' most of the ultrastructural changes depicted were collected from hamsters after swimming exercise, which markedly increased the intensity of t h e lesions. Acute, degenerative niuscular changes appeared to 1~ t h c predominant finding. which lvas probably related primarily to Caulfield's experimental design. Important ultrastructural findings observed in our studies included those interpreted to be regenerative arid those thought to be reparative changes. R e z r ~ i k 'has ~ described the typical morphologic changes that occur in regenerative niuscle, including proliferation of satellite cells and rriyoblasts. I n our study, the duplication of satellite cells, the formation of niyoblasts, and the presencc of increased numbei-s of mitochondria and sai-coplasrnic reticulum within the sarcoplasm of reactive muscle fibers I\ ere considered t o be evidence of the regenerative capacity of al'fected hamsters. Neither the degenerative nor the regenerative changes tvere characteristic of a particular agc group. For example, while fibrosis secondary to dystrophic muscle change was observed in one animal at five months, acute sarcoplasniic changes and rnyogenesis were observed in the extraocular m i d e s of animals u p t o seven months of age. These findings indicate that age is not a limiting factor in the evolution (and possible resolution) of muscle lesions. Ultrasrructural studies have bccri performed on extraocular muscles of certain strains of dystrophic mice. Changes described in the Re-129
MUSCLE & NERVE
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Figure 9. Medial rectus muscle from dystrophic hamster (#4025) examined at age 1.5 months. Within the sarcoplasm of degenerate muscle fibers are numerous mitochondria (m), cytoplasmx degradation products (arrow), and vacuolated areas Interpreted to be degenerating mitochondria. Other vacuolated areas are probably dilated sarcoplasmic reticulum. Proliferating myofibrils, with idenbfiable Z bands (z),are abundant in some segments. Bar = 2.5 p n
Figure 10 Medial rectus muscle from dystrophic hamster (#4022), age 2 months Dupkcation of myoblast nuclei, with numerous mitochondria and prominent sarcoplasmic rebculum IS seen (arrows) Note /denbhable satelhte cell (S) Bar = 2 5 pni
Extraocular Muscle of Dystrophic Hamster
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dy/dy strain include centrally located nuclei, sarcoplasmic vacuolation, mitochondria1 swelling, and lipid accumulation." Ultrastructural SI udies of' extraocular muscles of the C57 B1/6Jdy2j mouse emphasized changes observed at the neuromuscular j ~ n c t i o n .Similar ~ , ~ ~ changes were not observed in our preliminary studies, and the muscular lesions present in the UM-X7.1 hamster are presumably of myogenic origin; howrever, a more detailed ultrastructural study of the motor endplates is required. Recently, the acid phosphatase levels in dystrophic hamster muscle have been studied as an indicator of the concurrent sarcoplasmic degeneration,2 and elevated creatirie kinase is associated with the disease.6 Kuwabara and Lessell'" described the changes in extraocular muscle of two fatal cascs of human myotonic dystrophy. Abnormalities similar to those observed in our dystrophic-hamster tissue included centrally located nuclei, proliferation and irregular alignment of myofilaments, presence of large and irregular mitochondria, and irregular distribution of sarcoplasmic reticulum. They noted
the similarities between the abnormal fibers and the appearance of developing skeletal muscle cells. They suggested that the pathogenesis of the myopathy in myotonic dystrophy may be related to abnormal production and maintenance of myofibrils. We also have been impressed with the morphologic similarities between regenerating muscle fibers in dystrophic hamsters and normal myogenesis in control animals. However, structures such as degenerating organelles, mineralized debris, and infiltrating phagocytic cells were features seen only in the dystrophic animals. Cases of ocular myopathy associated with progressive muscular dystrophy have been observed in Croft et aP reviewed the recorded cases of ocular myopathy, or progressive external ophthalmoplegia, which also appears to be a hereditary disease. The changes described include mitorhondrial abnormalities in affected muscle fibers and other cells, cardiornyopathy, and pigmentary retinal degeneration. Similarly, in the UM-X7.1 dystrophic hamster, concurrent ocular lesions and cardiomyopathy are features oP the disease.
REFERENCES
1. Caulfield JB: Electron microscopic observations on the dystrophic- hamstcr muscle, Ann Y \i Acad Sci 138:151-159, 1966. 2. Cristie KN, Stoward PI: A cvtochernical study of acid phosphatase in dystrophic-hamster muscle. J Ultrktruct R k 58: 219-234, 1977. 3. Croft PR, Cuttin JC, Jewesbury EGO, Blackwood W, Mair WGP: Ocular myopathy (progressive external ophthalmoplegia) with neuropathic complications. Acta Neurol Scand 55:169-197, 1977. 4. Davidowitz J , Pachter- BR, Phillips G , Breinin GM: Structural alterations of the Junctional region in extraocular muscle of dystrophic mice. I. Modifications of sole-plate nuclei. Am J Pathol 82: 10 1 - 110, 1976. 5. Gertz EW: Cardiomyopathic Syrian hamster: a possible model of human disease. Prog Exp Tumor Res 16:242-260, 1972. 6. Hadlow WJ: Myopathies in animals. Pearson CM, Mostofi FK (Editors): The Striated Muscle. Williams & Wilkins, 1973, pp 364-409. 7. Hess A: T h e structure of vertebrate slow and twitch muscle fibers. Invest Ophthalmol 6217-228, 1967. 8. Jasmin G , Bajusz E: Polymyopathie et cardiomyopathie hkrtditaire chez le hamster d e Syrie. Inhibition stlective des lesions d u myocarde. Ann Anat Pathol (Paris) 18:49-66, 1973.
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Extraocular Muscle of Dystrophic Hamster
9. Kiloh LG, Nevin S: Progressive dystrophy of the external ocular muscles. Bruin 74:115-143, 1951. 10. Kuwabara T, Lessell S: Electron microscopic study of extraocular musc-les in inyotonic dystrophy. Am J Ophthalmol 82:303-309, 1976. 11. Nicolaissen B, Brodal A: Chronic progressive external ophthalmoplegia. Report of a rase with histopathologic examination of external eye muscle and skeletal muscle. Arch Ophthalmol 6 1:202-2 10, 1959. 12. Pachter BR, Davidowitz J, Breinin GM: A phase-electron microscopic study of extraocular muscle dystrophy in the mouse. Invest Ophthalmol 11:715-722, 1972. 13. Pachter BR, Davidowitz J , Breinin GM: Structural alterations of the junctional region in extraocular muscle of dystrophic mice. 11. Hypertrophy of the neuromusrular~junction apparatus. Am JPathol 82: 1 1 1- 118, 1976. 14. Peachey L: T h e structure of the extraocular muscle fibers of mammals. I n Bach-Y-Rita P, Collins CC (Editors): The Control OfEye Mnoements. New York, Academic Press, 1971, p p 47-66. 15. Reznik M: Current concepts of skeletal muscle rcgeneration. I n Pearson CM, Mostofi FK (Editors): The Striated iMuscb. Baltimore, Williams & Wilkins, 1973, p p 185-225. 16. Thakar JH, Percy DH, Strickland KP: Ocular ahnormalitics in the myopathic hamster (UM-X7.1). Invest Ophthalmol 16:1047-1052, 1977.
MUSCLE & NERVE
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