Skeletal Dysfunction PDF

CHAPTER 15 Skeletal Muscle A B C D 997 Figure 15.9 Pathologic Changes Resulting in Pale Skeletal Muscle. A, Pale streaks, necrosis and mineralization, degenerative myopathy, canine X-linked muscular dystrophy, diaphragm (left side), dog. B, Localized pallor, necrosis, injection site of an irritant s...

CHAPTER 15 Skeletal Muscle A B C D 997 Figure 15.9 Pathologic Changes Resulting in Pale Skeletal Muscle. A, Pale streaks, necrosis and mineralization, degenerative myopathy, canine X-linked muscular dystrophy, diaphragm (left side), dog. B, Localized pallor, necrosis, injection site of an irritant substance, semitendinosus muscle, cow. The irritant was injected just under the perimysium and caused necrosis and disruption of the myofibers. Some irritant seeped down between the fascicles to cause necrosis, but the fascicles of myofibers are still in place. C, Overall pale muscle with pale streaks from collagen and fat infiltration, denervation atrophy, equine motor neuron disease, horse. Equine motor neuron disease muscle (right) compared with normal muscle (left). D, Enlargement and pallor, steatosis, longissimus muscles, neonatal calf. The majority of the muscles have been replaced by fat. (A courtesy Dr. B.A. Valentine, College of Veterinary Medicine, Oregon State University. B and D courtesy Dr. M.D. McGavin, College of Veterinary Medicine, University of Tennessee. C courtesy Dr. A. de Lahunta, College of Veterinary Medicine, Cornell University.) muscle damage (see Fig. 15.35, A) or can simply reflect vascular stasis (hypostatic congestion) after death. Hemorrhagic streaks within the diaphragm often accompany death caused by acute exsanguination. A green discoloration can indicate either eosinophilic inflammation (Fig. 15.10) or severe putrefaction. Lipofuscin accumulation in old animals, especially cattle, can cause a tan-brown discoloration of muscle. Black discoloration of the fascia occurs in calves with melanosis as an incidental finding and in older gray horses with metastasis of dermal melanoma to muscle fascia. Evaluation of texture is also important. Severely thickened and often calcified fascia occurs in cats with fibrodysplasia ossificans progressiva. Fat infiltration or necrosis can result in abnormally soft muscle. Decreased or increased muscle tone can be caused by denervation. Decreased tone can also occur as a result of a lack of muscle conditioning or postmortem autolysis. Careful microscopic examination of multiple muscles is often required to detect lesions. In cases of suspected neuromuscular disease, multiple muscle samples should include active muscle (tongue, diaphragm, intercostal muscles, and masticatory muscles), proximal muscle (lateral triceps, biceps femoris, semimembranosus, semitendinosus, and gluteal), and distal muscle (extensor carpi radialis and cranial tibial). For purposes of a biopsy, certain muscles (e.g., lateral triceps, biceps femoris, cranial tibial, semimembranosus, and semitendinosus) are easier to sample because of their parallel myofiber orientation. The ideal samples will also vary, depending on the suspected disorder, such as a type 1 predominant postural muscle for diagnosis of equine motor neuron disease, a type 2 predominant locomotory muscle for diagnosis of equine polysaccharide storage myopathy and temporal or masseter muscle for diagnosis of masticatory myositis in dogs and masseter myopathy in horses. Short fibers, such as those in the intercostal muscle, are preferred for physiologic studies in which intact muscle fibers are necessary and for studies of neuromuscular junction zones. Sampling of Muscle for Examination Information on this topic is available at www.expertconsult.com. Microscopic Examination Information on this topic is available at www.expertconsult.com. Enzyme Histochemistry and Immunohistochemistry Information on this topic is available at www.expertconsult.com. Electron Microscopy Information on this topic is available at www.expertconsult.com. Other Methods of Evaluation Information on this topic is available at www.expertconsult.com. Dysfunction/Responses to Injury It is often said that the range of response of muscle to injury is limited, consisting primarily of necrosis and regeneration. Actually, muscle is a remarkably adaptive tissue, with a wide range of response CHAPTER 15 Skeletal Muscle To ensure proper fixation and orientation of sections prepared from fixed specimens, the sample should be a strip of muscle no more than 1 cm in diameter, with myofibers running lengthwise. Muscle maintains the ability to contract for some time after death, with the time varying, depending on the physiologic state. 997.e1 Contraction of muscle after contact with fixative is the most common cause of an artifact called contraction band artifact. Contraction can be prevented or at least minimized by use of a specially designed muscle clamp (E-Fig. 15.3, A) or by placing the sample on a rigid surface, such as a portion of a tongue depressor, and fixing the ends with sutures, staples, or clamps before submersion in the fixative (E-Fig. 15.3, B). A B C E-Figure 15.3 Techniques for Collection of Muscle Samples for Histologic Examination. Clamps are used to prevent contraction of a fresh muscle specimen when it is immersed in 10% neutral-buffered formalin or an electron microscopy fixative. A, Types of muscle clamps (from left to right): disposable plastic clamps (open and closed), stainless steel clamps (open and closed), a gallbladder clamp (unmodified), and a modified gallbladder clamp. The stainless steel clamps are autoclavable, the best but expensive. A suitable and economical clamp (not shown) can be made by welding a bar approximately 1 cm long, 3 to 5 mm wide, and 3 mm thick between the lower jaws of two small hemostats. B, Final excision stage. Initially two longitudinal incisions, approximately 5 mm apart and 15 mm long, are made into the muscle in the direction of the myofibers. A horizontal cut is made 3 to 4 mm below the surface to undermine a piece of muscle. One jaw of the clamp is inserted under the muscle until its tip just exits on the other side. The clamp is lifted several millimeters above the surface of the muscle to ensure that the muscle fibers are tense and then the jaws are clamped. The clamped piece of muscle is excised by cutting at each end adjacent to the clamp as shown above. The muscle sample, still in the clamps, is placed in the fixative, usually 10% buffered-neutral formalin, for histopathologic examination and fixed overnight. For fixation for electron microscopic examination, the muscle in the clamp is placed into an electron microscopy fixative for 1 to 2 hours. For histopathologic examination, the muscle is trimmed by freeing the strip of muscle between the clamps by cutting immediately adjacent to the clamp jaws. Then a transverse section is cut from one end of this sample, avoiding any crushed area, and the remainder of the sample is cut longitudinally in the direction of the myofibers. Both samples are desirable for histopathologic examination. For electron microscopy, after fixation for 1 to 2 hours, slivers 0.5 to 1 mm thick are shaved from the outside of the sample. These are cut into pieces 0.2 mm in diameter and 0.5 mm long, with the longer dimension being in the direction of the myofibers. This long sample facilitates embedment so that the fibers are oriented either in cross section or longitudinally. Both sections are required for electron microscopy. C, Pinning strips of muscle onto a rigid surface, such as a piece of tongue depressor before immersion in 10% neutral-buffered formalin, will also minimize fixation artifacts but is not as effective as the clamps shown above. (A and B courtesy Dr. M.D. McGavin, College of Veterinary Medicine, University of Tennessee. C courtesy Dr. B.A. Valentine, College of Veterinary Medicine, Oregon State University.) 997.e2 SECTION II Pathology of Organ Systems E-Table 15.1 Useful Special Stains and Enzyme Reactions Stain ROUTINE SECTIONS Masson trichrome Reticulin PTAH von Kossa Alizarin red S PAS PAS with amylase (diastase) FROZEN SECTIONS Modified Gomori’s trichrome NADH, SDH, cytochrome oxidase ATPase Acid phosphatase Nonspecific esterase Oil red O; Sudan black von Kossa, alizarin red S, PAS, PAS with amylase (diastase) digestion Use Differentiates collagen (blue) from other tissues, such as muscle and myelin (red) Stains reticular fibers of the muscle interstitium including endomysium, therefore outlines individual myofibers Stains cross-striations in longitudinal sections of muscle Stains carbonates and phosphates linked with calcium in mineralized fibers Stains calcium in necrotic and mineralized fibers Identifies glycogen and proteoglycans; also stains protozoal cysts Differentiates proteoglycans (amylase resistant) from glycogen (amylase sensitive) Stains mitochondria (red), nemaline rods (red), collagen (green); differentiates myelin (red) from collagen (green) in nerves Stains these mitochondrial enzymes Differentiates myofiber types Stains macrophages and denervated fibers Stains macrophages and denervated fibers; identifies neuromuscular junctions Stains lipid (only in frozen sections) Same as in routine stains (above) ATPase, Adenosine triphosphatase; NADH, nicotinamide adenine dinucleotide dehydrogenase; PAS, periodic acid–Schiff; PTAH, phosphotungstic acid hematoxylin; SDH, succinate dehydrogenase. Frequently, lesions in muscles can be detected and evaluated only by microscopic examination. Proper microscopic examination requires evaluation of both transverse and longitudinal sections. Myofiber diameters, cytoarchitectural changes, and the percentage of abnormal myofibers are most reliably evaluated in transverse sections. Longitudinal sections reveal the length of changes such as segmental necrosis or regeneration or deposition of storage material. Improperly oriented samples, which result in sections that have obliquely oriented myofibers and thus neither longitudinal nor transverse myofibers, are difficult to evaluate. Use of a magnifying glass or dissecting microscope can aid in determining the orientation of myofibers during trimming of muscle before sectioning. Routine stains, such as hematoxylin and eosin (H&E), run the risk of offering the pathologist a “vast pink wasteland” for evaluation and are often inadequate for detecting subtle myopathic changes, lesions within intramuscular nerves, or the presence of abnormal stored material. Various special stains, including reticulin, Masson trichrome, von Kossa, lipid (performed on frozen sections of fixed samples), and periodic acid–Schiff (PAS) for glycogen, are often invaluable in the evaluation of routinely processed skeletal muscle (E-Table 15.1). Examples of many of these disorders can be found in this chapter. Other valuable stains and reactions can only be performed on frozen sections of unfixed muscle samples (see E-Table 15.1). For many decades, myofiber typing could be done only on frozen sections using the myosin ATPase reaction. Recently, immunohistochemical staining of myosin has been developed for demonstration of myofiber types in formalin-fixed muscle. This is a major advantage because fiber-type staining is often essential for the complete evaluation of muscle. It is most useful in demonstrating preferential involvement of a fiber type and alteration of the fiber-type pattern, the result of denervation and reinnervation. There is no question that frozen section histochemistry of unfixed muscle samples is the “gold standard” of muscle pathology. Skeletal muscle may be the one tissue in which the morphology of cells and cellular components is best appreciated in frozen sections. Routine frozen section histochemistry on muscle includes a battery of stains applied to serial sections. Examples of many of these stains are illustrated in this chapter. Stains used include H&E, modified Gomori’s trichrome, ATPase for fiber typing, nicotinamide adenine dinucleotide dehydrogenase (NADH), succinate dehydrogenase (SDH), cytochrome oxidase, and other mitochondrial enzyme stains, PAS for glycogen, alizarin red S for calcium, alkaline phosphatase and nonspecific esterase for macrophages and denervated fibers, and lipid stains. When indicated, frozen sections also allow for immunostaining for cytoskeletal proteins, such as dystrophin (see Fig. 15.45) and the dystrophin-associated proteins. Certain abnormal structures, such as nemaline rods formed by expansion of Z bands, as seen in nemaline rod myopathy, are not visible in routine sections but are readily identified in frozen sections stained with modified Gomori’s trichrome. The major disadvantage of frozen section histochemistry is that unless a neuromuscular disease laboratory is readily available to immediately process unfixed muscle samples, careful preparation for overnight shipping, on ice, in a moist but not overly wet environment, is necessary. Any delay in shipment or overwetting or overheating of the sample results in nondiagnostic samples. In addition, preparation of frozen sections is time and labor intensive, and in most cases only a single transverse section approximately 1 cm in diameter is examined. This can create a significant sampling error when evaluating a small sample of a large muscle in which lesions may not be evenly distributed. Complete evaluation, which includes morphometric examination and calculation of the percentage and mean diameter of each fiber type, detects changes in the percentage of each fiber type and fiber atrophy or hypertrophy. But at this time, morphometric analysis is not routinely performed on samples submitted for diagnostic purposes. Frozen section histochemistry is always a powerful tool for evaluation of muscle disease. But in many disorders, it is possible to obtain diagnostic sections from routinely processed muscle samples when appropriate sample selection, handling, and processing are performed, and sections are examined by a pathologist familiar with muscle pathology. CHAPTER 15 Skeletal Muscle Although much of what used to be determined by electron microscopy has been supplanted by newer immunohistochemical procedures, electron microscopic evaluation of muscle is still important. Various structural alterations, such as abnormalities of neuromuscular junctions, mitochondria, sarcomeric disarray, sarcotubular dilatation, Z-line streaming, and cytoplasmic inclusions, may be best visualized, and in some cases only visualized, by this method. Sampling and handling methods to minimize contraction and other artifacts and to allow for precise transverse and longitudinal sections are imperative. 997.e3 Physiologic testing of isolated intact myofibers in vitro forms the basis for diagnosis of malignant hyperthermia (MH). Short fibers, such as from samples of intercostal muscle, are preferred. While maintained in a physiologic solution, myofiber bundles are exposed to various agents, such as caffeine and halothane, to detect abnormal contractural sensitivity. Biochemical and molecular biologic analysis of muscle samples can evaluate levels of muscle enzymes and other proteins, and genetic analysis can be performed to detect specific gene defects. These latter tests require fresh muscle samples snapfrozen in liquid nitrogen and maintained at −70° C until analysis. 998 SECTION II Pathology of Organ Systems A A * * * m B B Figure 15.10 Bovine Eosinophilic Myositis, Gluteal Muscles, Cow. A, Green discoloration of the muscle is due to inflammation that has abundant eosinophils (see B). The inflammation is attributed to degenerating Sarcocystis spp. For histopathologic findings, see Fig. 15.38. B, Note the large number of eosinophils (arrows) in the inflammatory exudate associated with degenerating muscle fibers (m). Hematoxylin and eosin (H&E) stain. (Courtesy Dr. M.D. McGavin, College of Veterinary Medicine, University of Tennessee.) to physiologic and pathologic conditions. Myofibers can add or delete sarcomeres to cause elongation or shortening of the entire muscle. In addition to necrosis and regeneration, myofibers can atrophy and hypertrophy, they can split, they can undergo a variety of cytoarchitectural alterations, and they can completely alter their physiologic functions when undergoing fiber-type conversion. To describe muscle response to injury as stereotypical does not do justice to this inherent plasticity. What is true, though, is that it is frequently not possible to determine the cause of muscle injury based on gross or histologic lesions alone. Supplementary tests and clinical histories are often essential. Necrosis and Regeneration Myofiber necrosis can accompany a variety of disorders. Because of their multinucleate nature, myofibers often undergo segmental necrosis, with involvement of only one or several contiguous segments within the cell. Global necrosis of the entire length of the myofiber occurs only under severe duress, such as extreme pressure to the entire muscle causing crush injury, or widespread ischemia because of pressure on, or thromboembolism of, a large artery. Necrotic portions of myofibers have several different histologic appearances. The earliest change is often segmental hypercontraction, resulting in segments of slightly larger diameter that are slightly darker staining (“large dark fibers”) that are best seen on transverse Figure 15.11 Myofiber Necrosis, Skeletal Muscle. A, Hypercontraction, transverse section. Large, deeply stained fibers (large, dark red fibers [arrow]) are hypercontracted segments of a myofiber, the initial stage of necrosis. Note the rounded outline of these myofibers compared with the polygonal outlines of normal myofibers. Formalin fixation, hematoxylin and eosin (H&E) stain. B, Segmental necrosis, monensin toxicosis, longitudinal section, horse. Segments of the myofibers have undergone hypercontraction (center of figure), and the remaining cytoplasm is fragmented (asterisks). Formalin fixation, H&E stain. (Courtesy Dr. M.D. McGavin, College of Veterinary Medicine, University of Tennessee.) sections (Fig. 15.11, A). On longitudinal sections, “twisting” or “curling” of affected fibers is often seen. But similar changes occur as an artifactual change in improperly handled samples. The cytoplasm of fully necrotic portions of the fiber is often homogeneously eosinophilic and pale (hyaline degeneration), with loss of the normal cytoplasmic striations and the adjacent muscle nucleus. The affected cytoplasm then becomes floccular or granular as that portion of the myofiber starts to fragment (Fig. 15.11, B; see Fig. 15.14, B). Increased intracellular calcium is a common trigger of necrosis in all cells, and myofibers contain a high level of calcium ions stored in the sarcoplasmic reticulum. Therefore, myofibers may be particularly sensitive to calcium-induced necrosis, either as a result of damage to the sarcolemma, causing influx of extracellular calcium, or from damage to the sarcoplasmic reticulum, releasing intracellular stores of calcium. Therefore, it is no surprise that necrotic myofibers are often prone to overt mineralization. Overtly mineralized myofibers appear as chalky white streaks on gross examination (see Fig. 15.9, A) and as basophilic granular to crystalline material within myofibers on histologic examination. Large deposits of mineral can induce a foreign body granulomatous response. Although the presence or absence of myofiber mineralization has sometimes been used as a diagnostic aid, the circumstances under which a necrotic myofiber segment can become mineralized are so diverse that myofiber mineralization must be considered a nonspecific response, indicative CHAPTER 15 Skeletal Muscle A A only of myofiber necrosis. Myofiber mineralization can be confirmed with histochemical stains, such as alizarin red S and von Kossa. Histochemical staining for calcium in frozen sections also detects increased intracytoplasmic calcium in damaged myofibers that are not overtly necrotic or mineralized (Fig. 15.12, A). Provided there is still an adequate blood supply, macrophages derived from transformation of blood monocytes rapidly infiltrate areas of myofiber necrosis (Fig. 15.12, B). Macrophages are able to traverse the basal lamina and rapidly clear cytoplasmic debris (Fig. 15.13, A). Other leukocytes, including neutrophils, eosinophils, and lymphocytes, can also be recruited to sites of extensive myonecrosis, presumably because of various cytokines released from damaged muscle. The infiltration of macrophages and other cells into areas of damaged muscle to clear away necrotic myofibers does not in any way constitute a form of myositis. Because myonuclei are unable to divide, regeneration of muscle relies on satellite cell activation. Muscle satellite cells are resistant to many of the insults that result in myofiber necrosis, and activation of satellite cells is triggered by necrosis of adjacent segments of that myofiber. Therefore, as macrophages are clearing cytoplasmic debris, satellite cells are becoming activated and begin to divide in preparation for regeneration of the affected myofiber segment. If the myofiber basal lamina is still intact, it will leave an empty cylindrical space known as a sarcolemmal tube. This name is clearly a misnomer, dating from the days when the term sarcolemma was applied to the tube formed by the basal lamina that remains after segmental myofiber necrosis. Clearly what is now termed the sarcolemma (plasmalemma) of necrotic fiber segments is lost, but this is a misnomer * * B Figure 15.12 Myofiber Necrosis, Skeletal Muscle, Transverse Section. A, There has been a massive influx of calcium (stained red-orange) into acutely necrotic fibers. Frozen section, alizarin red S stain. B, Macrophages with red-brown staining cytoplasm invading necrotic myofibers. Portions of intact fibers are in the lower left. Frozen section, nonspecific esterase stain. (A courtesy Dr. B.A. Valentine, College of Veterinary Medicine, Oregon State University. B courtesy Dr. B.J. Cooper, College of Veterinary Medicine, Oregon State University.) 999 B * C Figure 15.13 Segmental Necrosis and Regeneration. A, Monophasic segmental coagulation necrosis, skeletal muscle, longitudinal section of two myofibers. A segment of the upper fiber (right) and all the visible portion of the lower fiber have undergone necrosis, and macrophages (arrows) have invaded through the intact basal lamina and cleared the cytoplasmic debris. Satellite cells on the inner surface of the basal lamina of the lower fiber are activated, and one (arrowhead) is in mitosis. One-micron-thick plastic-embedded section, H&E stain. B, Polyphasic injury, segmental coagulation necrosis (arrows) and regeneration of myofibers, muscle, longitudinal section. Between each of the foci of coagulation necrosis in the lowest myofiber is a segment of small-diameter faintly basophilic cytoplasm lacking cross-striations, in which there is an internal chain of euchromatic nuclei (asterisks). This is a late stage of regeneration. Formalin fixation, hematoxylin and eosin (H&E) stain. C, Monophasic injury, late-stage regeneration, skeletal muscle, longitudinal section. The regenerating segment of the myofiber consists of myotubes, which have small diameters (asterisk), with slightly basophilic cytoplasm and internal rows of large euchromatic nuclei (arrowheads). Formalin fixation, H&E stain. (A courtesy Dr. A. Kelly, University of Pennsylvania. B courtesy Dr. B.A. Valentine, College of Veterinary Medicine, Oregon State University. C courtesy Dr. B.J. Cooper, College of Veterinary Medicine, Oregon State University.) 1000 SECTION II Pathology of Organ Systems that is firmly entrenched. The important concept to remember is that, if intact, the basal lamina forms a cylindrical scaffold to guide proliferating myoblasts and to keep fibroblasts out. Satellite cells may be seen undergoing mitosis, at which stage they are known as activated myoblasts, on the inner surface of this tube (see Fig. 15.13, A). Within hours, proliferating myoblasts will fuse end-to-end to form myotubes (Fig. 15.13, B and C), and within days the myotube produces thick and thin filaments and undergoes maturation to a myofiber, reestablishing myofiber integrity. If the basal lamina is ruptured, myotubes are said to be able to bridge gaps of 2 to 4 mm, and larger ones heal by fibrosis. The process of myofiber regeneration recapitulates embryologic development of skeletal muscle and is depicted schematically in Fig. 15.14. A percentage of dividing satellite cells Satellite cell Fibroblast Muscle nucleus Endomysium Basal lamina Plasmalemma A B Coagulation necrosis do not fuse with the forming myotube but instead become new satellite cells capable of future regeneration. In summary, the success of muscle regeneration depends on (1) the presence of an intact basal lamina and (2) the availability of viable satellite cells. The stages of successful muscle regeneration are summarized in Box 15.2. Thus, myofibers undergoing segmental necrosis in which the basal lamina is preserved, as in metabolic, nutritional, and toxic myopathies, regenerate very successfully. However, when large areas of satellite cells are killed (e.g., by heat, intense inflammation, or infarction), the situation is very different. In this case, a return to normal is not possible, and healing is chiefly by fibrosis. If the insult to the muscle is sufficient to disrupt the myofiber basal lamina but not enough to damage the satellite cells, regeneration attempts are ineffective. Because the basal lamina is not intact, there is no tube to guide the myoblasts proliferating from each end. Myoblast proliferation under these conditions results in formation of so-called muscle giant cells (Fig. 15.15). Thus, the presence of muscle giant cells indicates that conditions for regeneration have not been optimal after destructive lesions, such as those caused by trauma that transects myofibers, infarction, and intramuscular bacterial infection or injection of irritants. Muscle giant cells are often accompanied by fibrosis, which will unite the ends of the damaged myofibers. This also occurs in muscle damaged by invasive or metastatic sclerosing carcinomas. Cytokines released from damaged muscle fibers contribute to the signaling pathways that initiate Box 15.2 C Satellite cell Macrophage D Myoblast E F Figure 15.14 Segmental Myofiber Necrosis and Regeneration. A, Myofiber, longitudinal section. B, Segmental coagulation necrosis. C, The necrotic segment of the myofiber has become floccular and detached from the adjacent viable portion of the myofiber. The satellite cells are enlarging. D, The necrotic segment of the myofiber has been invaded by macrophages, and satellite cells are migrating to the center. The latter will develop into myoblasts. The plasmalemma of the necrotic segment has disappeared. E, Myoblasts have formed a myotube, which has produced sarcoplasm. This extends out to meet the viable ends of the myofiber. The integrity of the myofiber is maintained by the sarcolemmal tube formed by the basal lamina and endomysium. F, Regenerating myofiber. There is a reduction in myofiber diameter with central rowing of nuclei. There is early formation of sarcomeres (crossstriations), and the plasmalemma has re-formed. Such fibers stain basophilic with H&E stain. (Redrawn with permission from Dr. M.D. McGavin, College of Veterinary Medicine, University of Tennessee.)  Stages of Muscle Regeneration under Optimal Conditions Muscle nuclei disappear from the necrotic segment and the sarcoplasm becomes hyalinized (eosinophilic, amorphous, and homogeneous) because of the loss of normal myofibrillar structure (see Fig. 15.15, B). The necrotic portion may separate from the adjacent viable myofiber (see Figs. 15.12, B; 15.14, A and B; and 15.15, C). Within 24 to 48 hours, monocytes emigrate from capillaries, become macrophages, and enter the necrotic portion of the myofiber (see Figs. 15.13, B; 15.14, A; and 15.15, D). Concurrently, the satellite cells, located between the basal lamina and the sarcolemma, begin to enlarge (see Figs. 15.14, A, and 15.15, C and D), become vesicular with prominent nucleoli, and then undergo mitosis to become myoblasts. Myoblasts migrate from the periphery to the center of the sarcolemmal tube, admixed with macrophages (see Fig. 15.15, D). Macrophages lyse and phagocytose necrotic debris and form a clear space in the sarcolemmal tube, and the shape and integrity of the sarcolemmal tube are maintained by the basal lamina (see Fig. 15.14, A). Myoblasts fuse with one another to form myotubes, which are thin, elongated muscle cells with a row of central, closely spaced nuclei. Developing myotubes send out cytoplasmic processes in both directions within the sarcolemmal tube (see Fig. 15.15, E). When the processes contact each other or a viable portion of the original muscle fiber, they fuse. The regenerating fiber is characterized by (1) basophilia as a result of increased RNA content; (2) internal nuclei, often in rows, that have differentiated to myonuclei; (3) a lack of striations; and (4) a smaller-than-normal diameter (see Figs. 15.14, B and C, and 15.15, F). The fiber grows and differentiates. Its diameter increases, the sarcoplasm loses its basophilia, and longitudinal and cross-striations appear, indicating the formation of sarcomeres. In most species, within several days, the muscle nuclei of regenerating fibers move to their normal position at the periphery of the fiber, just under the sarcolemma. CHAPTER 15 Skeletal Muscle Box 15.3 1001  Findings Associated with Chronic Myopathic Change Excessive fiber-size (diameter) variation Internal nuclei Fiber splitting Other cytoarchitectural changes Fibrosis Fat infiltration Figure 15.15 Ineffectual Regeneration. Large, bizarre multinucleate muscle giant cells (arrow) are indicative of regeneration in an area in which the myofiber’s basal lamina has been damaged. Because the wall of the “myotube” of basal lamina is not intact, regenerating sarcoplasm exudes through the defect, and in cross section this appears as a “muscle giant cell.” Formalin fixation, hematoxylin and eosin (H&E) stain. (Courtesy Dr. M.D. McGavin, College of Veterinary Medicine, University of Tennessee; and Noah’s Arkive, College of Veterinary Medicine, The University of Georgia.) macrophage infiltration and regeneration, but they also contribute to interstitial fibroblast activation. Collagen is inelastic, and thus large areas of fibrosis inevitably reduce the ability of the muscle to contract and to stretch. Fibrosis within locomotory muscles often results in obvious alteration of the gait. Because segmental necrosis and regeneration are such a common result of a wide variety of insults (e.g., overexertion, selenium deficiency, and toxic injury), a histologic diagnosis of segmental necrosis is often not helpful in determining the cause of the disease. Pathologic classification of lesions according to distribution (e.g., focal, multifocal, locally extensive, and diffuse) and duration (e.g., acute, subacute, and chronic) has proven to be extremely useful in determining the possible causes of segmental muscle necrosis. Pathologic classification of degenerative myopathies is enhanced by use of the terms monophasic necrosis and polyphasic necrosis. Monophasic lesions are of the same duration, indicative of a single insult. Polyphasic lesions indicate an ongoing degenerative process. Thus, a focal monophasic lesion could be the result of a single traumatic incident such as an intramuscular injection (see Fig. 15.9, B). A multifocal monophasic lesion could represent a single episode of overly strenuous exercise (exertional myopathy) or a toxin being ingested on one occasion (e.g., a horse eating one dose of monensin; see Figs. 15.11, B, and 15.33, B). However, if the insult is repeated or ongoing, such as occurs in muscular dystrophy (see Fig. 15.44), selenium deficiency, or continuous feeding of a toxin, then new lesions (segmental necrosis) will form at the same time that regeneration is taking place; in other words, it will be a multifocal and polyphasic disease (see Fig. 15.13, B). Using this approach, it is sometimes possible to rule out a diagnosis (e.g., muscular dystrophy and selenium deficiency myopathy are typically polyphasic), but this is not an invariable rule. For example, in livestock with borderline concentrations of selenium, a sudden stress can cause a monophasic necrosis. The term rhabdomyolysis is often encountered, particularly in the clinical arena, and especially in association with exerciseinduced muscle injury (exertional rhabdomyolysis) in human beings, horses, and dogs. Technically, rhabdomyolysis simply means necrosis (lysis) of striated muscle. Rhabdomyolysis generally indicates the presence of a severe degenerative myopathy with a large degree of myofiber necrosis (see Fig. 15.35). In horses, the term exertional rhabdomyolysis has become firmly entrenched as a clinical entity in which exercise-induced muscle injury is the presenting sign. The term recurrent exertional rhabdomyolysis is often employed in cases in which repeated bouts of exercise-induced muscle damage have been documented. Alteration in Myofiber Size The normal myofiber diameter will vary, depending on fiber type, the muscle examined, the species, and the age of the animal. In some species (e.g., horses, cats, and human beings), there are three distinct populations based on diameter: type 1 fibers are the smallest, type 2B fibers are the largest, and type 2A fibers are intermediate in size. Different sizes in diameters are in part a reflection of the oxidative needs of the fibers; oxygen diffuses more readily into the interior of small-diameter fibers. In the dog, all fiber types are oxidative, and fiber-type diameter is much more uniform. A histogram generated from morphometric analysis of fiber diameters will reveal the characteristics of individual muscles in various species. Not surprisingly, this type of detailed information is more readily available for human patients than for animals. Even without morphometric analysis, however, a pathologist experienced in examination of muscle can often determine whether there is a normal fiber-size distribution (based on fiber diameter in transverse section) or whether there is an increase in fiber-size variation. The finding of increased fiber-size variation suggests that something is wrong but in itself does not give any indication of cause. Increased fiber-size variation can be a result of fiber atrophy, fiber hypertrophy, or both and is considered part of the spectrum of changes included in the term chronic myopathic change (Box 15.3). Atrophy The term atrophy is used to imply either a reduction in the volume of the muscle as a whole or a reduction in the diameter of a myofiber. In the early stages of atrophy, it may be difficult or impossible to detect loss of muscle mass by gross observation, and morphometric evaluation of myofiber diameters may be required. Several cellular physiologic processes can be activated to result in muscle atrophy. These include induction of lysosomal action to result in autophagy of cytoplasmic components, apoptosis (programmed cell death), and activation of the cytoplasmic ubiquitin-proteosomal machinery. Lysosomal activation is prominent in denervation atrophy and is the basis for the positive reaction of denervated fibers in alkaline phosphatase and nonspecific esterase preparations. The causes of muscle fiber atrophy include physiologic and metabolic processes and denervation. In most instances, muscle atrophy is reversible provided the cause is corrected. The type of fiber undergoing atrophy varies, depending on the cause; therefore, fiber typing is often required for a definitive diagnosis. Interestingly, type 2 fibers are the most likely to atrophy under a variety of circumstances (Box 15.4). Signaling molecules involved in muscle atrophy include tumor necrosis factor-α (TNF-α) and interleukin (IL)-1 and IL-6. 1002 Box 15.4 SECTION II Pathology of Organ Systems  Fiber Types Affected in Different Types of Muscle Atrophy Denervation: Type 1 and type 2 fibers; reinnervation leads to altered fiber-type patterns (fiber-type grouping) Disuse: Predominantly type 2 fibers; may vary, depending on the species and cause Endocrine disease: Predominantly type 2 fibers; associated with hypothyroidism and hypercortisolism Malnutrition, cachexia, and senility: Predominantly type 2 fibers Congenital myopathy: Often predominantly type 1 fibers Physiologic Muscle Atrophy. Decrease in myofiber diameter and therefore in the overall muscle mass is a physiologic response to lack of use (disuse atrophy), cachexia, and aging. Type 2 fibers are preferentially affected (E-Fig. 15.4). Disuse atrophy occurs relatively slowly, and only in muscles not undergoing normal contraction, as is caused by severe lameness or in muscles of a limb that is splinted or enclosed in a cast. The degree of disuse atrophy will be variable, but typically it is not as severe as the atrophy of cachexia or denervation. Disuse atrophy is often asymmetric. Muscle atrophy caused by cachexia can be profound, especially in cases of cancer cachexia, in which increased circulating levels of TNF alter the muscle metabolism, favoring catabolic processes rather than anabolic processes. Cachexia also develops relatively slowly and causes symmetric muscle atrophy. Starvation, malnutrition, neoplasia, and chronic renal and cardiac diseases are possible causes of cachexia. Atrophy Caused by Endocrine Disease. Preferential atrophy of type 2 fibers causing symmetric muscle atrophy also occurs because of various endocrine disorders. The most common are hypothyroidism and hypercortisolism in dogs. Aging horses with pituitary dysfunction or tumors (leading to equine Cushing’s syndrome) often develop type 2 muscle fiber atrophy. Myofibers contain a high concentration of surface receptors for several hormones, and atrophy caused by endocrine disease reflects the intimate interrelationship between the endocrine and the muscular systems. Denervation Atrophy. Denervation atrophy, also known by the misnomer neurogenic atrophy, is not uncommon in veterinary medicine. Maintenance of normal myofiber diameter depends on trophic factors generated by an intact associated nerve. Loss of neural input results in rapid muscle atrophy, and more than half the muscle mass of a completely denervated muscle can be lost in a few weeks. This trophic effect is not dependent on contractile activity because denervation atrophy is not a feature of neuromuscular junction disorders such as botulism and myasthenia gravis. In these disorders, there is a failure of neuromuscular transmission, but the nerve to the muscle is intact; therefore, the muscle is technically still innervated. Generalized neuropathies or neuronopathies, such as equine motor neuron disease, result in widespread and symmetric muscle atrophy. More commonly, however, only select nerve damage is present, resulting in asymmetric muscle atrophy. One example is equine laryngeal hemiplegia (roaring) secondary to damage to the left recurrent laryngeal nerve (Fig. 15.16). Note that purely demyelinating disorders of peripheral nerves can cause profound neuromuscular dysfunction, but axons are still intact. Associated myofibers are not technically denervated and therefore do not undergo denervation atrophy. After denervation, fibers become progressively smaller in diameter as peripheral myofibrils disintegrate. If an atrophic fiber is surrounded by normal fibers, it will be pressed into an angular shape, Figure 15.16 Denervation Muscle Atrophy, Left Cricoarytenoideus Dorsalis Muscle, Larynx, Dorsal Surface, Horse. Note the unilateral (left side) atrophy and pale gray to white discoloration of the muscle. This horse had a peripheral neuropathy, which led to laryngeal hemiplegia. (Courtesy Dr. J.F. Zachary, College of Veterinary Medicine, University of Illinois.) called an angular atrophied fiber. The angular atrophied fibers of denervation atrophy most often occur either singly or in small contiguous groups (small group atrophy) (Fig. 15.17, A). In more severe denervating conditions, in which many fibers within muscle fascicles are undergoing denervation atrophy, there are no normal fibers to cause compression and angularity, and affected fibers occur as larger groups of small-diameter, rounded fibers (large group atrophy; Fig. 15.17, B). Although myofibrils disappear rapidly, muscle nuclei do not do so at the same rate, and therefore denervation atrophy is often associated with a notably increased concentration of myonuclei. The breakdown of glycogen in the myofiber is an early change in denervation atrophy, and therefore denervated fibers stain faintly or not at all with the periodic acid–Schiff (PAS) reaction. A histologic diagnosis of denervation atrophy may be suspected, based on the characteristic features of routinely processed muscles, but is most reliably documented with histochemistry or immunohistochemistry to detect fiber types. The loss of a nerve fiber to a muscle results in atrophy of all myofibers innervated by that nerve. Because of the intermingling of motor units forming a mosaic pattern of fiber types, myofibers undergoing denervation atrophy are scattered in a section of muscle. Because the motor neuron determines the histochemical myofiber type and because denervating diseases typically involve both type 1 and type 2 neurons or nerves, atrophy of both type 1 and type 2 myofibers in muscle fasciculi is the hallmark of denervation atrophy (Fig. 15.18, A). In denervation atrophy, histologic examination of the intramuscular nerves is warranted because it may reveal axonal degeneration or loss of myelinated fibers. Masson trichrome stain can be useful here because it will differentiate myelin (red) from collagen (blue). If the nerve damage does not incapacitate the animal and the muscle can still be used (e.g., in locomotion), the remaining innervated myofibers often undergo notable hypertrophy because of increased workload. Often, the hypertrophied fibers in chronic denervation are type 1. Even without fiber typing, a pattern of severe small or CHAPTER 15 Skeletal Muscle A B E-Figure 15.4 Disuse Atrophy, Dog. A, Transverse section of normal biceps femoris muscle. B, Same muscle, same magnification, 60 days after disuse. Both type 1 (light) and type 2 (dark) fibers are atrophic, but type 2 fibers are more severely affected. Frozen section, ATPase pH 9.8. (Courtesy Dr. M.D. McGavin, College of Veterinary Medicine, University of Tennessee.) 1002.e1 CHAPTER 15 Skeletal Muscle A B Figure 15.17 Denervation Atrophy, Transverse Sections. Both sections are from horses with equine motor neuron disease. A, In relatively mild denervation, severely atrophied and angular fibers form small contiguous clusters indicative of small group atrophy (arrows). Formalin fixation, Masson trichrome stain. B, In severe denervation, entire fascicles of fibers undergo rounded atrophy characteristic of large group atrophy (lower left). Small group atrophy and admixed fiber hypertrophy are also present. A single pale stained fiber (arrow) is undergoing acute necrosis. There is also mild endomysial and perimysial fibrosis and mild fat infiltration (empty vacuoles in the upper right and lower left). Frozen section, modified Gomori’s trichrome stain. (Courtesy Dr. B.A. Valentine, College of Veterinary Medicine, Oregon State University.) large group atrophy (see Fig. 15.17, A), especially if associated with notable fiber hypertrophy (see Fig. 15.17, B), is strongly suggestive of denervation atrophy. A finding of damage in an associated peripheral nerve is definitive. Under many circumstances, denervated muscle fibers can be reinnervated by subterminal sprouting of axons from adjacent normal nerves. Reinnervation results in return to normal myofiber diameter, but reinnervation is often from sprouts of a different type of nerve. Because muscle fiber type is a function of the motor neuron, the newly innervated myofiber takes on the fiber type determined by that neuron. This process results in a loss of the normal arrangement of type 1 and type 2 myofibers and the formation of groups of the same fiber type adjacent to each other, called fiber-type grouping (see Fig. 15.18). Thus fiber-type grouping is the hallmark of denervation followed by reinnervation. What appears to be fiber-type grouping can also occur because of fiber-type conversion (most often to type 1 fibers) in chronic myopathic conditions. Careful evaluation of the structure and function of peripheral nerves helps distinguish neuropathic from myopathic changes. If previously reinnervated fibers are denervated again, the pattern includes large groups of atrophied fibers of a single fiber type, a process known as type-specific group atrophy. Type-specific group atrophy is far less common in animals than in human beings. Fiber-type grouping and type-specific group 1003 A B Figure 15.18 Denervation Atrophy and Reinnervation, Skeletal Muscle, Transverse Sections. A, Fiber typing reveals angular atrophy of both type 1 (light brown) and type 2 (dark brown) fibers, characteristic of denervation atrophy. In this case, there is also a loss of the normal mosaic pattern of fiber types, with groups of type 1 and of type 2 fibers indicative of reinnervation. This section is from a horse with laryngeal hemiplegia. Frozen section, ATPase pH 10.0. B, Fiber-type grouping in a dog indicative of denervation and reinnervation secondary to corticosteroid therapy. There is a loss of the normal mosaic pattern of fiber types, with grouping of type 1 (light red) and type 2 (dark red) fibers. The lack of angular atrophied fibers indicates that active denervation is not occurring at this time. Frozen section, ATPase pH 9.8. (A courtesy Dr. B.A. Valentine, College of Veterinary Medicine, Oregon State University. B courtesy Dr. M.D. McGavin, College of Veterinary Medicine, University of Tennessee.) atrophy can only be detected by methods that distinguish fiber types. Changes occurring as a result of denervation and reinnervation are illustrated in Fig. 15.19. Atrophy Caused by Congenital Myopathy. Congenital myopathy in children is often associated with selective type 1 fiber atrophy. This finding is less common in the congenital myopathies identified thus far in animals. Selective type 1 atrophy is, however, a feature of feline nemaline myopathy, an animal model of congenital nemaline myopathy in children. Hypertrophy Myofibers increase in diameter by the addition of myofilaments. Physiologic hypertrophy is the normal process of myofiber enlargement that occurs with exercise conditioning. Compensatory hypertrophy occurs because of pathologic conditions that (1) decrease the number of functional myofibers and therefore increase the load on remaining fibers or (2) interfere with normal cellular metabolic or other physiologic processes. Compensatory myofiber hypertrophy is therefore considered a relatively nonspecific response to a variety of 1004 SECTION II Pathology of Organ Systems * * * * A B Figure 15.20 Fiber Splitting of Hypertrophied Myofibers, Nemaline Myopathy, Skeletal Muscle, Transverse Section, Cat. Sarcolemmal ingrowth into the myofiber has resulted in multiple partitions with the formation of four myofibers (asterisks); however, all myofibers are enclosed by one basal lamina. Frozen section, modified Gomori’s trichrome stain. (Courtesy Dr. B.A. Valentine, College of Veterinary Medicine, Oregon State University.) C D Figure 15.19 Motor Units Undergoing Denervation and Reinnervation. A, Terminal axon branches innervate multiple myofibers, and myofiber type is determined by the electrical activity of the type of neuron innervating the myofiber. Normally the terminal axons of the motor units are intermingled, with the result that the differently stained myofiber types form a mosaic pattern. B, If a neuron (or axon) is damaged, the axon will undergo Wallerian degeneration, and the myofibers in that motor unit will undergo denervation atrophy. Small group atrophy is illustrated here. C, Axonal sprouts from a healthy neuron can reinnervate affected fibers and cause restoration of their normal diameter. The myofibers will assume the fiber type of the new motor unit, which often causes fiber-type conversion, leading to fiber-type grouping. D, If neuronal (or axonal) damage is progressive, denervation atrophy of large groups of fibers of a single type can occur, known as type-specific group atrophy. This type of atrophy is less common in animals than in human beings. (Redrawn with permission from Dr. B.A. Valentine, College of Veterinary Medicine, Oregon State University.) insults. Fibers undergoing compensatory hypertrophy can enlarge to more than 100 μm in diameter (normal is less than approximately 60 to 70 μm). Fiber hypertrophy often accompanies fiber atrophy, which contributes to increased fiber-size variation in various myopathic and neuropathic conditions. Compensatory hypertrophy can occur because of a decrease in the number of functional myofibers. Thus, in a partially denervated muscle, the remaining innervated fibers hypertrophy (see Fig. 15.17, B), presumably as a result of increased workload. Pathologically, hypertrophied fibers have less oxygen diffusion from interstitial capillaries to internal portions of the myofiber because of the increase in the distance from the capillary to the internal portions of the myofibers, which can lead to myofiber damage. Mechanical overloading of hypertrophied muscle fibers is also possible. For example, overloading of hypertrophied fibers can result in segmental necrosis of the hypertrophied fibers (see Fig. 15.17, B), or fibers can undergo longitudinal fiber splitting to generate one or more smallerdiameter “fibers,” all contained within the same basal lamina (Fig. 15.20). Serial sections of areas of fiber splitting generally reveal that splits do not extend the entire length of the myofiber. Fiber splitting is considered a form of cytoarchitectural alteration. Insulin-like growth factor 1 (IGF-1) is an important molecular signal involved in skeletal muscle hypertrophy. Genetic inactivation of the regulatory gene myostatin results in muscle hypertrophy caused by an increase in the number of myofibers. Cytoarchitectural Changes In addition to fiber splitting, a variety of other cytoarchitectural changes can occur within myofibers. Some are degenerative, the result of an insult that damages the myofiber but does not culminate in myofiber necrosis. Others reflect underlying ultrastructural alterations that may be either pathologic or compensatory. The functional significance of many of the myofiber cytoarchitectural changes is not known. Vacuolar Change Vacuolar change is a common cytoplasmic alteration. In formalinfixed paraffin-embedded sections or in any sample subjected to less than ideal handling, true vacuolar change can be very difficult to distinguish from artifacts. Vacuoles can be an early manifestation of processes leading to necrosis, they can reflect underlying sarcotubular dilatation as occurs in many myotonic conditions, they can be caused by abnormal storage of carbohydrate or lipid, or they can reflect underlying myofibrillar abnormalities. Additional studies are often necessary to determine the nature of the vacuoles. When severe, such as in glycogen storage diseases, the term vacuolar myopathy is often employed. Internal Nuclei Myonuclei of mature myofibers in domestic animals are normally found peripherally, just beneath the sarcolemma. Nuclei located one nuclear diameter or more from the sarcolemma are known as internal nuclei. (NOTE: The previously used term central nuclei is considered incorrect because few abnormally placed nuclei are exactly centrally located.) Internal nuclei are rare in normal mammalian muscle, but a small percentage can be found normally in avian and reptilian species. Rows of internal nuclei in small-diameter, slightly basophilic myofibers are characteristic of the myotubular stage of regeneration (see Fig. 15.13, B and C). In most species, myonuclei return to the normal peripheral location early in regeneration, within days of myotube formation. Rodents are the exception. In rodents, internal nuclei are retained after regeneration, which, in these species, provides a handy marker for identification of fibers that have undergone necrosis and regeneration. In other mammalian species, the presence CHAPTER 15 Skeletal Muscle 1005 A Figure 15.21 Chronic Myopathic Change, Medial Triceps Muscle, Horse. The variation in myofiber diameter and the presence of one or more internal nuclei in most myofibers (arrows) are indicative of a chronic myopathic change. Frozen section, hematoxylin and eosin (H&E) stain. (Courtesy Dr. B.A. Valentine, College of Veterinary Medicine, Oregon State University.) of internal nuclei in normal or hypertrophied fibers is a nonspecific finding indicative of chronic myopathic change (Fig. 15.21; see Box 15.3). In hypertrophied fibers, the migration of myonuclei to the internal portion of the myofiber can precede the sarcolemmal infolding that creates longitudinal fiber splitting. Whorled and Ring Fibers The cytoarchitectural rearrangements resulting in whorled and ring fibers are best appreciated in transverse sections. Whorled fibers contain spirals of cytoplasm with internally located nuclei. Whorled fibers can be seen in areas of chronic denervation and in areas in which myofiber necrosis with incomplete regeneration has occurred. Ring fibers (also known as ringbinden) contain a peripheral rim of sarcomeres oriented perpendicular to their normal orientation, resulting in peripheral radiating striations. Ring fibers are visible with many stains, both in frozen sections and in routinely processed sections. In either frozen or routine sections, they are best visualized in sections stained with PAS (Fig. 15.22, A) or iron hematoxylin. In human beings, ring fibers are common in a specific form of inherited muscular dystrophy known as myotonic dystrophy, but they are also seen in other myopathic and in neuropathic conditions and therefore are not specific for myotonic dystrophy. Similarly, there is no animal disorder in which ring fibers are specific, and these fibers can be seen in a variety of myopathic and neuropathic conditions such as ovine congenital muscular dystrophy. The presence of ring fibers can only be considered a chronic myopathic change. For example, numerous ring fibers were found in muscle from the contralateral weight-bearing limb from a horse with long-standing, non–weight-bearing foreleg lameness. Other Cytoarchitectural Changes Many other cytoarchitectural changes reflect alterations in mitochondrial density or integrity and are best appreciated on examination of frozen sections, in which mitochondria can be visualized, or on ultrastructural examination. The presence of peripheral aggregates of mitochondria, which stain red with modified Gomori’s trichrome stain, form the basis of “ragged red” fibers. Ragged red fibers are a hallmark of mitochondrial myopathy in human beings. In animals, however, ragged red fibers are common in various myopathic conditions and occur in normal dog and horse muscle. Mitochondrial abnormalities are also detected by oxidative enzyme reactions such as nicotinamide adenine dinucleotide dehydrogenase (NADH) (Fig. 15.22, B and C) and succinate dehydrogenase (SDH) in frozen sections. Nemaline rods, formed by expansions of the Z-line B C Figure 15.22 Cytoarchitectural Changes, Skeletal Muscle, Transverse Sections. A, Ring fiber, extensor carpi radialis muscle, horse. A ring fiber (center right) is characterized by a peripheral rim of sarcomeres, arranged circumferentially around a myofiber and with their length at right angles to the long axis of the myofiber. Frozen section, periodic acid–Schiff (PAS) reaction. B, Irregular mitochondrial distribution with peripheral aggregates of blue staining mitochondria, Labrador centronuclear myopathy, temporalis muscle, dog. Frozen section, NADH reaction. C, Irregularity of mitochondrial (blue-stained) distribution and “moth-eaten” fibers, polyneuropathy, dog. Fibers containing pale zones are characteristic of moth-eaten fibers. Frozen section, nicotinamide adenine dinucleotide dehydrogenase (NADH) reaction. (Courtesy Dr. B.A. Valentine, College of Veterinary Medicine, Oregon State University.) material, stain purple to red with modified Gomori’s trichrome stain in frozen sections. These rods can also be seen in animals with other myopathic conditions. Moth-eaten fibers contain multiple pale zones because of loss of mitochondrial oxidative enzyme activity on frozen sections and occur in denervating disorders and in myopathic conditions (Fig. 15.22, C). Sarcoplasmic masses are pale-staining zones usually at the periphery of myofibers but occasionally central. These zones can be seen in H&E-stained muscle sections and appear as light blue areas with few or no myofibrils. Ultrastructurally 1006 SECTION II Pathology of Organ Systems they often contain disarrayed myofilaments with or without degenerate mitochondria. Other less commonly encountered alterations in animal muscle are pale central cores visible with mitochondrial stains, tubular aggregates composed of sarcotubular membranes, and target fibers in which mitochondrial oxidative enzyme reactions reveal central clear zones surrounded by a thin rim of highly reactive cytoplasm. Other less commonly encountered alterations in H&E-stained sections of animal muscle are pale central cores. As demonstrated in sections stained by mitochondrial oxidative enzyme reaction (e.g., SDH), these are of three types: (1) cores rich in mitochondria and similar to the peripheral sarcoplasmic masses described previously, (2) target fibers so designated because of a pale center surrounded by a rim of densely staining mitochondria, and (3) aggregates of sarcotubular membranes that do not stain with mitochondrial stains. progressive denervation are implicated as possible causes of sarcopenia in aging human beings. Aged animals often exhibit mild to severe muscle atrophy. To what degree this atrophy in animals is due strictly to age-related changes rather than to underlying chronic organ dysfunction (e.g., renal failure, cardiac disease, and neoplasia) is most often unknown. Sarcopenia and cachexia can occur concurrently in aged animals and people. Old cattle can accumulate lipofuscin within skeletal muscle, which can cause a tan-brown discoloration, but there is no apparent clinical significance to this change. Aged cattle have also been found to have angular atrophy of myofibers, lymphocytic inflammation, and cytoarchitectural changes (i.e., changes in mitochondrial distribution, reduction in mitochondrial enzymes, vacuolation, and accumulation of myostatin and amyloid precursor proteins). Chronic Myopathic Change Portals of Entry/Pathways of Spread Evaluation of abnormal skeletal muscle often reveals chronic myopathic change, which includes alterations in myofiber diameter, cytoarchitectural alterations, and interstitial fibrosis and fat infiltration (see Box 15.3). Chronic myopathic change accompanies a variety of myopathic and neuropathic conditions. In particularly severe cases, a definitive cause may not be identified. Chronic inflammation or denervation and chronic degenerative myopathy resulting in repeated bouts of myonecrosis and regeneration often cause diffuse endomysial and perimysial fibrosis (see Figs. 15.17, B, and 15.47, B). Interstitial infiltration of muscle by mature adipocytes is less common than fibrosis and occurs most commonly in chronically denervated muscle (see Fig. 15.17, B), particularly neonatal muscle that lacks appropriate innervation (Fig. 15.23). Fat infiltration can also occur because of severe chronic degenerative myopathy. A chronically damaged or denervated muscle that develops profound fibrosis and/or fat infiltration can be grossly enlarged, despite atrophy or loss of myofibers—a condition known as pseudohypertrophy (see Fig. 15.9, D). Portals of entry and pathways of spread are summarized in Box 15.5. Injury to muscle can occur secondary to trauma or infection. Muscle lying superficially can be damaged by penetrating wounds, including those created by intramuscular injections (Fig. 15.24; see also Fig. 15.9, B), which can also allow entry of infectious agents. Muscles located deeply are often injured after bone fracture. Crush injuries from external forces cause extensive muscle damage, and excessive Aging Aging changes in skeletal muscle are well documented in human beings but less well documented in domestic animals. The term sarcopenia refers to generalized reduction in muscle mass, strength, and function related to aging, in the absence of underlying disease. In contrast, cachexia is generalized muscle atrophy caused by underlying disease or malnutrition. Changes in mitochondrial function and Figure 15.23 Lipomatosis (Steatosis), Calf. Lost myocytes have been replaced by mature adipocytes (clear [nonstaining] areas). Islands of remaining myofibers have groups of angular atrophied fibers admixed with hypertrophied fibers, suggestive of denervation atrophy. Formalin fixation, hematoxylin and eosin (H&E) stain. (Courtesy Dr. M.D. McGavin, College of ­Veterinary Medicine, University of Tennessee.) Box 15.5  Portals of Entry and Pathways of Spread into the Muscular System DIRECT Penetrating wounds Intramuscular injections Bone fracture causing trauma to adjacent muscle External pressure causing crush injury HEMATOGENOUS Bloodborne pathogens, toxins, autoantibodies, and immune complexes Cytotoxic lymphocytes causing immune-mediated damage Other inflammatory cells Figure 15.24 Inflammation and Myofiber Necrosis, Injection Site, Muscles, Lateral Thigh, Cow. Necrotic muscle has been stained green by the injected material, which has spread distally down the fascial plane between the two muscles from the original injection site (top right). (Courtesy Dr. M.D. McGavin, College of Veterinary Medicine, University of Tennessee.) CHAPTER 15 Skeletal Muscle tension can cause muscle tearing. Muscles are endowed with an extensive vascular network that can allow entry of bloodborne pathogens, immune complexes, antibodies and toxins, and inflammatory cells. Other routes by which muscle can become dysfunctional are summarized in Box 15.6. Some muscular disorders are genetically determined. Inherited or acquired dysfunction of motor neurons or nerves causes muscle injury in the form of atrophy. Toxins or an altered endocrine or electrolyte status can affect muscle, and physiologic damage can be caused by exhaustive or overexuberant exercise. Defense Mechanisms/Barrier Systems Defense mechanisms and barrier systems are summarized in Box 15.7. The thick encircling fascia (epimysium) of many muscles provides some protection from penetrating injuries and from extension of adjacent infection. This fascia can, however, also contribute to injury under circumstances that lead to increased intramuscular pressure causing hypoxia (compartment syndrome). Tissue macrophages are not typically found in normal muscle but are recruited rapidly from circulating monocytes in the vasculature. Macrophages can cross even an intact basal lamina and effectively clear debris from damaged portions of myofibers, allowing for rapid restoration of the myocyte through satellite cell activation. Neutrophils and Box 15.6  Other Causes of Muscle Dysfunction PHYSIOLOGIC Excessive muscle tension causing muscle rupture Exercise-induced damage to myofibers Loss of innervation Loss of blood supply Endocrine and electrolyte abnormalities GENETIC Inborn errors of metabolism Genetic defects of myofiber structural components Developmental defects NUTRITIONAL/TOXIC Deficiency of selenium and/or vitamin E Toxic plants or plant products Feed additives (ionophores) Other toxins (e.g., some snake venoms) Box 15.7  Defense Mechanisms and Barrier Systems of Skeletal Muscle SKIN, SUBCUTIS, AND FASCIA Form structural barriers to protect against external injury VASCULATURE Collateral circulation to protect against ischemia Recruitment of monocytes that become tissue macrophages Recruitment of neutrophils and other inflammatory cells Capillary endothelium resistant to tumor metastasis IMMUNOLOGIC RESPONSES Innate humoral and cellular immunologic responses OTHER Adequate tissue antioxidant concentrations Physiologic adaptation (e.g., hypertrophy, fiber type alteration) Regenerative capacity of myofibers 1007 other inflammatory cells are also recruited from the bloodstream in response to injury or infection. The extensive vascular network of muscle includes extensive collateral circulatory pathways that render muscle relatively resistant to ischemic damage caused by thrombosis or thromboembolism. Despite the high vascular density of muscle, metastasis of neoplasms to muscle is rare. There is evidence that the capillary endothelium of skeletal muscle is inherently resistant to neoplastic cell adhesion and invasion. Diseases Affecting Multiple Species of Domestic Animals Adequate muscle function is essential for the survival of any species. Many domestic animals have been selectively bred for improved musculature for meat production, performance, or appearance. Therefore, muscle disease in animals can have a significant economic impact. In some cases, it is selection pressure imposed by human beings that has resulted in development and perpetuation of various myopathic conditions in animals. It is likely that continued selection for what appears to be a phenotypically desirable trait will lead to the recognition of new genetic mutations and myopathic conditions in the future. It is interesting to compare the effects of muscular disorders that affect human beings and animals. The four-footed stance of animals allows for greater stability, which can allow an animal to remain ambulatory for some time, when a similarly affected person would be confined to a wheelchair. However, disorders that result in recumbency, even if it is transitory, can be devastating in livestock. It is much more difficult to nurse a large animal through a period of recumbency than it would be to nurse a hospitalized human being or small animal. The most common and important muscle disorders of animals are discussed by species because this is the way diseases are considered clinically. The same disease may occur in different species. Details of less common muscle disorders are presented in E-Appendix 15.1. Types of Muscle Disease Classification of muscle diseases based on lesions alone is not very satisfactory, and many classifications are based on cause (e.g., toxic myopathy or nutritional myopathy). An example of such a classification is given in Table 15.2. Myopathic conditions can be inherited or acquired. Inherited disorders can affect muscle metabolism or myofiber structure. Acquired muscle disease in livestock is often associated with nutritional deficiency or with ingestion of myotoxins, whereas acquired muscle disease in the dog is most often caused by immune-mediated inflammatory conditions. Other causes of acquired myopathies include ischemia, infectious agents, hormonal or electrolyte abnormalities, and trauma. There are also many neuropathic conditions that result in denervation atrophy (see section on Dysfunction/Responses to Injury, Alteration in Myofiber Size, Atrophy, Denervation Atrophy). More information on most of the disorders described in this section can also be found under the appropriate species heading or in E-Appendix 15.1. Degenerative Degenerative myopathies are those resulting in segmental or global myofiber necrosis, in which inflammatory cells are not the cause of the myofiber damage. Disturbance of Circulation. Given the numerous capillary anastomoses and rich collateral circulation of skeletal muscle, only disorders that result in occlusion of a major artery or that cause widespread intramuscular vascular damage will result in myofiber necrosis