Morphological changes in the muscle of vitamin C-deficient guinea pigs

의학과/박사[한글] [영문] In recent years it has been established that, at least in some circumstances, vitamin E is a necessary factor for the preservation of the integrity of skeletal muscle fibers. This has been demonstrated, with study of specific skeletal muscle lesions in vitamin E-deficient animals. The lesions of skeletal muscle fibers have been demonstrated in a number of species of animals, including guinea pigs (Goettsch et al., 1931, Madsen, 1936. Shimotori et al., 1939, Chor et al., 1939), rabbits (Goettsch et al., 1931, Morgulis and Spencer, 1936, Mackenzie and McCollum, 1939, Morris, 1939), goats and sheep (Madsen et al., 1932), mice (Pappenheimer, 1942), rats (Olcott, 1938, Einarson and Ringste, 1938, Pappenheimer, 1939, Goettsch and Ritzmann, 1939), and dogs (Anderson et al., 1939, Brinkhous and Warner, 1941). And the lesions of advanced vitamin E deficiency wer so strikingly similar to those of plasmocid intoxication and of "virus myositis" that the same disorder of some metabolic factor in muscular activity in each disease seemed to be probable. A few investigators (Milhorat et al., 1945, Rabinovitch et al., 1951) have reported suggestive evidence that lare amounts of vitamin E and synthetic alpha-tocopherol are beneficial in polymyositis and in "menopausal muscle dystrophy." Mackenzie and McCollum (1940) and Mackenzie (1942) have studied the effect of alpha-tocopherol in nutritional muscular dystrophy in rabbits and the influence on the metabolism of creatine. Furtheromore, Fitch and dinning (1964) have studied the metabolism of creatine-1-C**14 in vitamin E-deficient monkeys. Sekizima (1941) and Betz (1951) have demonstrated that the muscular lesions developed in the animals on vitamin A-free diet resembled in many respects to the findings muscle dystrophy due to lack of vitamin E. Hitherto, Krakower and Axtmayer (1940) have reported that the muscular lesions studied in vitamin A-deficient rats can be prevented by the administration of alpha-tocopherol and hence are not solely related to the vitamin A-deficient state. The muscles of the trunk and extremities of rabbits have been found to degenerate after the administration of massive doses of cortisone and the pathogenesis was considered by Ellis(1953, 1956). Moreover, Kendal and Hart(1959), Maclean and Schurr (1959) and williams(1959) have pointed out that a reversible myopathy of similar nature with widespread weakness of muscle fibers has been seen in man following the administration of cortisone derivatives. Glaser and Stark (1958) have found that potassium deficiency produced a similar lesion in the skeletal muscle. The lesion differed from that of the cortisone-treated animals in that the myocardium was also affected. But the prolongation of the refractory period, typical of potassium deficiency, was absent in cortisone myopathy. The skeletal muscle lesions produced by vatimin C deficiency were first described by Dalldorf(1929), and in 1941 Sekizima studied comparative observations on changes of skeletal muscles in avitaminosis A, B, and C. These studies are few and were confined to the skeletal muscle lesions in vitamin C deficiency only. The majority of the studies have dealt mainly with the morphological changes of the muscle in a few types of avitaminosis. And the mechanisms involved in the metabolic relationship between the muscle lesions and avitaminosis is still unknown. Moreover, very little attention has been paid to the probable mechanism of the development of the muscle lesions produced by vitamin C-deficiency in guinea pigs. Therefore, a study of the pattern of muscle lesions involved in various experimental conditions and of the muscle chemical contents seems to be appropriate and important. Furthermore, there are still very few theories-first, as to the mode of action of the various factors influencing the muscle lesions in avitaminosis, and secondly as to the factors in progressive muscular dystrophy in human, the cause of which is still unknown. The purpose of this study is to characterize the patterns and the nature of muscle lesions in experimental vitamin C-deficiency in guinea pigs. It seeks to clarify the C-deficiency in relation to potassium deficiency, the administration of cortisone, of vitamin B, of potassium, and of synthetic alpha-tocopherol, and also it demonstrates a factor modifying the changes of water, lipid and potassium in skeletal muscle. Materials and Method Healthy guinea pigs of both sexes weighing from 250 Grams to 350 Grams were used. Although a total of 48 guinea pigs were subjected to the experiment, only 41 were used in the final study. The animals were divided into 8 groups; Group Ⅰ consisted of 6 guinea pigs as control: Group Ⅱ consisted of 5 vitamin C-deficient guinea pigs; Group Ⅲ consisted of 5 guinea pigs receiving glucose, saline and vitamin C following vitamin C deficiency; Group Ⅳ consisted of 5 guinea pigs receiving cortisone and vitamin C following vitamin C deficiency; Group Ⅴ consisted of 5 guinea pigs receiving cortisone following vitamin C deficiency; Group Ⅵ consisted of 4 guinea pigs subjected to potassium deficiency; Group Ⅶ consisted of 5 guinea pigs receiving potassium, thiamine and vitamin C-deficient diet; Group Ⅷ consisted of 6 guinea pigs receiving alpha-tocopherol and vitamin C-deficient diet. The essential vitamin C-deficient diet was prepared as follows; After the Purina Rabbit Pellet was placed within the oven at 60℃ for 36 hours, it was exposed to sunlight on a copper plate for 48 hours. For the supplemental vitamin B complex in the diet 5% brewer's yeast was added. The special potassium deficient diet was prepared as follows; 64.2% corn starch, 30.0% casein, 3.5% butter fat 1.3% calcium carbonate, 1.0% sodium chloride, and multivitamins. Lowry and Histings methods(1942) were used, for the measurement of the content of water, lipid, and potassium in the skeletal muscle, and the methods (Gradwohl 1956) for the determination of the concentration of serum ascorbic acid and potassium were used. The animals were sacrificed at 6 weeks, though some of the animals died before the experimental period came to an end. During the experiment animal behavior was carefully observed. Skeletal muscles from anterior and posterior thigh, and the organs were grossly inspected and fixed in 10 per cent formalin. Paraffin sections were prepared for hematoxylin-eosin, van Gieson, Trichrome stainings, and periodic acid Shiff's reaction. Results 1. Control Group (Group Ⅰ): The interstitial tissue showed no particular changes, such as edema, proliferation of fibroblasts, infiltration of inflammatory cells, hemorrhage, and fatty infiltration. The size & shape of the muscle fibers are almost equal. No abnormalities, such as hyaline degeneration, necrosis, fragmentation, vacuolation, atrophy or pigments were observed. The endomysium and cross striations were well preserved. The sarcolemmal nuclei showed the normal staining quality without pyknosis. No particular abnormalities were observed in this group. 2. Vitamin C-deficiency Group (Group Ⅱ): The muscle fibers showed marked morphological changes, which were patchy in distribution. The edema of the intramuscular connetive tissue was generally marked and present in varying severity. Fibrosis was scarcely present. Minimal infiltration of small and large mononuclear cells was variably found. The caliber of muscle fibers was very variable, polygonal and round or oval, and the muscle fibers was tortuous with partial or marked disappearance, hyaline degeneration, swelling or marked atrophy. Sometimes there were unusually dense myofibrils separated by cleftlike spaces,a nd fragmentation. Marked obscuration of cross striation was seen. The sarcolemmal nuclei were slightly increased in number, and mostly pyknotic, varying in shape and size. There were also many clumps of nuclei, calcification foci, but only few large, oval and vesicular nuclei were noted. 3. Glucose, saline and vitamin C treated Group following vitamin C deficiency (Group Ⅲ): Changes were generally similar to those of Group Ⅱ, but less marked. Edema was present in a variable degree, but less prominent than in Group Ⅱ. Fibrosis was variably present in patchy distribution. The changes of the muscle fibers appeared to be less marked than in those of Group Ⅱ, but degenerative and atrophic changes of muscle fibers were marked. The sarcolemmal nuclei were somewhat increased in number. Many showed varible sized and shaped pyknotic nuclei. Many areas revealed clumps of macrophages in minimal degree around the degenerated and atrophic muscle fibers. 4. Cortisone and vitamin C treated Group following vitamin C deficiency (Group Ⅳ): Edema was also variably present, similar in nature to that in group Ⅱ. Fibrosis was present in minimal degree in patchy distribution, the degree being less than that in Group Ⅲ. The changes of the muscle fibers and sarcolemmal nuclei were generally similar to those of Group Ⅱ, but the number of pyknotic nuclei was less than those of Group Ⅱ. There were areas of focal aggregations of macrophages around the degenerated muscle fibers, and a very small number of nuclear clumps and calcification in the necrotic area, and a degree of regenerative activity were noted. 5. Cortisone treated Group following vitamin C deficiency (Group Ⅴ): Edema was also present to a variable degree, and the changes were similar to the results found in Group Ⅱ and Ⅳ Fibrosis was present in minimal degree in patchy distribution, but the degree of fibrosis appeared to be less than that of Group Ⅲ. The changes of muscle fibers and sarcolemmal nuclei were generally similar to those of Group Ⅱ, and regenerative activity occassionally observed. 6. Potassium deficiency Group (Group Ⅵ): Edema were also variably present, less than in Group Ⅱ. Fibrosis was present in minimal degree in a patchy distribution. The changes of muscle fibers and sarcolemmal nuclei were generally similar in nature to those in Group Ⅱ, and Ⅳ and indistinguishable from those in Group Ⅳ, but less in degree than in Group Ⅱ. There were also focal areas of aggregation of macrophages around the degenerated muscle fibers, pyknotic nuclei, moderate number of nuclear clumps, and large oval vesicular nuclei were rarely found. 7. Potassium, thiamine and vitamin C-deficient diet treated Group (Group Ⅶ): Edema was variable and similar to findings in Group Ⅱ. Fibrosis was moderate. The changes of muscle fibers and sarcolemmal nuclei were generally similar in nature to those of Group Ⅱ and Ⅵ. Nuclear clumps and pyknosis were similar or slightly less in number than in Group Ⅱ. Regenerative activity was occasionally found. 8. Vitamin E and vitamin C-deficient diet treated Group (Group Ⅷ): The interstitial tissue showed minimal to nearly normal degree of edema. Fibrosis was rarely seen. The caliber of the muscle fibers was almost normal, but in several scattered foci, swollen or atrophic, or tortuous muscle fibers were found in a minimal degree and number, and were accompanied by an accumulation of macrophage. The muscle fibers showed areas of hyaline degeneration. There was decrease in visualization of cross striation. However, these changes were rarely seen. The number of sarcolemmal nuclei appeared to be slightly increased as compared with the control group, and were almost normal in appearance. There were also several nuclear clumps in the degenerated muscle fibers. Conclusions 1. Guinea pigs maintained on vitamin C-deficient and potassium-deficient diets, and large doses of cortisone developed profound morphological lesions in their skeletal muscles. These changes were morphologically identical among the groups. However, morphological lesions in their cardiac muscles were slight or almost absent. 2. There were no definite relationships between the potassium level in the skeletal muscle and serum ascorbic acid level in potassium deficient animals, but it appears that there are certain interrelationships between the alteration of serum ascorbic acid level and alpha-tocopherol in vitamin C-deficient animals. 3. The lesions of the muscle being produced by a vitamin C-deficient diet can be prevented by the administration of vitamin E, but cannot be prevented by the administration of thiamine or potassium. It appears that the mechanisms involving the prevention of the lesion of the muscles developed by vitamin C deficiency by the administration of alpha-tocopherol are difficult to interpret. However, they may suggest the possibility of interaction of vitamins in the processes of metabolism within the muscle mass.restrictio