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Muscle Biopsy in Veterinary Practice
Symposium on Orthopedic Diseases
Muscle Biopsy in Veterinary Practice M.D. McGavin, M.V.Sc., Ph.D.*
Most skeletal muscles in mammals consist of three distinct physiologic fiber types: (1) slow contracting, slow fatiguing, oxidative (SO), (2) fast contracting, slow fatiguing, mixed oxidative-glycolytic (FOG), and (3) fast contracting, fast fatiguing, glycolytic (FG). Despite this, in paraffin-embedded histologic sections of muscle stained with hematoxylin and eosin (H&E), muscle fibers appear much the same (Fig. lA), apart from some variation in fiber diameter. Over 20 years ago, histochemical techniques were developed that allowed identification of these physiologic fiber types in frozen sections. 10 Such stains included myosin-ATPase, succinic dehydrogenase (SDH) for mitochondria, and NADH-TR, which stains chiefly mitochondria but also intermyofibrillar network. 10 The ATPase reaction is the most useful for the identification of different fiber types (Fig. IB). In normal muscle, many of the fibers in the same fasciculus are innervated by the same neuron, from the same ventral horn cell in the spinal cord, to form part of a motor unit. The innervation determines the physiologic type of muscle fiber and thus determines whether a fiber is SO, FOG, or FG. In sections stained by the myosin-ATPase reaction at a pH of 9.4, these fibers are light, medium, and dark, respectively, and are usually designated types I, Ila, and lib. 10 Damage to a neuron or to its axon may cause lesions in muscle fibers that are innervated by that nerve. Muscle fibers of a motor unit are scattered randomly through one or more muscle fasciculi and cannot be identified in hematoxylin and eosin-stained sections, but myosinATPase-stained sections will reveal that changes in the muscle are all of the same fiber type (see Fig. lA). Also, certain diseases preferentially involve one of the different fiber types. Thus, histochemical fiber-typing revolutionized muscle pathology because it made possible the differentiation between lesions that were primarily in the muscle, the so-called myopathic changes (for example, myositis) and muscle lesions secondary to changes in the neuron or nerves, the so-called neuropathic lesions. Since the devel~ opment of histochemical stains, the techniques that should be used for full histologic and histochemical examination of muscles have been defined and include such methods as H&E, modified Gomori' s trichrome, myosinATPase's incubated at different pH's, NADH-TR, and SDH as a mini*Diplomate, American College of Veterinary Pathologists; Professor, Department of Pathobiology, University ofTennessee College of Veterinary Medicine, Knoxville, Tennessee
Veterinary Clinics of North America: Small Animal Practice-Vol. 13, No. 1, February 1983
135
M. D. McGAVIN
136
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Figure 1. Canine superficial digital flexor muscle. Serial sections stained with hematoxylin and eosin (A) and myosin-ATPase (B). Note that in the H&E-stained section, it is possible to recognize small fibers (arrows), but only in the myosin-ATPase-stained section (pH=9.4) can it be seen that the atrophic fibers are all type II fibers which are also reduced in frequency. Diagnosis: type II atrophy.
mum. 8• 10 Some of these techniques are capricious, and their use is confined to specialized laboratories that do chiefly neuromuscular pathology. Originally such laboratories were primarily located in medical schools but later spread to institutions doing muscle research. Because of the cost and the fact that the selection of muscle for examination must be done accurately, specialized pathologic examinations of muscle are usually undertaken in association with clinical departments rather than in pathology departments of diagnostic laboratories.
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"How useful are these techniques directly or indirectly to veterinary practitioners?" "How can they avail themselves of these techniques and what samples should they send?" It is certainly nothing new for muscle to be removed, either at necropsy or biopsy, formalin-fixed, and forwarded to a pathology laboratory. Unfortunately, the series of histochemical stains required for full evaluation of muscle depends on active enzymes, and thus can be done only on frozen samples, not on formalin-fixed tissues. Histologic examination of formalin-fixed muscle has limited usefulness, chiefly because the differentiation of physiologic muscle fiber types (types I, Ila, and lib) cannot be demonstrated. Consequently, the full evaluation of pathologic changes in muscles usually requires a specialized neuromuscular laboratory. Nevertheless, histologic examination of formalin-fixed tissue is useful for the recognition of inflammation, some degenerations, necrosis, and regenerations as well as large changes (atrophy or hypertrophy) in fiber diameters. However, both histochemical and histopathologic techniques are required for optimal evaluation so that the fiber type involved can be recognized. There are several excellent accounts of the histopathologic examination of muscle. 7• 8• 10 The difficulties of these methods will be discussed in this article so that the reader can understand the limitations of the techniques and how muscle should be obtained and fixed so that routine histopathologic examination will yield the maximum amount of information. MUSCLE BIOPSY
Surgical techniques for the biopsy of a wide variety of body tissues have now been described, and many were reviewed in this series. 20 Probably the most widely biopsied tissue in veterinary medicine is skin, but as judged by the accessions at pathology laboratories, liver and kidney, gastrointestinal tract, and uterus are now frequently biopsied. Muscle makes up only a small percentage of these specimens, and many of these samples are unfortunately unsuitable for the pathologist to obtain the maximum amount of information. Artifacts are the major problem. They occur in many samples of other tissues from a variety of causes: compression by forceps, crushing by scissors during excision, and stretching. Compression, crushing, and stretching artifacts in many tissues result in pyknosis and nuclear streaming and, if extensive, may make the histopathologic sections uninterpretable. However, in biopsies of skin, these defects are usually localized to those portions of the sample that have been mistreated. Artifacts usually do not obscure lesions unless the sample is extremely small or has been handled brutally, as, for example, if the edge of the skin has been grasped in several different places by forceps. However, muscle is not so forgiving, and even what would be considered normal surgical handling for other tissues will cause muscle to contract or overcontract, producing artifacts that will not be confined to one edge but will be present ~oughout the specimen. Thus the prevention of artifacts is essential for reliable histopathologic examination. The basic principle is to realize that muscle is designed to contract when stimulated, and it will do so when excised at biopsy and sometimes at necropsy. Also, ntuscle will contract when placed in most fixatives, including 10 per cent buffered neutral
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formalin . The factor that decides whether muscle is able to contract when stimulated is the availability of an energy supply in the muscle cells. Gordon and Texidor were able to prevent contraction of canine and human muscles by applying a pair of ligatures to each end of an isolated cylinder of muscle still attached to the muscle belly. 11 The outer ligatures produced ischemia after two minutes and reduced contraction when the sample was placed in fixative. After death, oxygen is no longer available and contraction will depend on glycogen stores to form ATP for contraction. Glycogen is slowly broken down, but some muscle fibers in well-nourished animals are still alive and able to contract several days after death if the body is held at 4°C. 17 It is these fibers that produce contraction artifacts when muscle is placed into fixative. Muscles from emaciated animals with little or no glycogen stores will soon cease to contract after death. Thus, the fresher the muscle, the more numerous the artifacts. This is one case when a tissue fixed immediately after death may appear histologically worse than one fixed some hours or even days after death, particularly if the temperature is cool. Also, diseased muscle fibers and fibers from very young animals are highly irritable and readily produce contraction artifacts at fixation. Techniques for reducing or eliminating these artifacts are described below. Indications Adams cites five different clinical problems in which muscle biopsy may be useful in human medicine. 2 1. Progressive muscular atrophy-to determine if the disease is neurogenic or myogenic. 2. Localized or diffuse myositis-to determine the type of inflammation (for example, eosinophilic myositis, suppurative myositis) or sometimes to detect the etiologic agent (as in toxoplasmosis). 3. Diffuse "vascular" or "collagen" diseases"-such as the immunemediated diseases. 4. Following injury to the nerve and blood vessels to a limb-to assess the state of the muscle and intermuscular-intramuscular nerves and thus determine the prognosis. 2 An example of this would be damage to some of the fibers of the sciatic nerve during fracture of the pelvis or by compression by an orthopedic plate during repair of a fracture. 5. Metabolic diseases-such as hypothyroidism, administration of excess steroids. Obviously, some of these conditions can be better evaluated by other clinical and laboratory techniques. However, muscle biopsy is particularly useful in the evaluation of muscular atrophy to determine whether it is neurogenic or myogenic and to determine the type of myositis whether this is due to an infectious disease or to an immune-mediated disease such as polymyositis. However, muscle biopsy is best undertaken only after complete clinical evaluation and, where possible, after electromyography, in order to clinically characterize the lesion and to locate the affected muscle. Selection of Muscle for Biopsy
Identifying Affected Muscle. Selection of a suitable muscle for biopsy is not as simple as it would seem and depends on the nature of the disease
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affecting the muscles. For example, is the disease generalized, localized, bilaterally symmetric, proximal or distal? Thus, identifying the affected muscles requires a clinical examination that includes a neurologic evaluation. If the disease is generalized or symmetric, muscles on one side can be examined by electromyography and biopsies taken from the contralateral muscles. Muscle used for electromyography should be avoided for biopsies, as the needle punctures may evoke an inflammatory response resulting in artifacts that may be misinterpreted by the pathologist. Selection of Muscle in Early Stages of the Disease. Muscles with chronic lesions (for example, following acute eosinophilic myositis in the canine temporalis muscle) are seldom useful for histologic examination. The end-stage of many muscle diseases is fibrosis and fiber atrophy. The characteristic changes are frequently seen only early in the disease. Size of Muscle and Direction of Fibers. To prevent artifacts many muscle biopsies are removed in isometric clamps. These clamps must be applied to parallel fibers-thus the muscles must be large enough to accommodate the clamps and also not be so small that the muscle mass removed constitutes a significant loss of functional fibers. In dogs and cats, muscles such as the semitendinosus, semimembranosus, biceps femoris, long digital extensor and tibialis cranialis, triceps brachii-lateral head, superficial digital extensor, and deltoid 12 have parallel fibers and are easier to biopsy. The vastus lateralis, a popular muscle for biopsy, has fibers which, although parallel to the long axis of the femur, are not parallel to the lateral surface of the muscle but run obliquely from the lateral fascial sheath toward the femur. Only in the distal third of the caudal border are the fibers approximately parallel to the surface, making this a suitable site for biopsy. Muscles such as the gastrocnemius and many of the flexors caudal to the antebrachium are pennate or semipennate, making bundles of long parallel fibers difficult or impossible to locate in small animals. Normal Data for Muscle Known. Extreme departures from normal in fiber size and the percentage of each of the different fiber types are relatively easy to recognize. In determining a full histochemical muscle fiber profile, however, the means and standard deviations of fiber diameters for type I and II fibers and also the percentage of each fiber type in the muscle are estimated. These data cannot be evaluated unless the laboratory has normal control data against which to compare the findings. The percentage of each fiber type and mean diameters of each muscle are dependent upon: the specific muscle, variation between different areas of the muscle itself, age, species, exercise (effect of conditioning): At the present time, accurate baseline data are known for a limited number of muscles in different species. 3 • 4 Most laboratories have developed data for muscles such as the vastus lateralis, biceps femoris, triceps brachii, and pectineus. Recently, Armstrong et al. reported the percentages of fiber types in 9llocomotory muscles in the dog. 27 However, availability of normal data is not likely to be a problem for the practitioner, because the samples he selects will have to be fixed in 10 per cent buffered neutral formalin and thus no histochemical profile can be attempted.
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Figure 2. Muscle fixed in 10 per cent buffered neutral formalin without clamps, paraffinembedded and stained with hematoxylin and eosin. A , The majority of the fibers, cut in crosssection, are polygonal, except for one that is rounded and deeply eosinophilic. This is a contracted fiber and corresponds to the extremely contracted portion of the fibers (arrows) shown in longitudinal sections (B).
To summarize, the following are the difficulties that occur during the histologic examination of muscle: l. The wrong muscle (that is, the unaffected muscle) is taken. 2. Muscle is diseased to an extent that renders it useless for histopathologic examination. 3. The characteristic lesions-for example, eosinophilic myositis-may have disappeared. 4. With muscles that are too small, it is difficult to apply clamps and too great a proportion of its contractile mass is removed. Most of the muscle removed will be replaced by fibrous tissue and will not be functional contractile tissue. 5. With muscles that do not have parallel fibers-for example, the temporalis, gastrocnemius, and many of the flexors on the caudal aspect of the antebrachium-clamping is difficult. To obtain the best sample, the surgeon must know the direction of fibers in the muscle before making the initial skin incision. Trial dissection of cadavers is strongly advocated. Clamps should be applied at a site where the majority of fibers are parallel. 6. Whereas stretching or compression of other tissues usually ruins only the portion of the sample that is touched, muscle is so irritable that artifacts may occur anywhere throughout the sample. Specific artifacts include extreme contraction bands of Nageotte, shredding, rounded hyalinized fibers (Fig. 2), sarcoplasmic masses, and cracking. 2• 16 Contraction
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bands may not only obscure lesions in that portion of the muscle, but may also result in the formation of artifactual sarcoplasmic masses in adjacent segments of the fibers. These artifacts can be confused with genuine sarcoplasmic masses formed by accumulations of sarcoplasm immediately under the sarcolemma and seen in some dystrophies. Also, contraction of some of the ·fibers or connective tissue can cause adjacent muscle fibers to have a zigzag pattern as they are passively shortened. All of these artifacts are the results of contraction of the still-irritable muscle on contact with fixative . To avoid these artifacts, it is desirable that: (I) the muscle be prevented from contracting with some form of clamp, and (2) stimuli be minimized during the actual excision. Clamping Devices
Muscle samples are best prevented from contracting by the application of some type of device before the muscle is excised. Some of these isometric clamps are commercially available, 21 · 26 and Rayport has developed a disposable plastic clamp. 22 However, others have used tongue-holding forceps, 5 modified Collins tongue-holding forceps, modified Judd-DeMartel gallbladder forceps, 19 Kelly clamps9 or hemostats 13 connected by a bar and modified ovarian clamps. 6 Bradley has also used so-called bulldog paper clips.5 The Price 21 and Staheli26 clamps are available in two sizes to take samples of approximately 1 or 2 em in length. The smaller-sized clamp is really designed for collecting samples for electron microscopy but is suitable for sampling small muscles such as those of the cat. However, these clamps are expensive and their cost probably cannot be justified in general practice. Eight-inch Judd-DeMartel gallbladder clamps with l-inch-diameter circular jaws modified by cutting off the outer half of each circular jaw19 are quite economical, and we use many of them for holding muscle in our necropsy laboratory. Recommendations for preventing muscle-contraction artifacts have changed over the years. The original methods did not suggest that the muscle be clamped before the sample was removed. Rather, the recommendation was to excise a cylinder of muscle, place it on a block of wood or piece of cardboard, straighten to its original length, and either allow it to adhere by drying2• 10• 15• 24 or ligate it to the wood. 18 Pathologists frequently recommend placing biopsy specimens of skin ,I intestine, 20 stomach, liver, and kidney on thin cardboard. They should be straightened gently to lie flat and allowed to dry for 30 seconds to two minutes so that they can adhere to the card, before being placed in fixative. This technique has also been proposed for muscle, 14 but in our experience it does not always prevent their contraction. Because of its unreliability, this method should be used only when no fixation devices are available and optimal results are not expected. In the absence of any clamps, the simplest method is to make two parallel -incisions, 3 to 6 mm apart, into the belly of the muscle in the same direction as the muscle fibers and suture each end of the isolated portion of muscle to a wooden stick, such as an applicator. 25 The muscle and stick are removed together and placed in the fixative . However, wood is not suitable for use in some fixatives, such as osmium tetroxide. This method has the advantage of being simple, inexpensive, and effective with such
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fixatives as 10 per cent buffered neutral formalin, but it takes more time and manipulation than the clamps and thus has the inherent danger of producing more artifacts. Techniques This has been described in detail by several authors. 5 • 6 • 12• 21 The basic requirement is to isolate a cylinder of muscle with parallel fibers, clamp it, then excise it. Thus, the first step is to identify that portion of the muscle with parallel fibers. If the surgeon is unfamiliar with the topography of the fibers, he or she should dissect muscles of a cadaver to determine the best site of sampling. Lack of familiarity with the direction of fibers will result in unnecessary handling of the muscle and the inevitable development of artifacts. One secret in obtaining good muscle biopsy samples is to minimize handling of the muscle. The portion that is to be excised should not be touched at all, except by the scalpel or razor blade. 12• 23 Blunt dissection, manipulation by forceps, stretching, and even pressure from fingers or sponges should be completely avoided, as even relatively minor stimuli will cause contraction. Price et al. have recommended the use of muscle cylinders 1.5 to 2 em long, 1 em wide, and 0.5 to 0.8 em thick for light microscopy and 1 to 1.5 em long, 0.5 em wide, and 0.5 em thick for electron microscopy. The procedures can be summarized as follows: 1. Expose the belly of the muscle at the site known to have parallel fibers. 2. Incise and gently retract the fascia from the muscle. 3. Make two incisions 2 to 6 mm apart and 1. 0 to 2. 5 em long, parallel to the direction of the muscle fibers. The length will depend on the size of the clamps used and the size of the muscle. 4. At this point, some authors describe a third cut to isolate the strip of muscle from the underlying belly of the muscle. Others apply the clamps first, then make the third cut. In our experience, artifacts occur less often if the clamp is applied before undercutting the strip of muscle. 5. The isolated cylinder of muscle in clamps is removed by cutting transversely across the muscle fibers a few millimeters beyond each end of the clamp (Fig. 3). 6. The muscle and clamps are placed in fixative, agitated to remove blood and serum, and then allowed to fix. CONCLUSIONS The surgical technique for obtaining muscle biopsy and the full histochemical and histopathologic evaluation of the specimen are expensive. When this type of evaluation is indicated, the most suitable procedu.r e is to take the patient to one of the laboratories that offer this service. This is more successful than transporting muscle specimens in liquid nitrogen or dry ice, as specimens may be delayed and may deteriorate in shipment, resulting in unnecessary expense. Muscle specimens fixed in 10 per cent buffered neutral formalin can be forwarded to a laboratory, but the surgeon must take steps to avoid artifacts. Every effort should be made to avoid stretching, pulling, or
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Figure 3. Muscle biopsy techniques. Canine left vastus lateralis muscle. The surgeon has exposed the caudal border of the vastus lateralis, reflected the fascial sheath and made two parallel incisions approximately 3 mm apart, applied isometric clamps and is now about to make the final three cuts to remove the clamped cylinder of muscle.
compression, and some form of clamp or device must be used to prevent contraction. Otherwise, artifacts may be so severe as to obscure lesions or even to prevent the pathologist from determining if there are any lesions. Thus, any irritable muscle, including one taken at necropsy, must be clamped prior to fixation because this irritability can continue for several days after death, if the body is held in a refrigerator at approximately 4°C. Unless the energy stores (glycogen and ATP) have been completely exhausted from the muscle either before death or by delay after death, muscles can still be irritable and thus contract when they contact the fixative. For optimal evaluation, histopathologic, histochemical, and even electron microscopic examination are required. The veterinary practitioner should carefully evaluate whether useful information would be obtained by the processing of fixed tissue. This is relatively inexpensive and suitable for diagnosing inflammatory conditions such as polymyositis. However, for a complete evaluation of histologic changes in muscle, the patient should be referred to a neuromuscular disease laboratory.
REFERENCES 1. Ackerman, A. B.: Histologic Diagnosili of Inflammatory Skin Diseases: A Method by Pattern Analysis. Philadelphia, Lea and Febiger, 1978. 2. Adams, R. D. : Diseases of Muscle: A Study in Pathology. Hagerstown, Maryland, Harper and Row, 1975. 3. Bradley, R.: Sketetal muscle biopsy techniques in animals for histochemical and ultrastructural examination and expecially for the diagnosis of myodegeneration in cattle. Br. Vet. J., 134:434-!44, 1978.
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4. Bradley, W. G.: Disorders of Peripheral Nerves. Oxford, Blackwell Scientific Publications, 1974. 5. Braund, K. G., Dillon, A. R., Mikeal, R. L., eta!.: Subclinical myopathy associated with hyperadrenocorticism in the dog. Vet. Pathol., 17:134-148, 1980. 6. Braund, K. G., Shires, P. K., and Mikeal, R. L.: Type I fiber atrophy in the vastus lateralis muscle in dogs with femoral fractures treated by hyperextension. Vet. Pathol., 17:164-176, 1980. 7. Brooke, M. H.: The pathologic interpretation of muscle histochemistry. In Pearson, C. M., and Mostofi, F. K. (eds.): The Striated Muscle. Baltimore, Williams and Wilkins Company, 1973. 8. Cancilla, P. A. : Techniques of muscle biopsy and staining methods with particular emphasis on commonly encountered artifacts. In Pearson, C. M., and Mostofi, F. K. (eds.): The Striated Muscle. Baltimore, Williams and Wilkins Company, 1973. 9. Dikeman, M. E. , Melton, C. C., Tuma, H. J., eta!.: Biopsy sample analysis to predict bovine muscle palatability with emphasis on tenderness. J. Anim. Sci., 34:49--54, 1972. 10. Dubowitz, V. , and Brooke, M. H. : Muscle Biopsy: A Modern Approach. London, W. B. Saunders Company, 1973. 11. Gordon, E . E., and Texidor, T. A. : Problems in muscle biopsy: An experimental study. Arch. Phys. Med., 45:396-402, 1964. 12. Griffith, I. R. , Duncan, I. D. , McQueen, A., et a!.: Neuromuscular diseases in dogs: Some aspects of its investigation and diagnosis. J. Small Anim. Pract., 14:533-554, 1973. 13. Harriman, D. G. F .: Muscle. In Blackwood, W., and Corsellis, J.A.N . (eds.): Greenfield's Neuropathology. Edition 3. London, Edward Arnold, 1976. 14. Kalulas, C. A., Adams, R. D., and McGee, D. A.: The control of artifact in biopsied muscle tissue. In Proceedings of the Fifth International Neuropathological Congress. International Congress Series 100. Amsterdam, Excerpta Media, 1965. 15. Klinkerfuss, G. H.: An electron microscopic study of myotonic dystrophy. Arch. Neurol., 16:181-193, 1967. 16. McGavin, M. D .: Muscle biospy: Fruits and frustrations . In Proceedings of the Fourth Kal Kan Symposium. Columbus, Ohio State University, 1980. 17. McGavin, M. D. , and Baynes, I. D. : A congenital progressive ovine muscular dystrophy. Pathol. Vet., 6:513-524, 1969. 18. Mussini, 1., Di Mauro, S., and Angelina, C. : Early ultrastructural and biochemical changes in muscle in dystrophia myotonica. J. Neurol. Sci., 10 : 58~, 1970. 19. Namba, T., Shapiro, M. S., and Grob, D.: A simple clamp for skeletal muscle biopsy. Am. J. Clin. Pathol., 50:250, 1968. 20. Osborne, C. A. (ed.): Symposium on Biopsy Techniques. VET. CLIN. NORTH AM., 4:211467, 1974. 21. Price, H. M., Howes, E . L., Sheldon, D. B., eta!. : An improved biopsy technique for light and electron microscopic studies of human skeletal muscle. Lab. Invest., 14:194199, 1965. 22. Rayport, M.: A disposable isometric muscle biopsy clamp. J.A.M.A. , 210:1451-1452, 1965. 23. Report of a Sub-committee on quantitation of muscle biopsy findings. J. Neurol. Sci., 6:179--188, 1968. 24. Santa, T.: Fine structure of the human skeletal muscle in myopathy. Arch. Neurol. , 20:479--489, 1969. 25. Schroeder, J. M., and Becker, P. E.: Anomalien des T-System und des sarkoplasmatischen Reticulums bei der Myotonie, Paramyotonie und Adynamie. Virchows Arch. [Pathol. Anat.], 357:319--344, 1972. 26. Staheli, L. T.: A clamp for isometric muscle biopsies. Surgery, 59:1154-1155, 1966. 27. Armstrong, R. B., Saubert, C. W., IV, Seeherman, H. J., eta!.: Distribution of fiber types in locomotory muscles of dogs. Am. J. Anat. , 163:87-98, 1982. College of Veterinary Medicine University of Tennessee Knoxville, Tennessee 37901-1071
Muscle Biopsy in Veterinary Practice M.D. McGavin, M.V.Sc., Ph.D.*
Most skeletal muscles in mammals consist of three distinct physiologic fiber types: (1) slow contracting, slow fatiguing, oxidative (SO), (2) fast contracting, slow fatiguing, mixed oxidative-glycolytic (FOG), and (3) fast contracting, fast fatiguing, glycolytic (FG). Despite this, in paraffin-embedded histologic sections of muscle stained with hematoxylin and eosin (H&E), muscle fibers appear much the same (Fig. lA), apart from some variation in fiber diameter. Over 20 years ago, histochemical techniques were developed that allowed identification of these physiologic fiber types in frozen sections. 10 Such stains included myosin-ATPase, succinic dehydrogenase (SDH) for mitochondria, and NADH-TR, which stains chiefly mitochondria but also intermyofibrillar network. 10 The ATPase reaction is the most useful for the identification of different fiber types (Fig. IB). In normal muscle, many of the fibers in the same fasciculus are innervated by the same neuron, from the same ventral horn cell in the spinal cord, to form part of a motor unit. The innervation determines the physiologic type of muscle fiber and thus determines whether a fiber is SO, FOG, or FG. In sections stained by the myosin-ATPase reaction at a pH of 9.4, these fibers are light, medium, and dark, respectively, and are usually designated types I, Ila, and lib. 10 Damage to a neuron or to its axon may cause lesions in muscle fibers that are innervated by that nerve. Muscle fibers of a motor unit are scattered randomly through one or more muscle fasciculi and cannot be identified in hematoxylin and eosin-stained sections, but myosinATPase-stained sections will reveal that changes in the muscle are all of the same fiber type (see Fig. lA). Also, certain diseases preferentially involve one of the different fiber types. Thus, histochemical fiber-typing revolutionized muscle pathology because it made possible the differentiation between lesions that were primarily in the muscle, the so-called myopathic changes (for example, myositis) and muscle lesions secondary to changes in the neuron or nerves, the so-called neuropathic lesions. Since the devel~ opment of histochemical stains, the techniques that should be used for full histologic and histochemical examination of muscles have been defined and include such methods as H&E, modified Gomori' s trichrome, myosinATPase's incubated at different pH's, NADH-TR, and SDH as a mini*Diplomate, American College of Veterinary Pathologists; Professor, Department of Pathobiology, University ofTennessee College of Veterinary Medicine, Knoxville, Tennessee
Veterinary Clinics of North America: Small Animal Practice-Vol. 13, No. 1, February 1983
135
M. D. McGAVIN
136
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Figure 1. Canine superficial digital flexor muscle. Serial sections stained with hematoxylin and eosin (A) and myosin-ATPase (B). Note that in the H&E-stained section, it is possible to recognize small fibers (arrows), but only in the myosin-ATPase-stained section (pH=9.4) can it be seen that the atrophic fibers are all type II fibers which are also reduced in frequency. Diagnosis: type II atrophy.
mum. 8• 10 Some of these techniques are capricious, and their use is confined to specialized laboratories that do chiefly neuromuscular pathology. Originally such laboratories were primarily located in medical schools but later spread to institutions doing muscle research. Because of the cost and the fact that the selection of muscle for examination must be done accurately, specialized pathologic examinations of muscle are usually undertaken in association with clinical departments rather than in pathology departments of diagnostic laboratories.
MUSCLE BIOPSY IN VETERINARY PRACTICE
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"How useful are these techniques directly or indirectly to veterinary practitioners?" "How can they avail themselves of these techniques and what samples should they send?" It is certainly nothing new for muscle to be removed, either at necropsy or biopsy, formalin-fixed, and forwarded to a pathology laboratory. Unfortunately, the series of histochemical stains required for full evaluation of muscle depends on active enzymes, and thus can be done only on frozen samples, not on formalin-fixed tissues. Histologic examination of formalin-fixed muscle has limited usefulness, chiefly because the differentiation of physiologic muscle fiber types (types I, Ila, and lib) cannot be demonstrated. Consequently, the full evaluation of pathologic changes in muscles usually requires a specialized neuromuscular laboratory. Nevertheless, histologic examination of formalin-fixed tissue is useful for the recognition of inflammation, some degenerations, necrosis, and regenerations as well as large changes (atrophy or hypertrophy) in fiber diameters. However, both histochemical and histopathologic techniques are required for optimal evaluation so that the fiber type involved can be recognized. There are several excellent accounts of the histopathologic examination of muscle. 7• 8• 10 The difficulties of these methods will be discussed in this article so that the reader can understand the limitations of the techniques and how muscle should be obtained and fixed so that routine histopathologic examination will yield the maximum amount of information. MUSCLE BIOPSY
Surgical techniques for the biopsy of a wide variety of body tissues have now been described, and many were reviewed in this series. 20 Probably the most widely biopsied tissue in veterinary medicine is skin, but as judged by the accessions at pathology laboratories, liver and kidney, gastrointestinal tract, and uterus are now frequently biopsied. Muscle makes up only a small percentage of these specimens, and many of these samples are unfortunately unsuitable for the pathologist to obtain the maximum amount of information. Artifacts are the major problem. They occur in many samples of other tissues from a variety of causes: compression by forceps, crushing by scissors during excision, and stretching. Compression, crushing, and stretching artifacts in many tissues result in pyknosis and nuclear streaming and, if extensive, may make the histopathologic sections uninterpretable. However, in biopsies of skin, these defects are usually localized to those portions of the sample that have been mistreated. Artifacts usually do not obscure lesions unless the sample is extremely small or has been handled brutally, as, for example, if the edge of the skin has been grasped in several different places by forceps. However, muscle is not so forgiving, and even what would be considered normal surgical handling for other tissues will cause muscle to contract or overcontract, producing artifacts that will not be confined to one edge but will be present ~oughout the specimen. Thus the prevention of artifacts is essential for reliable histopathologic examination. The basic principle is to realize that muscle is designed to contract when stimulated, and it will do so when excised at biopsy and sometimes at necropsy. Also, ntuscle will contract when placed in most fixatives, including 10 per cent buffered neutral
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formalin . The factor that decides whether muscle is able to contract when stimulated is the availability of an energy supply in the muscle cells. Gordon and Texidor were able to prevent contraction of canine and human muscles by applying a pair of ligatures to each end of an isolated cylinder of muscle still attached to the muscle belly. 11 The outer ligatures produced ischemia after two minutes and reduced contraction when the sample was placed in fixative. After death, oxygen is no longer available and contraction will depend on glycogen stores to form ATP for contraction. Glycogen is slowly broken down, but some muscle fibers in well-nourished animals are still alive and able to contract several days after death if the body is held at 4°C. 17 It is these fibers that produce contraction artifacts when muscle is placed into fixative. Muscles from emaciated animals with little or no glycogen stores will soon cease to contract after death. Thus, the fresher the muscle, the more numerous the artifacts. This is one case when a tissue fixed immediately after death may appear histologically worse than one fixed some hours or even days after death, particularly if the temperature is cool. Also, diseased muscle fibers and fibers from very young animals are highly irritable and readily produce contraction artifacts at fixation. Techniques for reducing or eliminating these artifacts are described below. Indications Adams cites five different clinical problems in which muscle biopsy may be useful in human medicine. 2 1. Progressive muscular atrophy-to determine if the disease is neurogenic or myogenic. 2. Localized or diffuse myositis-to determine the type of inflammation (for example, eosinophilic myositis, suppurative myositis) or sometimes to detect the etiologic agent (as in toxoplasmosis). 3. Diffuse "vascular" or "collagen" diseases"-such as the immunemediated diseases. 4. Following injury to the nerve and blood vessels to a limb-to assess the state of the muscle and intermuscular-intramuscular nerves and thus determine the prognosis. 2 An example of this would be damage to some of the fibers of the sciatic nerve during fracture of the pelvis or by compression by an orthopedic plate during repair of a fracture. 5. Metabolic diseases-such as hypothyroidism, administration of excess steroids. Obviously, some of these conditions can be better evaluated by other clinical and laboratory techniques. However, muscle biopsy is particularly useful in the evaluation of muscular atrophy to determine whether it is neurogenic or myogenic and to determine the type of myositis whether this is due to an infectious disease or to an immune-mediated disease such as polymyositis. However, muscle biopsy is best undertaken only after complete clinical evaluation and, where possible, after electromyography, in order to clinically characterize the lesion and to locate the affected muscle. Selection of Muscle for Biopsy
Identifying Affected Muscle. Selection of a suitable muscle for biopsy is not as simple as it would seem and depends on the nature of the disease
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affecting the muscles. For example, is the disease generalized, localized, bilaterally symmetric, proximal or distal? Thus, identifying the affected muscles requires a clinical examination that includes a neurologic evaluation. If the disease is generalized or symmetric, muscles on one side can be examined by electromyography and biopsies taken from the contralateral muscles. Muscle used for electromyography should be avoided for biopsies, as the needle punctures may evoke an inflammatory response resulting in artifacts that may be misinterpreted by the pathologist. Selection of Muscle in Early Stages of the Disease. Muscles with chronic lesions (for example, following acute eosinophilic myositis in the canine temporalis muscle) are seldom useful for histologic examination. The end-stage of many muscle diseases is fibrosis and fiber atrophy. The characteristic changes are frequently seen only early in the disease. Size of Muscle and Direction of Fibers. To prevent artifacts many muscle biopsies are removed in isometric clamps. These clamps must be applied to parallel fibers-thus the muscles must be large enough to accommodate the clamps and also not be so small that the muscle mass removed constitutes a significant loss of functional fibers. In dogs and cats, muscles such as the semitendinosus, semimembranosus, biceps femoris, long digital extensor and tibialis cranialis, triceps brachii-lateral head, superficial digital extensor, and deltoid 12 have parallel fibers and are easier to biopsy. The vastus lateralis, a popular muscle for biopsy, has fibers which, although parallel to the long axis of the femur, are not parallel to the lateral surface of the muscle but run obliquely from the lateral fascial sheath toward the femur. Only in the distal third of the caudal border are the fibers approximately parallel to the surface, making this a suitable site for biopsy. Muscles such as the gastrocnemius and many of the flexors caudal to the antebrachium are pennate or semipennate, making bundles of long parallel fibers difficult or impossible to locate in small animals. Normal Data for Muscle Known. Extreme departures from normal in fiber size and the percentage of each of the different fiber types are relatively easy to recognize. In determining a full histochemical muscle fiber profile, however, the means and standard deviations of fiber diameters for type I and II fibers and also the percentage of each fiber type in the muscle are estimated. These data cannot be evaluated unless the laboratory has normal control data against which to compare the findings. The percentage of each fiber type and mean diameters of each muscle are dependent upon: the specific muscle, variation between different areas of the muscle itself, age, species, exercise (effect of conditioning): At the present time, accurate baseline data are known for a limited number of muscles in different species. 3 • 4 Most laboratories have developed data for muscles such as the vastus lateralis, biceps femoris, triceps brachii, and pectineus. Recently, Armstrong et al. reported the percentages of fiber types in 9llocomotory muscles in the dog. 27 However, availability of normal data is not likely to be a problem for the practitioner, because the samples he selects will have to be fixed in 10 per cent buffered neutral formalin and thus no histochemical profile can be attempted.
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Figure 2. Muscle fixed in 10 per cent buffered neutral formalin without clamps, paraffinembedded and stained with hematoxylin and eosin. A , The majority of the fibers, cut in crosssection, are polygonal, except for one that is rounded and deeply eosinophilic. This is a contracted fiber and corresponds to the extremely contracted portion of the fibers (arrows) shown in longitudinal sections (B).
To summarize, the following are the difficulties that occur during the histologic examination of muscle: l. The wrong muscle (that is, the unaffected muscle) is taken. 2. Muscle is diseased to an extent that renders it useless for histopathologic examination. 3. The characteristic lesions-for example, eosinophilic myositis-may have disappeared. 4. With muscles that are too small, it is difficult to apply clamps and too great a proportion of its contractile mass is removed. Most of the muscle removed will be replaced by fibrous tissue and will not be functional contractile tissue. 5. With muscles that do not have parallel fibers-for example, the temporalis, gastrocnemius, and many of the flexors on the caudal aspect of the antebrachium-clamping is difficult. To obtain the best sample, the surgeon must know the direction of fibers in the muscle before making the initial skin incision. Trial dissection of cadavers is strongly advocated. Clamps should be applied at a site where the majority of fibers are parallel. 6. Whereas stretching or compression of other tissues usually ruins only the portion of the sample that is touched, muscle is so irritable that artifacts may occur anywhere throughout the sample. Specific artifacts include extreme contraction bands of Nageotte, shredding, rounded hyalinized fibers (Fig. 2), sarcoplasmic masses, and cracking. 2• 16 Contraction
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bands may not only obscure lesions in that portion of the muscle, but may also result in the formation of artifactual sarcoplasmic masses in adjacent segments of the fibers. These artifacts can be confused with genuine sarcoplasmic masses formed by accumulations of sarcoplasm immediately under the sarcolemma and seen in some dystrophies. Also, contraction of some of the ·fibers or connective tissue can cause adjacent muscle fibers to have a zigzag pattern as they are passively shortened. All of these artifacts are the results of contraction of the still-irritable muscle on contact with fixative . To avoid these artifacts, it is desirable that: (I) the muscle be prevented from contracting with some form of clamp, and (2) stimuli be minimized during the actual excision. Clamping Devices
Muscle samples are best prevented from contracting by the application of some type of device before the muscle is excised. Some of these isometric clamps are commercially available, 21 · 26 and Rayport has developed a disposable plastic clamp. 22 However, others have used tongue-holding forceps, 5 modified Collins tongue-holding forceps, modified Judd-DeMartel gallbladder forceps, 19 Kelly clamps9 or hemostats 13 connected by a bar and modified ovarian clamps. 6 Bradley has also used so-called bulldog paper clips.5 The Price 21 and Staheli26 clamps are available in two sizes to take samples of approximately 1 or 2 em in length. The smaller-sized clamp is really designed for collecting samples for electron microscopy but is suitable for sampling small muscles such as those of the cat. However, these clamps are expensive and their cost probably cannot be justified in general practice. Eight-inch Judd-DeMartel gallbladder clamps with l-inch-diameter circular jaws modified by cutting off the outer half of each circular jaw19 are quite economical, and we use many of them for holding muscle in our necropsy laboratory. Recommendations for preventing muscle-contraction artifacts have changed over the years. The original methods did not suggest that the muscle be clamped before the sample was removed. Rather, the recommendation was to excise a cylinder of muscle, place it on a block of wood or piece of cardboard, straighten to its original length, and either allow it to adhere by drying2• 10• 15• 24 or ligate it to the wood. 18 Pathologists frequently recommend placing biopsy specimens of skin ,I intestine, 20 stomach, liver, and kidney on thin cardboard. They should be straightened gently to lie flat and allowed to dry for 30 seconds to two minutes so that they can adhere to the card, before being placed in fixative. This technique has also been proposed for muscle, 14 but in our experience it does not always prevent their contraction. Because of its unreliability, this method should be used only when no fixation devices are available and optimal results are not expected. In the absence of any clamps, the simplest method is to make two parallel -incisions, 3 to 6 mm apart, into the belly of the muscle in the same direction as the muscle fibers and suture each end of the isolated portion of muscle to a wooden stick, such as an applicator. 25 The muscle and stick are removed together and placed in the fixative . However, wood is not suitable for use in some fixatives, such as osmium tetroxide. This method has the advantage of being simple, inexpensive, and effective with such
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fixatives as 10 per cent buffered neutral formalin, but it takes more time and manipulation than the clamps and thus has the inherent danger of producing more artifacts. Techniques This has been described in detail by several authors. 5 • 6 • 12• 21 The basic requirement is to isolate a cylinder of muscle with parallel fibers, clamp it, then excise it. Thus, the first step is to identify that portion of the muscle with parallel fibers. If the surgeon is unfamiliar with the topography of the fibers, he or she should dissect muscles of a cadaver to determine the best site of sampling. Lack of familiarity with the direction of fibers will result in unnecessary handling of the muscle and the inevitable development of artifacts. One secret in obtaining good muscle biopsy samples is to minimize handling of the muscle. The portion that is to be excised should not be touched at all, except by the scalpel or razor blade. 12• 23 Blunt dissection, manipulation by forceps, stretching, and even pressure from fingers or sponges should be completely avoided, as even relatively minor stimuli will cause contraction. Price et al. have recommended the use of muscle cylinders 1.5 to 2 em long, 1 em wide, and 0.5 to 0.8 em thick for light microscopy and 1 to 1.5 em long, 0.5 em wide, and 0.5 em thick for electron microscopy. The procedures can be summarized as follows: 1. Expose the belly of the muscle at the site known to have parallel fibers. 2. Incise and gently retract the fascia from the muscle. 3. Make two incisions 2 to 6 mm apart and 1. 0 to 2. 5 em long, parallel to the direction of the muscle fibers. The length will depend on the size of the clamps used and the size of the muscle. 4. At this point, some authors describe a third cut to isolate the strip of muscle from the underlying belly of the muscle. Others apply the clamps first, then make the third cut. In our experience, artifacts occur less often if the clamp is applied before undercutting the strip of muscle. 5. The isolated cylinder of muscle in clamps is removed by cutting transversely across the muscle fibers a few millimeters beyond each end of the clamp (Fig. 3). 6. The muscle and clamps are placed in fixative, agitated to remove blood and serum, and then allowed to fix. CONCLUSIONS The surgical technique for obtaining muscle biopsy and the full histochemical and histopathologic evaluation of the specimen are expensive. When this type of evaluation is indicated, the most suitable procedu.r e is to take the patient to one of the laboratories that offer this service. This is more successful than transporting muscle specimens in liquid nitrogen or dry ice, as specimens may be delayed and may deteriorate in shipment, resulting in unnecessary expense. Muscle specimens fixed in 10 per cent buffered neutral formalin can be forwarded to a laboratory, but the surgeon must take steps to avoid artifacts. Every effort should be made to avoid stretching, pulling, or
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Figure 3. Muscle biopsy techniques. Canine left vastus lateralis muscle. The surgeon has exposed the caudal border of the vastus lateralis, reflected the fascial sheath and made two parallel incisions approximately 3 mm apart, applied isometric clamps and is now about to make the final three cuts to remove the clamped cylinder of muscle.
compression, and some form of clamp or device must be used to prevent contraction. Otherwise, artifacts may be so severe as to obscure lesions or even to prevent the pathologist from determining if there are any lesions. Thus, any irritable muscle, including one taken at necropsy, must be clamped prior to fixation because this irritability can continue for several days after death, if the body is held in a refrigerator at approximately 4°C. Unless the energy stores (glycogen and ATP) have been completely exhausted from the muscle either before death or by delay after death, muscles can still be irritable and thus contract when they contact the fixative. For optimal evaluation, histopathologic, histochemical, and even electron microscopic examination are required. The veterinary practitioner should carefully evaluate whether useful information would be obtained by the processing of fixed tissue. This is relatively inexpensive and suitable for diagnosing inflammatory conditions such as polymyositis. However, for a complete evaluation of histologic changes in muscle, the patient should be referred to a neuromuscular disease laboratory.
REFERENCES 1. Ackerman, A. B.: Histologic Diagnosili of Inflammatory Skin Diseases: A Method by Pattern Analysis. Philadelphia, Lea and Febiger, 1978. 2. Adams, R. D. : Diseases of Muscle: A Study in Pathology. Hagerstown, Maryland, Harper and Row, 1975. 3. Bradley, R.: Sketetal muscle biopsy techniques in animals for histochemical and ultrastructural examination and expecially for the diagnosis of myodegeneration in cattle. Br. Vet. J., 134:434-!44, 1978.
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4. Bradley, W. G.: Disorders of Peripheral Nerves. Oxford, Blackwell Scientific Publications, 1974. 5. Braund, K. G., Dillon, A. R., Mikeal, R. L., eta!.: Subclinical myopathy associated with hyperadrenocorticism in the dog. Vet. Pathol., 17:134-148, 1980. 6. Braund, K. G., Shires, P. K., and Mikeal, R. L.: Type I fiber atrophy in the vastus lateralis muscle in dogs with femoral fractures treated by hyperextension. Vet. Pathol., 17:164-176, 1980. 7. Brooke, M. H.: The pathologic interpretation of muscle histochemistry. In Pearson, C. M., and Mostofi, F. K. (eds.): The Striated Muscle. Baltimore, Williams and Wilkins Company, 1973. 8. Cancilla, P. A. : Techniques of muscle biopsy and staining methods with particular emphasis on commonly encountered artifacts. In Pearson, C. M., and Mostofi, F. K. (eds.): The Striated Muscle. Baltimore, Williams and Wilkins Company, 1973. 9. Dikeman, M. E. , Melton, C. C., Tuma, H. J., eta!.: Biopsy sample analysis to predict bovine muscle palatability with emphasis on tenderness. J. Anim. Sci., 34:49--54, 1972. 10. Dubowitz, V. , and Brooke, M. H. : Muscle Biopsy: A Modern Approach. London, W. B. Saunders Company, 1973. 11. Gordon, E . E., and Texidor, T. A. : Problems in muscle biopsy: An experimental study. Arch. Phys. Med., 45:396-402, 1964. 12. Griffith, I. R. , Duncan, I. D. , McQueen, A., et a!.: Neuromuscular diseases in dogs: Some aspects of its investigation and diagnosis. J. Small Anim. Pract., 14:533-554, 1973. 13. Harriman, D. G. F .: Muscle. In Blackwood, W., and Corsellis, J.A.N . (eds.): Greenfield's Neuropathology. Edition 3. London, Edward Arnold, 1976. 14. Kalulas, C. A., Adams, R. D., and McGee, D. A.: The control of artifact in biopsied muscle tissue. In Proceedings of the Fifth International Neuropathological Congress. International Congress Series 100. Amsterdam, Excerpta Media, 1965. 15. Klinkerfuss, G. H.: An electron microscopic study of myotonic dystrophy. Arch. Neurol., 16:181-193, 1967. 16. McGavin, M. D .: Muscle biospy: Fruits and frustrations . In Proceedings of the Fourth Kal Kan Symposium. Columbus, Ohio State University, 1980. 17. McGavin, M. D. , and Baynes, I. D. : A congenital progressive ovine muscular dystrophy. Pathol. Vet., 6:513-524, 1969. 18. Mussini, 1., Di Mauro, S., and Angelina, C. : Early ultrastructural and biochemical changes in muscle in dystrophia myotonica. J. Neurol. Sci., 10 : 58~, 1970. 19. Namba, T., Shapiro, M. S., and Grob, D.: A simple clamp for skeletal muscle biopsy. Am. J. Clin. Pathol., 50:250, 1968. 20. Osborne, C. A. (ed.): Symposium on Biopsy Techniques. VET. CLIN. NORTH AM., 4:211467, 1974. 21. Price, H. M., Howes, E . L., Sheldon, D. B., eta!. : An improved biopsy technique for light and electron microscopic studies of human skeletal muscle. Lab. Invest., 14:194199, 1965. 22. Rayport, M.: A disposable isometric muscle biopsy clamp. J.A.M.A. , 210:1451-1452, 1965. 23. Report of a Sub-committee on quantitation of muscle biopsy findings. J. Neurol. Sci., 6:179--188, 1968. 24. Santa, T.: Fine structure of the human skeletal muscle in myopathy. Arch. Neurol. , 20:479--489, 1969. 25. Schroeder, J. M., and Becker, P. E.: Anomalien des T-System und des sarkoplasmatischen Reticulums bei der Myotonie, Paramyotonie und Adynamie. Virchows Arch. [Pathol. Anat.], 357:319--344, 1972. 26. Staheli, L. T.: A clamp for isometric muscle biopsies. Surgery, 59:1154-1155, 1966. 27. Armstrong, R. B., Saubert, C. W., IV, Seeherman, H. J., eta!.: Distribution of fiber types in locomotory muscles of dogs. Am. J. Anat. , 163:87-98, 1982. College of Veterinary Medicine University of Tennessee Knoxville, Tennessee 37901-1071