295

Journal of the Neurological Sciences, 1978, 38:295-315 © Elsevier/North-Holland Biomedical Press

T H E EARLY EFFECTS MITOCHONDRIA

OF

ISCHEMIA

UPON

SKELETAL

MUSCLE

R. R. HEFFNER and S. A. BARRON Dent Neurologic Institute; Division of Neuropathology and Department of Neurology, State University of New York, School of Medicine, Buffalo, N Y (U.S.A.)

(Received 7 February, 1978) (Accepted 6 June, 1978)

SUMMARY The effects of early ischemia were studied in the anterior tibial muscle of Sprague-Dawley rats after 2-24 hr of tourniquet compression at the thigh. Ragged-red fibers, moth-eaten fibers, cores and targets were seen in tissue examined by enzyme histochemistry and electron microscopy. Giant mitochondria, abnormalities of cristal arrangement, crystalloids, osmiophilic inclusion bodies and myeloid figures were dominant features of the mitochondrial reaction. The results of this experiment indicate that early ischemia induces a variety of changes described in other neuromuscular conditions such as dystrophy and the "mitochondrial myopathies." The pathogenesis of these changes and their relationship to human disease of muscle is discussed.

INTRODUCTION Intensive investigation of the effects of acute ischemia in the central nervous system has been in progress for several years (Garcia 1975); by comparison, considerably less is known about similar effects in one of its important end-organs, skeletal muscle. Perhaps this is because, unlike the brain where the results of anoxia are often almost immediate and catastrophic, muscle is widely regarded as more resistant to long periods of ischemia. This "resistance" is only relative and is due in Author addresses and affiliations: Reid R. Heffner, M.D., Neuropathology Laboratory, Dent Neurologic Institute, 3 Gates Circle, Buffalo, NY 14209, U.S.A. ; Associate Professor (Neuropathology), State University of New York, Buffalo, NY, U.S.A. Stephen A. Barron, M.D., Dent Neurologic Institute, 3 Gates Circle; Assistant Professor (Neurology), State University of New York, Buffalo, NY, U.S.A.

296 part to the rich collateral circulation of muscle (Clark and Blomfield 1945), especially in the extremities, which renders the development of ischemia unlikely in the human patient without underlying vascular disease and the achievement of ischemia in the experimental animal more difficult than in most other organs (Scully, Shannon and Dickersin 1961). Further knowledge concerning the effects of ischemia ale particularly relevant at this time for at least two reasons. Peripheral vascular disease in association with atherosclerosis, diabetes, vasculitis and embolization (Heffner 1971) has become a common medical problem resulting in profound functional abnormalities in the neuromuscular system of the extremities (Strandness 1969), yet the pathological explanation for these abnormalities is incomplete. Secondly, there is continuing evidence that ischemia may play a role in the pathogenesis of muscular dystrophy (Rowland 1976). Morphologic studies comparing ischemic myocyte injury and dystrophy have focused on a stage of disease when fiber necrosis is widespread. Not surprisingly, these studies have demonstrated similar, non-specific changes in both processes and have, therefore, been inconclusive. If more specific changes do occur, it is likely they will be apparent early, before cell necrosis is advanced. For this reason we have chosen to examine an experimental model of muscle ischemia in the rat during the first 24 hours of anoxic cell damage. We have concentrated our observations on the mitochondria which, because of their role in aerobic respiration, would be expected to represent a more sensitive indicator of early ischemic change than perhaps any other component of the muscle cell. MATERIALS AND METHODS A total of 60 male Sprague-Dawley rats weighing 250-300 g were used in this investigation.

Ischemic animals A group of 50 rats were anesthetized by intraperitoneal Nembutal injection. lschemia of the left hind leg was achieved employing the technique described by Moore, Ruska and Copenhaver (1956) in which a rubber band tourniquet was placed around the thigh. During the post-ligation period the animals dragged the left hind limb which remained cyanotic. Rats were sacrificed in groups of 10 at 2, 6, 12, 18 and 24 hr after ligation. Control animals Ten rats underwent Nembutal anesthesia without the application of a tourniquet. They were sacrificed 24 hr after the administration of anesthesia. Tissue preparation Animals were anesthetized by intraperitoneal injection of Nembutal and biopsies of ischemic and control tibialis anterior muscles were obtained for light and electron microscopy. Specimens for light microscopy were frozen in isopentane

297 cooled by immersion in liquid nitrogen. Serial sections which were cut in a cryostat were stained with hematoxylin and eosin (H&E), phosphotungstic acid hematoxylin (PTAH), periodic acid-Schiff (PAS), modified Gomori trichrome (Engel and Cunningham 1963), myofibrillar adenosine triphosphatase, pH 9.4 (ATPase), reduced nicotinamide adenine dinucleotide-tetrazolium reductase (NADH-TR), and phosphorylase. Specimens for electron microscopy were maintained at resting length in a Price isometric clamp and immediately fixed in 2.5 ~ glutaraldehyde buffered in Millonig's solution. Post-fixation in 2 ~ osmium tetroxide was followed by embedding in Epon 812. Ultra-thin sections were cut using a Reichert OMU-3 ultra-microtome and examined with a Zeiss EM-10 electron microscope. RESULTS Light-microscopic observations In control animals the normal tibialis anterior muscle was composed of 3 fiber types when subjected to histochemical staining. Type 1 fibers had low ATPase activity, moderate oxidative activity, and a variable phosphorylase content. Type 2A fibers were characterized by a high ATPase and oxidative activity and a weak phosphorylase reaction. In type 2B fibers ATPase activity was moderate, oxidative enzyme reactions were weak and phosphorylase concentration was high. The deep portion of the tibialis anterior muscle contained larger numbers of type 1 and 2A fibers than the superficial part v~here fibers having low ATPase activity were rarely found. Moth-eaten, core/targetoid and target areas were never present in control specimens, nor were ragged-red fibers. (1) 2 hours ischemia: No histological changes were observed by light microscopy. (2) 6 hours ischemia: Increasing numbers of moth-eaten fibers were seen up to 18 hr of ischemia (Table 1). These were not visible in H&E stains and were best

TABLE 1 PATHOLOGIC CHARACTERISTICS OF RAGGED-RED, MOTH-EATEN AND CORE] TARGETOID FIBERS IN ACUTE SKELETAL MUSCLE ISCHEMIA Pathologic appearance

Light microscopy PAS PTAH Gomori trichrome NADH-TR ATPase phosphorylase Electron microscopy densityofmitochondria

ragged-redareas

moth-eatenareas

core/targetoidareas

normal-pale blue-magenta red dark normal-pale normal-pale

pale smudged smudged very pale pale pale

pale smudged smudged very pale pale pale

low; disruption and lysis absent absent marked

low; disruption and lysis absent absent marked

high; excessive crowding giant mitochondria present crystalloids present myofibrillardisorganization absent

298

Fig. 1. Six hr ischemia. Typical moth-eaten fiber at right. Patchy loss of oxidative enzyme activity within sarcoplasm and disorganization of myofibrillar network. NADH-TR, , 800.

demonstrated in N A D H - T R reactions in which multiple, small, irregularly-shaped foci of decreased oxidative enzyme activity were distributed in patchy fashion within affected fibers (Fig. 1). In some fibers such foci became confluent, resulting in larger moth-eaten areas measuring up to 30 /~m in diameter. Confluent foci were more conspicuous at 12 and 18 hr of ischemia. Moth-eaten areas appeared smudged, indicating myofibrillar disorganization, in PTAH and Gomori stains where they also were more darkly stained than the neighboring more normal portions of the involved fiber. These areas generally stained poorly with PAS and were pale or unstained in phosphorylase and ATPase reactions. (3) 12 hours ischemia: Scattered ragged-red fibers were identified by the presence of subsarcolemmal collections which were intensely red in modified Gomori trichrome stains (Fig. 2A). These red, often raised, lobular foci, which were typically found at the periphery of fibers resulting in an irregular (ragged) cell border, assumed a bluemagenta color after P T A H staining (Fig. 2B) and reacted strongly in N A D H - T R preparations. Ragged-red fibers were more abundant following 18 and 24 hr of ischemia. At that time, the internal sarcoplasm of some contained numerous minute granular structures, often in clusters, with similar staining properties as the more prominent, peripheral ragged-red areas. (4) 18 hours ischemia: Core/targetoid foci were first detected at 18 hours and became more prevalent after 24 hr of ischemia. In cross-sections these structures, best

299

Fig. 2. Twelve hr ischemia. A: 2 ragged-red fibers. Top fiber is more classical in appearance with irregular red (dark in this photograph) cell border. Modified Gomori trichrome, x 400. B: ragged-red fiber with multiple magenta (dark in this photograph) subsarcolemmal loci. PTAH, x 600.

300

Fig. 3. Eighteen hr ischemia. Large central core/targetoid with marked loss of oxidative enzyme activity. N A D H - T R , × 600.

Fig. 4. Two hr ischemia. Subsarcolemmal zone of a muscle fiber. Many mitochondria are swollen and rounded with lucent inner compartments compared to isolated relatively intact mitochondrion (M). Fracture, lysis and loss of cristae is apparent. Occasional membranous profiles (arrows) are present. × 25,000.

301 viewed in N A D H - T R reactions, appeared as single, central (Fig. 3) or slightly eccentric, r o u n d to oval zones o f decreased or absent oxidative enzyme activity with s m o o t h borders. Althougb some variability in size was noted, most were large with diameters reaching 25-75 ~ o f the respective fiber diameter. The largest measured up to 60 # m in diameter. Core/targetoids possessed staining properties quite similar to those of moth-eaten areas. Phosphorylase activity and PAS staining were reduced. A smudged appearance and increased staining intensity was noted in P T A H and G o m o r i preparations. In longitudinal sections, core/targetoids typically extended over 5-10 sarcomeres rather than the entire length o f the fiber and were oriented with their long axes in the direction o f the fiber long axis. Occasionally, target fibers exhibiting 3 distinct zones in contrast to the 2 zone structure of core/targetoids were seen. The target center which had an identical appearance to the core/targetoid focus was surrounded by an intermediate zone where ATPase and oxidative activity was abnormally increased. The outer portions o f muscle fibers having targets and core/targetoid foci appeared relatively normal in all preparations by light microscopy. (5) 24 hours ischemia: Moth-eaten fibers were only rarely seen. Ragged-red fibers and core/targetoid lesions were regularly found.

TABLE 2 ULTRASTRUCTURAL ASPECTS OF MITOCHONDRIAL REACTIONS TO ACUTE ISCHEMIA IN SKELETAL MUSCLE Changes were graded from 0-4 as follows (0: no fibers affected; 1 : less than 10 ~ of fibers affected; 2:10-20 % of fibers affected; 3:20-50 ~ of fibers affected; 4:50-75 ~ of fibers affected). Mitochondrial changes

Increased numbers (excessive crowding) Decreased numbers (disruption and lysis) Increased size swollen, rounded giant Matrix lucencies Inclusions flocculent densities glycogen osmiophilic bodies lipid-like bodies Cristal abnormalities expansion of intermembranous spaces fracture and lysis membranous profiles (myeloid figures) trilaminar plates crystalloids (paracrystalline inclusions) cristal membrane whorling

Time (hr) 2

6

12

18

24

0 0

0 1

1 3

2 3

2 3

3 0 2

3 0 3

3 0 3

4 1 4

4 1 4

1 0 0 0

2 0 0 0

3 1 0 0

2 2 1 1

2 2 1 1

1 2 1 0 0 0

2 3 1 1 0 0

2 3 2 2 0 0

2 4 2 1 1 1

2 4 3 1 1 1

302

Electron-microscopic observations Mitochondrial changes with respect to time are summarized in Table 2. The prevalence of each change at 5 time intervals is given a numerical grade ranging from 0-4 depending upon the percentage of fibers exhibiting that change (0: no fibers affected; 1: less than 1 0 ~ of fibers affected; 2: 1 0 - 2 0 ~ of fibers affected; 3:20-50 ~ of fibers affected; 4 : 5 0 - 7 5 ~ of fibers affected). (1) 2 hours ischemia: Many muscle cells appeared completely normal from an ultrastructural standpoint. Approximately 5 0 ~ of fibers contained mitochondria which were swollen and excessively rounded (Fig. 4). Such mitochondria were most often found in the subsarcolemmal portions of the fibers but could also be seen internally in some cells. In swollen mitochondria that were enlarged up to 4 times normal size the inner compartment was almost invariably lucent. This appearance was accentuated by the fracture, lysis and loss of cristae. The remaining cristae were often abnormally straight and arranged in parallel. Separation of cristal membranes occurred as focal, rounded or oval, bleb-like expansions of the intermembranous space (Fig. 5). Probably as a result of degenerative changes, the cristae became hazy in outline (Fig. 6). Within the matrix flocculent densities were observed. These were single or multiple, amorphous, irregular in shape and not membrane-bound. (2) 6 hours isehemia: Mitochondria were markedly swollen and rounded in both subsarcolemmal and internal portions of many fibers. Rupture of the outer membranes was sometimes evident. The inner compartments were lucent except for the presence of numerous flocculent densities. Fracture, lysis and loss of cristae were widespread. Most of the remaining cristae appeared hazy, poorly defined and abnormally straight. Expansion of the intracristal spaces was more prevalent than at 2 hr. Membranous profiles, first seen at 2 hr, were more abundant within the matrix (Fig. 6). These usually appeared as collections of 2-5 membranes, concentrically arranged, with a round or oval shape. The average thickness of these membranes, 60 A, corresponded closely to that of the inner mitochondrial membrane to which these profiles were sometimes attached. Within subsarcolemmal mitochondria of necrotic fibers scattered trilaminar plates were observed (Fig. 7). Plates seemed to be the result of a deposition of amorphous material within the intracristal space, often simulating a membrane measuring 50-80 A in thickness. Such a membrane-like structure was typically interposed between two widened parallel cristal membranes which comprised a truncated, abnormally straightened cristal segment. At the ultrastructural level the moth-eaten fiber was identified by the presence of numerous focal areas of mitochondrial and myofibrillar disruption in a muscle fiber which was otherwise intact. The sarcolemmal membrane, nucleus and much of the sarcoplasm including its contractile elements, sarcoplasmic reticulum, glycogen stores and mitochondria were relatively well-preserved. In moth-eaten areas myofibrillar disorganization, myofilament dissolution, expansion of intermyofibrillar spaces, and Z band streaming or zig-zagging produced alteration and destruction of the normal cross-banded pattern of the sarcomeres. Glycogen was absent from these areas in contrast to normal portions of the fiber where it tended to be abundant. No normal mitochondria remained within moth-eaten areas. Many were lost or could no longer

303

Fig. 5. Mitochondrion after 2 hr ischemia in which focal, bleb-like intracristal expansions (arrows) are noted, x 63,000.

Fig. 6. Mitochondrion after 2 hr ischemia showing swelling, rounding and matrix lucency. Loss of eristae is extensive and remaining cristae are hazy appearing. Small membranous profile is seen (arrow). × 50,000.

304

Fig. 7. Six hr ischemia. Swollen subsarcolemmal mitochondrion. Trilaminar plates with linear, membrane-like intracristal densities (arrows) are evident. Parallel arrangement of remaining cristae is a common accompanying finding, x 50,000.

be positively identified due to rupture and lysis. Others were reduced to membranous fragments which could still be recognized as mitochondrial vestiges. Less severely involved mitochondria exhibited the same changes as those already described at 6 hr of ischemia, although the changes in moth-eaten areas were more extreme. (3) 12 hours ischemia: The mitochondrial changes described at 6 hr of ischemia were more widespread and intense. Rupture and loss of mitochondria were far more prevalent. In ragged-red fibers abnormally large subsarcolemmal aggregations of mitochondria were seen. The mitochondria were swollen, rounded and closely-packed together leaving room for little or no intervening sarcoplasm. Inner compartments were lucent and contained prominent flocculent densities. There was fracture and lysis of cristae. Remaining cristae were abnormally straight, often arranged in parallel, and sometimes transformed into trilaminar plates which were less evident in ragged-red areas than elsewhere. Membranous profiles within mitochondria were larger and more numerous, especially in ragged-red areas. Such profiles often had the appearance of myeloid figures composed of poorly-defined, smudged lamellae with greatly variable width and a tendency toward a spiral arrangement. At times myeloid figures attained a size sufficient to fill the inner compartment or escape from its confines into the neighboring sarcoplasm (Fig. 8).

305

Fig. 8. Intramitochondrial myeloid figures seen at 12 hr ischemia. Larger myeloid figure (arrow apparently extending from mitochondrion into sarcoplasm. × 40,000.

Fig. 9. Large, rounded, intenselyosmiophilic inclusionbodies within mitochondria after 18 hr ischemia. x 25,000.

306 (4) 18 hours ischemia: Ragged-red fibers were easily identified as cells containing large masses of mitochondria in subsarcolemmal and internal locations. In ragged-red fibers, as well as elsewhere, a number of mitochondria contained either finely granular or large, dense osmiophilic inclusions. The former were focal accumulations, not clearly membrane-bound, of rounded densities varying in diameter from 120-350 A, consistent with the appearance of glycogen. These were not accompanied by similar accumulations in the sarcoplasm. The latter inclusions measuring 0.4-2.1 #m in diameter, were usually singular within the mitochondrion, intensely osmiophilic and homogeneous in density. They were frequently round in shape, less commonly oval or elliptical, with smooth external borders (Fig. 9). Core/targetoid areas were manifested as large, usually single, central or slightly eccentric regions of mitochondrial and myofibrillar disruption within muscle fibers which were otherwise relatively intact. Core/targetoid areas closely resembled motheaten areas in that myofibrillar disorganization, myofilament dissolution, expansion of intermyofibrillar spaces, and Z band streaming or zig-zagging were associated with loss of glycogen and mitochondrial damage. Mitochondria were swollen and rounded, their inner compartments were lucent, and flocculent densities were abundant. Reduced numbers of cristae were seen while the remaining cristae appeared shortened, excessively straight and hazy. Many mitochondria were fragmented or markedly reduced in number in core/targetoid areas. In target fibers there was a transitional zone between the central zone, which was identical to the core/targetoid area, and the more normal peripheral part of the involved fiber. Mitochondrial and myofibrillar changes were less severe in the transitional zone than in the central zone. Occasionally mitochondria in the transitional zone were increased in number and closely-spaced. (5) 24 hours ischemia: Giant mitochondria, first detected at 18 hours of ischemia, now became more apparent both in ragged-red fibers and less often in other fibers. Giant mitochondria were distinguished from swollen, rounded mitochondria by their larger size which at times exceeded that of the nucleus and their better state of preservation. Consequently matrix lucency, flocculent densities, and destruction of cristae were not part of the reaction which was, instead, epitomized by an abnormal arrangement of cristae. The most common pattern was that in which the cristal membranes were increased in number and oriented in a concentric configuration (Fig. 10). These membranes were seen as whorling, parallel pairs with periodic narrowing at 30-50 A intervals. Ragged-red fibers contained mitochondria which were abnormal in shape, often square or rectangular. Typically paracrystalline inclusions were present within such mitochondria (Fig. 11). The appearance of these crystalloids was determined by their orientation to the plane of section. In the most consistently-observed view, inclusions were composed of stacks of parallel, electron-dense lamellae measuring about 60 A in thickness. Lamellae, commonly in groups of 4, were separated from each other by a distance of about 50 ./k represented by lucent spaces. Many lamellae exhibited a beaded appearance due to the presence of electron-dense granules approximately 70 A in diameter arranged in periodic fashion along the membrane at constant intervals of 70-80 A.

307

Fig. 10. Whorling, concentric membrane pairs filling giant mitochondrion in ragged-red fiber at 24 hr ischemia, x 40,000.

Fig. 11. Ragged-red fiber after 24 hr ischemia in which several subsarcolemmal mitochondria contain paracrystallineinclusions, x 31,500.

308 Scattered mitochondria at 18 and 24 hours of ischemia contained lipid-like bodies. These were round, intermediately electron-dense, well demarcated but not definitely membrane-bound structures similar in appearance to lipid bodies found in the sarcoplasm of normal striated muscle. DISCUSSION A number of human neuromuscular diseases have been described in which mitochondrial abnormalities of voluntary muscle are a prominent pathologic finding. One such disease, first reported by Ernster, Ikkos and Luft (1959), is characterized by euthyroid hypermetabolism associated with loosely coupled oxidative phosphorylation in muscle mitochondria (Luft, Ikkos, Palmieri, Ernster and Afzelius 1962; Afifi, Ibrahim, Bergman, Haydar, Mire, Bahuth and Kaylani 1972). Shy and his co-workers (Shy and Gonatas 1964; Shy, Gonatas and Perez 1966) showed that myopathies having striking mitochondrial abnormalities may exist in the absence of metabolic implications. These authors proposed the terms "megaconial" and "pleoconial" to indicate the predominant morphologic change affecting the mitochondria. For a time, the idea that all diseases of skeletal muscle which had anomalous mitochondrial structure were a unified group of "mitochondrial myopathies" gained acceptance. The designation "mitochondrial myopathies" is still valid in a descriptive sense. However, as more cases have been studied, it has become evident that mitochondrial abnormalities may be seen in many diverse conditions including Kearns-Sayre (Berenberg, Pellock, DiMauro, Schotland, Bonilla, Eastwood, Hays, Vicale, Behrens, Chutorian and Rowland 1977) or Kearns-Shy syndrome (Karpati, Carpenter, Larbrisseau and Lafontaine 1973), carnitine deficiency (Engel, Banker and Eiben 1977), polymyositis (Chou 1969), thyroid dysfunction (Engel 1966; Norris and Panner 1966), steroid myopathy (Engel 1966), and Leigh's disease (Crosby and Chou 1974). Ultrastructurally, these changes can be classified under 3 categories: (1) increase in the number and size of mitochondria; (2) abnormalities in the shape and arrangement or structure of cristae; (3) presence of inclusions such as glycogen, lipid, and crystalloids. All 3 types of change were observed in our model of ischemic myopathy supporting the fact, already indicated by the heterogeneity of conditions in which mitochondrial aberrations are found, that mitochondria react in a limited and stereotypic manner after injury. Our studies also show that, although the mitochondrial changes common to the so-called "mitochondrial myopathies" often occur in subacute or chronic processes suggesting perhaps a similar slow evolution of the mitochondrial changes, the entire spectrum of change can actually develop very rapidly within the course of a few hours. As has been demonstrated in mitochondria of other organ systems - - particularly heart (Ganote, Seabra-Gomes, Nayler and Jennings 1975), kidney (Glaumann, Glaumann, Berezesky and Trump 1975) and brain (Brierley, Meldrum and Brown 1973) - - mitochondria of voluntary muscle are highly susceptible to hypoxic injury. The vulnerability of mitochondria during muscle ischemia was shown by Stenger, Spiro, Scully and Shannon (1962) in the canine sartorius and by Boehme, Themann and Gold (1966) in arteriosclerotic vascular disease of humans, although these earlier

309 ultrastructural studies were focused primarily on the contractile elements of muscle. In the present experimental model, during the first 6 hr of ischemia many of the mitochondrial changes, more numerous in necrotic myocytes, were of a destructive nature not usually found in human "mitochondrial myopathies". Mitochondria in the subsarcolemmal regions became swollen and abnormally rounded after 2 hr of ischemia. Similar observations were made by Karpati, Carpenter, Melmed and Eisen (1974) in rat soleus muscle after aortic ligation. This change, which spread to the inner portions of the cell with time, remained prominent during all phases of ischemia up to 24 hr. Condensation of the mitochondrial matrix so commonly seen in kidney (Glaumann et al. 1975) was not appreciated in skeletal muscle. On the other hand, matrix lucency, presumably due in part to an increase in inner compartment fluid (Moore et al. 1956), was widespread and seemed to be accentuated by cristal intermembrane vesiculation (Makitie and Teravainen 1977a), fracture and lysis. After 6 hr of ischemia greater numbers of mitochondria in necrotic cells showed signs of irreversible injury - - loss of cristae, flocculent densities and rupture of outer membranes. Based upon the relative integrity of their sarcolemma, contractile elements and organelles, a population of cells seemed to have received a sublethal injury. Some of these assumed the light microscopic appearance of ragged-red fibers containing large collections of mitochondria as described by Olson, Engel, Walsh and Einaugler (1972). These were identified in frozen sections as subsarcolemmal zones with irregular (ragged) red borders in trichrome stains which reacted intensely after incubation for oxidative enzymes. In some cells mitochondrial collections were not restricted to the subsarcolemmal zones but were dispersed throughout the myocyte. Ultrastructurally ragged-red fibers contained aggregations of closely-spaced mitochondria, some of which were swollen and rounded with matrix lucencies, cristal disruptions and flocculent densities. In other aggregations giant and abnormally-shaped mitochondria were seen. These were better preserved and larger than swollen rounded forms and lacked matrix lucency, cristal lysis and flocculent densities. In giant mitochondria the orderly arrangement of cristae was often replaced by a concentric whorling pattern. Many abnormally-shaped mitochondria were square or rectangular and contained paracrystalline inclusions essentially identical to those described by Karpati et al. (1973) in the Kearns-Shy syndrome and quite similar to those reported in other human "mitochondrial myopathies" (Chou 1969; DiMauro, Schotland, Bonilla, Lee, Gambetti and Rowland 1973; Schotland, DiMauro, Bonilla, Scarpa and Lee 1976; Shibasaki, Santa and Kuroiwa 1973; Toppet, Telerman-Toppet, Szliwowski, Vainsel and Coers 1977). Such mitochondrial abnormalities were not associated with pathological accumulations of lipid or glycogen within the sarcoplasm as may be seen in ragged-red fibers occurring in human disease. The derivation of paracrystalline inclusions remains obscure. Recently one variant of these was reported to be composed of protein, possibly arising from the polymerization of creatine kinase. This variant, similar in many ways to the paracrystalline inclusions we have observed, differed in its more simplified double membrane structure which under high resolution electron microscopy appeared to be constructed of helically wound filaments

310 (Hanzlikova and Schiaifino 1977). Paracrystailine inclusions were distinct from intracristal plates which were less commonly seen in ragged-red fibers but which were prevalent in necrotic fibers where swollen disrupted mitochondria were abundant. Intracristal plates were usually found in fragmented, abnormally straightened cristae and were composed of amorphous material simulating an intercristal membrane. They have been described in ischemic skeletal muscle (Karpati et al. 1974) and heart (Ganote et al. 1975). These plates are thought to be the result of a degenerative phenomenon and to originate from protein deposits between cristal membranes rather than from membrane duplication or proliferation (Smith and Klima 1976). Raggedred fibers have not previously been demonstrated in either human or animal muscle after ischemia (Makitie and Teravainen 1977b), although mitochondrial clustering under hypoxic circumstances has been noted by electron microscopists (Reznik 1967). Of interest is a recent publication describing ragged-red fibers in rats after the administration of pharmacologic agents known to uncouple mitochondrial oxidative phosphorylation (Melmed, Karpati and Carpenter 1975), especially in view of the association of this phenomenon and mitochondrial abnormalities in Luffs disease. The fine structural features of that experiment differed somewhat from those in the present study, however. Intracristal plates were conspicuous within the mitochondria of ragged-red fibers while alterations in cristal architecture and paracrystalline inclusions were not encountered. Crystalloids similar to those in our experiment have been observed in a current investigation of human muscle in intermittent claudication, although they were only rarely present and ragged-red fibers were not seen (Teravainen and Makitie 1977). Membranous profiles resembling myelin (myeloid figures) have been noted in many diseases of diverse etiologies in which muscle destruction is severe. Myopathies induced by vincristine (Clarke, Karpati, Carpenter and Wolfe 1972) and chloroquine (Macdonald and Engel 1970) are prototypes of this type of damage. The genesis of myeloid figures is thought to be lysosomal (Macdonald et al. 1970), sarcotubular (Clarke et al. 1972) and possibly mitochondrial (Macdonald et al. 1970). After 6 hr of ischemia in our experimental model myeloid figures were progressively more numerous in both necrotic and intact cells. Many of these lay adjacent to or were surrounded by mitochondria, while others were clearly within mitochondria from which they seemed to take origin. A few appeared to escape the confines of the parent mitochondria into the neighboring sarcoplasm, indicating a probable pathogenesis for at least some of the flee-lying myeloid figures. The ubiquity of this reaction and its importance in terms of the pathogenesis of myeloid figures has not been previously recognized in ischemic myopathy. In skeletal muscle mitochondria subjected to ischemia several types of inclusions were evident. These were more regularly situated in degenerating or necrotic cells, except for those intraconial inclusions composed of glycogen which were nearly restricted to ragged-red fibers. Flocculent densities were widespread in necrotic cells when mitochondria were swollen and exhibited cristal effacement. Such densities are familiar in ischemic heart (Ganote et al. 1975) and kidney (Glaumann et al. 1975) in which they are regarded as an expression of irreversible injury (Jennings 1976). Their

311 chemical composition is disputed and may be either protein or lipid in nature (Jennings 1976). Particles of calcium phosphate arranged in rosettes around a hollow center are consistently found in mitochondria of anoxic myocardium (Shen and Jennings 1972), but are not seen in voluntary muscle judging from these experiments and past ultrastructural studies. Not previously described in ischemic skeletal muscle was the presence of large, rounded, intensely osmiophilic intraconial bodies of uncertain origin. Because of their electron density they are less likely to be purely protein in composition and probably contain significant amounts of mineral. Structures with similar morphologic characteristics have been identified in human "mitochondrial myopathies" (Afifi et al. 1972). Moth-eaten fibers have been described in many diseases including myotonic dystrophy, inflammatory myopathies, polymyalgia rheumatica and Parkinson's disease (Dubowitz and Brooke 1973; Dubowitz 1974) but they have not been adequately appreciated in acute ischemic myopathy. Moreover, the ultrastructural features of moth-eaten fibers have received little attention in the medical literature (Roy and Dubowitz 1970; Morris and Raybould 1971). In this experimental model the major fine structural change in moth-eaten areas was severe mitochondrial disruption, accounting for the patchy reduction of oxidative enzyme activity in histochemical preparations. Mitochondrial changes were accompanied by a loss of glycogen and myofibrillar disorganization reflected in a lack of PAS staining and ATPase activity of involved areas. Unlike ragged-red areas, mitochondria did not appear to be increased in number or closely-approximated and neither giant mitochondria nor paracrystalline inclusions were seen. Cores and targets have not previously been reported in acute ischemia of skeletal muscle. We have chosen the term "core/targetoid" after Engel, Brooke and Nelson (1966) to refer to all core-like structures, recognizing the unresolved controversy over the exact definition of cores, multicores (Heffner, Cohen, Duffner and Daigler 1976), and targetoids. For the most part, these foci were large, single areas of decreased oxidative enzyme activity oriented with the long axis in the same plane as that of the involved fiber. In most respects the core/targetoid and moth-eaten foci were quite similar at both the light and electron microscopic levels, the essential difference being the patchy, multifocal distribution of abnormal areas within the moth-eaten fiber. It is possible that some core/targetoids have their genesis in moth-eaten fibers as these patchy areas coalesce. Areas of what were interpreted as early, incomplete coalescence could be seen in a few moth-eaten fibers. If coalescence does occur, it might explain the scarcity of moth-eaten fibers after 18 hr of ischemia at a time when core/targetoids initially appeared. Ultrastructurally target fibers were characterized by a three zone architecture, the center appearing "unstructured" and surrounded by a transitional zone where filament disruption was less severe (Neville 1973). While mitochondrial changes in the center zone resembled those in core/targetoids, mitochondria in the transitional zone were better preserved but, more importantly, closely-spaced, accounting perhaps for the increased oxidative enzyme activity in the transitional zone. Target fibers are most commonly encountered in denervation (Engel 1961), although they have been

312 recognized in other processes such as tenotomy (Engel et al. 1966). In our model of acute ischemia it seems unlikely that denervation could occur rapidly enough to effect such a change. Despite considerable research in recent years, the pathogenesis of muscular dystrophy remains speculative. Since a major impetus has been directed toward Duchenne muscular dystrophy, this disorder is often considered a model for a more complete understanding of the entire group of myopathies. The idea that muscular dystrophy may follow ischemic events was proposed by Kur6 and Okinaka in t930 and has been revived by Hathaway, Engel and Zellweger (1970) who produced necrotic and regenerative foci in rabbit muscle similar to those in Duchenne dystrophy after embolic occlusion of the muscle microvasculature. Focal lesions resembling these have recently been observed in human peripheral vascular disease (Engel and Hawley 1977). Mendell, Engel and Derrer (197t) achieved comparable results in rats by creating functional ischemia of muscle when a combination of aortic ligation and serotonin administration was used, implying that vasoactive biogenic amines might play a role in the pathogenesis of Duchenne dystrophy. It has since been shown that in Duchenne dystrophy platelet transport of serotonin is impaired (Murphy, Mendell and Engel 1973) and fluorescent material, possibly biogenic amines, accumulates in skeletal muscle (Wright, O'Neill and Olson 1973). However, evaluation of muscle blood flow (Paulson, Engel and Gomez 1974), morphometric analysis of intramuscular blood vessels (Jerusalem, Engel and Gomez 1974) and determinations of vasogenic amine excretion in human dystrophy (Mendell, Murphy. Engel, Chase and Gordon 1972) have not supported the theory that dystrophy is related to an ischemic phenomenon. The majority of our findings, which extend the observations of Karpati et al. (1974), seem to offer further evidence against functional ischemia being a major factor in the development of Duchenne dystrophy. In 22 cases of Duchenne dystrophy which we have examined by light and electron microscopy we have never observed ragged-red fibers or target fibers. Ultrastructurally, mitochondrial swelling and cristal lysis may be seen in necrotic fibers but these changes are not as conspicuous as in ischemia. Moreover, we have not found giant mitochondria, significant abnormalities in mitochondrial shape and cristal arrangement, or intraconial inclu3ions resembling crystalloids and osmiophilic bodies. We have not seen intracristal plates in Duchenne dystrophy, although they have been mentioned as an infrequent occurrence by Karpati et al. (1974). Of interest is the presence of core/targetoids and moth-eaten fibers in experimental ischemic myopathy, since both are reported in the muscles of Duchenne dystrophy carriers (Roy and Dubowitz 1970; Morris and Raybould 1971). Whether these abnormalities which are not consistent features of clinically manifest Duchenne dystrophy have any relevance to a possible ischemic etiology in muscular dystrophy is unclear at this time.

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