CHANGES IN SKELETAL MUSCLE ASSOCIATED WITH CACHEXIA

OSCAR S. M. MARIN, M.D.,* AND D. DENNY.BROWN, M.D. From the Neurological Unit, Boston City Hospital, and the Department of Neurology, Harvard Medical School, Boston, Mass. The and friability of skeletal muscles in states of cachexia are well known. The classic descriptions of of muscle not only mention the progressive atrophy of muscle, but also note fatty granular changes in many of the muscle fibers."2 Deposits of lipochrome have also been noted, but their greater prominence in older patients has led them to be regarded as a senile alteration.3 4 Increasing awareness of states of "polymyositis" in relation to carcinomatosis in recent years,5'6 and the intriguing problem as to the relationship of simple reversible atrophy and the degeneration of muscle fibers in these states has led us to investigate this problem. We have examined the skeletal muscles in a series of i8 consecutive necropsy cases where cachexia was a prom- inent feature. In most such material simple atrophy was the prominent finding, but in 3 cases degenerative changes were generalized and severe, and these are the subject of this report. METHODS Samples of many muscles, usually including sternocleidomastoid, diaphragm, deltoid, psoas, abdominal wall, quadriceps, adductor of thigh and gastrocnemius, were taken at necropsy and fixed in Zenker's solution. Pieces of muscular nerve were also obtained. After paraffin embedding, these tissues were sectioned and stained with hematoxylin and eosin, phosphotungstic acid hematoxylin (PTAH), or eosin- methylene blue. When degenerative changes were present, periodic acid-Schiff (PAS), Feulgen, Turnbull, and von Kossa reactions were done on the relevant material, and frozen sections were stained for fat by oil red 0. Tables I and II summarize the relevant data, including the nature of the verified cause of cachexia, and the chief histologic features in the skeletal muscles. Any muscular change related to denerva- tion or to direct invasion by carcinoma or other pathologic alteration are excluded. The first 4 cases are reported in detail below. Since it was necessary to relate the degenerative changes to those of nutritional atrophy, we made counts of fiber area in ioo fibers in representative transverse sec- tions in each of the 4 most remarkable cases, and in 2 control cases-a normal 35-year-old man without nutritional impairment, killed by subarachnoid hemorrhage, and a woman of 78 years who died of acute cerebral hemorrhage without nutritional disorder. The estimations of area were made by planimeter on an enlargement of Accepted for publication, February 6, I962. *Research Fellow in Neurology, Harvard Medical School; Senior Trainee, National Institute of Neurological Diseases and Blindness, National Institutes of Health, United States Public Health Service. Present address: Department of Medicine (Neurology), Catholic University School of Medicine, Santiago, Chile. 23 24 MARIN AND DENNY-BROWN Vol. 4z, No. I

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ax 0go fI ea r CaLd C' a *Uco U m ._I N o~~~1 14 164 26 MARIN AND DENNY-BROWN Vol. 4I, No. z transverse sections. Since we shall be chiefly concerned with the smallest fibers, the distribution of fiber size in these 6 cases was plotted in semi-logarithmic paper (Text- figs. i and 2). REPORTS OF CASES The general observations in all cases examined are set out in Table I. Degeneration of muscular or intramuscular nerve bundles was not pres-

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AREA OF CROSS SECTION IN TEXT-FIGURE I. Frequency curves of muscle fiber size in a normal man aged 35 years, a woman aged 78 without cachexia, and case i, on semilogarithmic scale, to show the shift to smaller fiber size. ent in any of the material reported. Mild arteriosclerotic vascular lesions were present in cases i, 2, 6, 8, 9, I3, 14, I5 and i6. The atrophic and degenerative lesions were generalized in all muscles examined in every instance except case I5, where the alterations were more pronounced in distal muscles. The 4 cases, I3 to i6 (Table II), were controls for age without cachexia, and all presented a mild atrophy with slight increase in the number of sarcolemmal nuclei per low power field, but without degenerative change in muscle fibers. A plot of the fiber area scatter in cross section from case I 5, a woman 78 years of age who died of a cere- July, Z962 SKELETAL MUSCLE IN CACHEXIA 27 bral hemorrhage, is compared in Text-figure i with that of a normal man aged 35 years. Whereas the normal adult muscle shows the great majority of fibers between 200 and 700 square t,f with few fibers ranging down to 40 to ioo square jp (Fig. iA), senile muscle exhibits a large peak at 50 square ,. and a few fibers up to i00 square , (Fig. iB). Simple atrophy of this type presents a shift of frequency from large fiber size

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AREA OF CROSS SECTION IN p t TEX-FIGURE 2. Frequency curves of muscle fiber size in cases 2, 3 and 4, to be com- pared with Text-figure i. to smaller, without other significant change. In the presence of cachexia, additional changes were found; these will be described in relation to 4 cases typifying different degrees of further alteration. Case I W.M., 63 years of age, had suffered from a squamous cell carcinoma in the middle third of the esophagus, which had caused progressive dysphagia for 8 months. At necropsy the tumor was found to have caused a tracheo-esophageal fistula. There were metastases to the right lung and to both kidneys. There was extreme emaciation and cachexia, with a weight-height ratio of 0.62 (Table I). 28 8MARIN AND DENNY-BROWN Vol. 4z, No. z The microscopic appearance was the same in all muscles examined. A typical low power view, Figure IC, presented a remarkable mixture of fibers of two predominant types. The plot of fiber size frequency (Text-fig. I) showed wide separation of the groups with one distinct peak at ioo square ,1, and another at 20 square ,&. The smaller fibers stood out by reason of their dark staining with all stains used and their prismatic shape in cross section (Fig. 2). Yet in longitudinal section these small fibers exhibited normal myofibrils and general architecture (Fig. 2 B). Their muscle nuclei were of normal appearance, and the ap- parent increase in number could be accounted for by the reduction in fiber size. The myofibrils were of natural appearance, though so closely packed that it was not possible to count them in cross section. Under oil immersion, groups of 6 or more myofibrils commonly appeared to be fused into one dark mass in which the exact number of components was uncertain. The appearance could be accounted for by the almost total disappearance of sarcoplasm. An early granular change was found in the myofibrils of only one or two of these small muscle fibers out of many hundreds examined. There was no evidence of fragmentation of muscle fibers or of the clumping of nuclei or nuclear remnants found in degen- eration of muscle. No abnormality was detected in the endomysium. Case 2 P.F., a 77-year-old man who had been a stonecutter for over 30 years, was admitted to the hospital because of cough and dyspnea on exertion. Repeated sputum and gastric cultures were negative for tubercle bacilli. Pulmonary silicosis was diagnosed. In spite of various treatments, his general health deteriorated in the last 6 months of life. Necropsy examination, restricted to the chest, revealed extremely severe cachexia, severe pulmonary silicosis with bronchiectasis and emphysema, and ter- minal acute bronchitis. There was no evidence of tuberculosis. Samples of pectoralis, sternocleidomastoid, deltoid and biceps muscles were alone available for study in this case. All muscles were greatly reduced in bulk and those examined at necropsy were of grayish brown color and friable consistency. Histologic examination revealed that about 70 per cent of the muscle fibers showed degenerative changes in addition to a general loss of fiber size (Figs. iD and 3A). The largest fibers had a cross-section area equal to the smallest fibers in a normal control (Text-fig. 2). There were large numbers of fibers ranging from IO to 40 square I in area that stained poorly in transverse section and showed few myofibrils with phospho- tungstic acid stain. There was a small peak at IOO to I50 square ,z. The most striking feature was the presence of clumps of darkly staining muscle nuclei within these small fibers in longitudinal section, and in places in pale segments of the larger fibers (Fig. 3). Some masses of clumped, shrunken and deformed nuclear substance lay in the connective tissue. The cytoplasm of the pale thin fibers and of the pale segments July, Z92 SKELETAL MUSCLE IN CACHEXIA 29 of larger fibers contained a dusty, finely granular material that stained pink with eosin and with Masson trichrome (Figs. 3B and 3C) but not at all with PTAH. In some fibers, as in Figure 3B, fragments of myo- fibrils could be observed in various stages of rupture. The Feulgen method stained the nuclear masses intensely, and some of the larger granules. Coarse lipochrome granules were frequently seen next to the poles of the sarcolemmal nuclei in otherwise healthy fibers, but were in- frequent in the pale areas of sarcoplasm. No histiocytic or other infiltra- tion was found. There was no floccular and no evidence of re- generation of muscle or other nuclear change other than progressive clumping and . In places (Fig. 3A) hyaline fragments of a larger fiber suggested recent ante-mortem rupture, without cellular reaction. The slight prominence of endomysium could be accounted for by con- densation of tissue, and there was no fibroblastic or other connective tissue reaction. Case 3 E.B., a 74-year-old woman who had been in poor general health for many weeks, was admitted to the hospital for disorientation and confusion, with rectal bleeding. Rectal biopsy disclosed mucinous adenocarcinoma. Within a short period intestinal obstruction developed, and the patient succumbed soon after a laparotomy. Necropsy examination confirmed the rectal , with widespread metastases to periton- eum, adrenals and liver. There was extreme emaciation (weight ioo pounds). Samples of biceps, deltoid, quadriceps, pectineus, psoas and sternocleidomastoid muscles were obtained, with various nerves. All muscles appeared greatly reduced in bulk, friable, and of brown-gray color. In cross section the appearance of all muscles examined was similar, presenting, besides atrophy of all fibers, two contrasting types, a few larger, rounded, and more darkly staining, the others pale, shrunken and poorly outlined (Figs. iE and 4). The great majority of fibers were approximately io square /L in area (Text-fig. 2). In longitudinal section the larger fibers appeared structurally intact, with myofibrils of natural appearance and the usual flattened peripheral sarcolemmal nuclei. In all the smaller fibers the myofibrils were either no longer recognizable or reduced to small segmented fragments. The cytoplasm was filled with a fine granular substance that stained pink with eosin and, in places, more coarsely pink with Masson trichrome (Fig. 4), but only faintly with PTAH. In addition, scattered single granules of coarse lipochrome pigment lay free in the cytoplasm or closely related to clumps of nuclei. Some of these gave a positive PAS reaction (Fig. 5A). In moderately affected fibers the myofibrils could be seen to be undergoing discoid de- generation (Figs. 4B and 4C), with shrunken, dark-staining muscle nuclei that were elongated in early stages of the process but later became rounded and small, finally aggregating in amorphous clumps. All stages 30 MARIN AND DENNY-BROWN Vol. 4z, No. z of disintegration of myofibrils into eosinophilic granules were present (Fig. 4). No histiocytes were present, and the floccular feature of fiber necrosis was absent. The connective tissue was not altered, and no vascular alter- ation was present except for occasional atheroma in large vessels. Frac- ture of the larger muscle fibers was frequent in longitudinal section, but in the absence of reactive changes this was interpreted as a postmortem artifact, reflecting the friability of the tissue. The degenerative change was present in the whole length of each affected fiber. Case 4 D.C., a 53-year-old woman, complained of abdominal enlargement and pain for one year prior to admission. Following a diagnosis of ovarian carcinoma and treatment with nitrogen mustard (total dosage, 30 mg.), the patient failed to improve and ex- pired some i5 months after the onset of symptoms. Necropsy examination revealed severe cachexia (weight go pounds). A left ovarian tumor proved to be a poorly differentiated adenocarcinoma. There was widespread metastasis to the peritoneum and liver. All muscles were greatly reduced in bulk and of gelatinous gray-brown appearance. The muscle fascicles could no longer be distinguished. Samples of muscle from abdominal wall, diaphragm and psoas were examined microscopically. All muscle fibers were found to be greatly reduced in diameter (Fig. IF), with the largest number being IO to 40 square ,u in cross section area (Text-fig. 2). The largest fibers still retained their circular outline, but the smaller fibers were polygonal or threadlike, containing few if any myofibrils. In longitudinal section the same dusty granular appear- ance of the cytoplasm noted in cases 2 and 3 was present in the smallest fibers, with scattered clumps of brown lipochrome pigment (Figs. 5B and 5C). Fragments of myofibrils could be observed in fibers in an early stage of dissolution. Lipochrome pigment was regularly present near the poles of muscle nuclei even in otherwise intact muscle fibers in this case and was also present in cardiac muscle and liver cells. Intermediate stages of fragmentation of myofibrils were found in early stages of the process in fibers of intermediate size, but this aspect was not so promi- nent as in case 3. In all but the largest fibers there was proliferation of muscle nuclei. With the disappearance of myofibrils, many of the nuclei showed regressive changes, becoming shrunken and pyknotic where the myofibrils were no longer recognizable. The fine granular pigment per- sisted even in the smallest fibers, being then intermingled with larger lipochrome granules. There was no evidence of regeneration, floccular necrosis or of phagocytosis or connective tissue reaction. DISCUSSION A survey of the skeletal muscles of patients dying of various diseases associated with cachexia reveals pronounced alteration of skeletal July, Z962 SKELETAL MUSCLE IN CACHEXIA 31 muscle fibers. The most severe type of change, granulo-fatty degenera- tion, was described by Klippel 1 in I889 in a Paris thesis. His material included 7 cases of chronic tuberculosis, 3 of uterine carcinoma, and 5 others of senile cachexia or cirrhosis. The muscle changes were not asso- ciated with any degeneration in the muscular nerves and were irregu- larly distributed. Evidence of regeneration was lacking. He noted also the marked irritability of such muscles to mechanical stimulation (pinch- ing, percussion) during life, in contrast to their reduced electrical ex- citability. These changes were described in greater detail by Durante.2 The granular degeneration was found to be limited to the few discrete fibers in each muscle in the beginning but eventually became generalized, with pronounced fatty changes. The connective tissue became increased terminally and contained pigment granules, giving the muscles a brown- ish coloration. In the years following these descriptions the condition has aroused little interest. Jewesbury and Topley3 examined the skeletal muscles in a number of general diseases and noted the irregularity in the size of muscle fibers in cachexia. They were not impressed by the discoloration and friability described by Durante,2 but found marked nuclear aggre- gations in muscle fibers in 30 of their 40 cases with chronic . Only a few presented marked granularity and fatty changes, but in two cases of advanced cachexia from malignant disease there was fat in the inter- stitial tissue of the muscles as well as in the muscle fibers. The most severely affected muscles varied from case to case. Our own material confirms the observations of these earlier authors. In cases with the most severe cachexia some degree of the alteration termed granulo-fatty degeneration by Klippel 1 was found. The degree of degenerative change varied from case to case, without direct relation to the weight loss suffered by the patient, but was present in some degree in the most severely cachectic patients. The least degree of change, and the most common feature in cachectic patients, was the separation of muscle fibers (already small by reason of inanition) into two groups. One group retained a rounded form and moderately small diameter of 25 to 35 j.&, characteristic of senile, disuse, or inanition atrophy. The other became greatly shrunken and prismatic in section and stained darkly with basic stains. Owing to their prismatic shape, their relative size is best expressed as cross section area, which presents a second peak in graphic area/frequency representation. It is surprising that these fibers can remain structurally intact in spite of their very severe atrophy. The next change was a fragmentation of the contents of the smallest muscle fibers, beginning sometimes in small segments, sometimes throughout the fiber length. This was associated with proliferation of 32 MARIN AND DENNY-BROWN Vol. 4z, No. z the muscle nuclei, which became dispersed throughout the fragmenting columns of myofibrils; eventually these became shrunken, pyknotic and clumped into darkly staining masses. Stages of this fragmentation were observed in cases 2 and 4 and were particularly frequent in case 3. The myofibrils broke up into coarse granules, at first appearing to segment across the Z line. At this stage they lost their affinity for hematoxylin and became eosinophilic. As the pattern of striation was lost, the eosino- philic granules became smaller and smaller, eventually becoming a fine dust at the stage when the nuclei had become clumped and inert. At the same time a few larger granules made their appearance. Some of these were brown in color and PAS positive. Others stained intensely for neu- tral fat (oil red 0). In some cases large granules of collected in the sarcoplasm near the poles of nuclei in otherwise intact fibers and later in rows between myofibrils. When granular degeneration of the smaller fibers occurred in such cases, the lipofuscin pigment was added to the granular debris. In this way pigmentary change in some cases was precocious, but the fragmentation of myofibrils was otherwise the same. When lipochrome pigment was prominent, it was seen in fibers that had not as yet shown disintegration of myofibrils, and it was present in the heart muscle of such cases. Lipochrome therefore appeared to arise in a different manner than the fatty granular change. Case 4, in which lipo- chrome pigment was universally present, had been treated with a heavy dosage of nitrogen mustard, but lipochrome and all the other alterations we have described occurred in cases without such treatment. Our material thus has provided a perspective of changes in the course of cachexia. Fatty-granulo-pigmentary degeneration of this type is unique among the histologic transformations of skeletal muscle, and deserves more attention. In our material, fragmentation of the columns of myofibrils was clearly the primary event and presumably reflected some fundamental change in the chemistry of myosin. The proliferative changes in the muscle nuclei followed by degenera- tion, associated with this change, were identical to those observed in the muscle fiber in other types of injury. Proliferation of this type is asso- ciated with disappearance of the nucleolus and dispersal of the nuclear chromatin throughout the nucleoplasm.7 The shrunken nuclear clumps remained strongly Feulgen-positive long after all appearance of viability was lost. As Durante2 observed, the absence of regenerative changes in the muscle fibers in cachectic degeneration is most remarkable. Fatty- granulo-pigmentary degeneration is a most destructive lesion. In our material any alteration in the connective tissues would be accounted for by the condensation consequent upon loss of muscle fibers. July, Z962 SKELETAL MUSCLE IN CACHEXIA 33 It is of particular interest that at all stages of the process the changes affected only a proportion of the muscle fibers. There was always a pro- portion of larger fibers that remained structurally intact, some of them of almost normal size and appearance (Figs. iE and 2). As the histo- grams (Text-fig. 2) show, the proportion of these larger fibers steadily lessened as degenerative changes became more generalized. Though the curves shift to the left with increasing severity of degeneration, the alterations can be approximately separated into (a) change from medium-sized fibers to small fibers; (b) change from small fibers to degenerating fibers; (c) change from degenerating fibers to empty tubes filled with granules; and (d) fragmentation into short tubes, nuclear and pigmentary residue. In simple senile atrophy none of these features were found. The increased irritability to percussion of the muscles in cachectic patients, noted by Klippel 1 and others, was found by Denny-Brown and Pennybacker 8 to be associated with slowly transmitted contractile waves in the fiber contents, without action potential. We have not attempted to correlate this phenomenon with the histologic lesions reported here, though it obviously requires the presence of intact myofibrils, and these are lost in the second stage of the cachectic alterations. In recent years attention has been directed to myasthenic features of the muscular weak- ness in many cases of carcinomatosis,9-12 particularly with bronchogenic carcinoma. The relation of such change to the increased mechanical ir- ritability is atpresent unknown. The muscular changes we have observed in other cases of carcinomatosis have sometimes the characteristics of acute or chronic polymyositis. It remains to be determined how fre- quently the alterations due to cachexia are responsible for progressive muscular disease in patients with carcinomatosis, independently of poly- myositis. Polymyositis of more banal type can certainly be associated with carcinomatosis, but in irregular fashion, seldom running a course parallel with the other systemic manifestations of the carcinoma.","8 Cachectic changes in the muscles, on the other hand, approximate in severity the other changes of carcinomatosis, if that is the cause. CONCLUSIONS The association of the fatty-granulo-pigmentary degeneration of muscles described by Klippel 1 with severe cachexia has been confirmed. The degeneration was associated with severe loss of sarcoplasm, fol- lowed by fragmentation of myofibrils, which are the primary source of the sarcoplasmic granules. The sarcoplasmic granules appeared to pass through a fatty lipid stage in the process of dissolution, with conversion of some to free unsaturated fats. 34 MARIN AND DENNY-BROWN Vol. 4I, No. z The process was irreversible and was associated with proliferation and degeneration of the muscle nuclei. Great shrinkage of some muscle fibers, due to loss of sarcoplasm, was the first stage of the process, which appeared reversible up to this point. REFERENCES I. KLIPPEL, M. Des amyotrophies dan les maladies generales chroniques et de leurs relations avec les lesions des nerfs periph6riques. These de Paris, No. 177, I889, I67 PP. 2. DURANTE, G. Anatomie pathologique des muscles. In: Manuel d'Histologie Pathologique. CORNIL, V., and RANVIER, L. (eds.) Felix Alcan, Paris, I902, Vol. 2, PP. I-477. 3. JEWESBURY, R. C., and TOPLEY, W. W. C. On certain changes occurring in the voluntary muscles in general diseases. J. Path. & Bact., I9I2-I9I3, I7, 432- 453. 4. ADAMS, R. D.; DENNY-BROWN, D., and PEARSON, C. M. Diseases of Muscle. A Study in . Paul B. Hoeber, Inc., New York, I953, 556 pp. 5. DENNY-BROWN, D. Primary sensory neuropathy with muscular changes asso- ciated with carcinoma. J. Neurol. Neurosurg. & Psychiat., 1948, II, 73-87. 6. WALTON, J. N., and ADAMS, R. D. Polymyositis. E. & S. Livingstone, Edin- burgh, and Williams & Wilkins Co., Baltimore, I958, 270 PP. 7. DENNY-BROWN, D. Experimental Studies Pertaining to , Regen- eration and Degeneration. In: Neuromuscular Disorders (The Motor Unit and Its Disorders). Proc. A. Res. Nerv. & Ment. Dis., I958. Williams & Wilkins Co., Baltimore, I960, Vol. 38, pp. I47-I96. 8. DENNY-BROWN, D., and PENNYBACKER, J. B. Fibrillation and fasciculation in voluntary muscle. Brain, I938, 6i, 3II-334. 9. MACKENZIE, I., and HALL, J. M. Bronchial neoplasm with myasthenia. (Letter to the editor) Lancet, I954, I, IO8. IO. SHAPAR, J. Bronchial neoplasm with myasthenia. (Letter to the editor) Lancet, 1954, I, IO9-IIO. II. HENSON, R. A.; RUSSELL, D. S., and WILKINSON, M. Carcinomatous neurop- athy and myopathy: a clinical and pathological study. Brain, I945, 77, 82-I2I. I2. EATON, L. M., and LAMBERT, E. H. Electromyography and electrical stimula- tion of nerves in diseases of motor unit. Observations on myasthenic syn- drome associated with malignant tumors. J.A.M.A., I957, I63, III7-II24. 13. DENNY-BROWN, D. The nature of polymyositis and related muscular diseases. Tr. & Stud. Coll. Physicians Philadelphia, I960, 28, 14-29.

LEGENDS FOR FIGURES FIG. I. Representative transverse sections. A. Normal control. B. Senile control. C. Case I. D. Case 2. E. Case 3. F. Case 4. The progressive appearance of smaller fibers and their degeneration in this series of cases is shown. A, hematoxylin and eosin stain; all others, phosphotungstic acid-hematoxylin (PTAH). X 140. July, 1962 SKELETAL MUSCLE IN CACHEXIA 35

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FIG. 4. Case 3. A general view of a severely affected muscular fasciculus. A. Clumping of degenerated pyknotic nuclei. B and C. Various stages of fragmentation of myofibrils are seen, with the final granular change in the uppermost fiber. A. Hematoxylin and eosin stain. X 280. B and C. Masson trichrome stain. X 640. July, I_962 SKELETAL MUSCLE IN CACHEXIA 39

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FIG. 5. A. Case 3; coarse PAS-positive granules are evident. B. Case 4; similar coarse granules are stained for fat in frozen section with oil red 0. C. Case 4; Masson trichrome stain shows granules of lipofuscin. A and B. X 640. C. X 800.