Proceedings des 36èmes Journées Annuelles de l Association Vétérinaire Equine Française
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1 Close this window to return to IVIS Proceedings des 36èmes Journées Annuelles de l Association Vétérinaire Equine Française 9-11 Octobre Reims, France Reprinted in IVIS with the Permission of the Meeting Organizers
2 S07-05 THE CONTRIBUTION OF MUSCLE BIOPSIES TO THE DIAGNOSIS OF NEUROMUSCULAR DISEASE IN THE HORSE C. HAHN DVM MSc PhD DipECEIM DipECVN MRVCS, Neuromuscular Disease Laboratory, Royal (Dick) School of Veterinary Studies, EASTER BUSH, MIDLOTHIAN, EH25 9RG, Abstract: Somatic motor function is supplied by the motor unit, consisting of the neuronal soma, its axon, the neuromuscular junction and between a few to hundreds of muscle fibres supplied by each neuron. Each myofiber is a multinucleate syncytium formed by fusion of immature myoblasts. Myofibres are broadly divided into type I and type II fibres distinguished by their physiologic and histochemical properties. Slow, oxidative type I fibres are predominant in postural muscles such as the sacrocaudalis dorsalis medialis muscle and fast glycolytic type II fibers, including the gluteal muscles and semitendinosus muscles, are prevalent in locomotory muscles. Diseases of muscle are classified into two broad categories. Neurogenic myopathies are caused by lesions in lower motor neurons secondarily resulting in muscle fiber atrophy and angulation. Examples include peripheral nerve and nerve root trauma, Australian stringhalt and equine motor neuron disease. Type I fibres are targeted in equine motor neuron disease. Myopathic diseases primarily affect the muscle and include various forms of congenital, inflammatory and degenerative disorders, recurrent exertional rhabdomyolysis, muscle trauma, polysaccharide storage myopathy, white muscle disease and rarely malignant hyperthermia, inflammatory myopathies and myotonic dystrophy. Of particular note in Europe is atypical myopathy (atypical myoglobinuria), a devastating, enigmatic, pasture associated myodegenerative disease that has caused considerable morbidity and mortality in some areas of Europe. Type II fibres are selectively atrophic in a variety of unrelated myopathic conditions including metabolic myopathies and disuse atrophy. Introduction: biopsies play an integral role in evaluation of the patient with neuromuscular disease and it is an essential element in the assessment of a horse with suspected myopathy. In addition to being indispensable for the evaluation of muscle diseases, muscle biopsy is also involved in the evaluation of suspected neuropathic disease, particularly in the distinction of an atypical neurogenic disorder from a primary myopathic one. A basic knowledge of muscle physiology and myopathic diseases is important to allow clinicians to arrive at a rational differential diagnosis by synthesizing information obtained from the clinical history, physical examination, laboratory and ectrodiagnostic studies. A much broader range of procedures can be undertaken on cryostat frozen fresh samples rather than formalin fixed samples as biopsies are ideally processed using histochemistry, enzyme histochemistry and immunohistochemistry techniques. Experienced pathologists can then use the clinical features to assist in the interpretation of the constellation of pathologic findings in the biopsy. basics: Somatic motor function is supplied by the motor unit, consisting of the neuronal soma, its axon, the neuromuscular junction and from a few to hundreds of muscle fibres supplied by an individual neuron. s are composed of numerous bundles (fascicles) of muscle fibres and each myofibre is a multinucleate syncytium formed by fusion of immature myoblasts. The sarcoplasm of each myofiber is occupied largely by 132 -
3 actin and myosin myofibrils which form the contractile apparatus of the cell. There are two basic myofiber types, type I and type II which are distinguished by their physiologic and histochemical properties: muscle fibre type is determined by the firing frequency of the innervating somatic lower motor neuron. Each muscle has a characteristic ratio of type I to type II myofibres: type I myofibres in horses are smaller than type II fibres and have a slow contraction time following electrical stimulation. They are used for sustained, low-level activity required particularly in postural muscles, and are equipped with numerous large mitochondria and abundant intracellular lipid for oxidative metabolism. The sacrocaudalis dorsalis medialis (tail head) muscle is a type I predominant muscle accessible for biopsy. Type II myofibres have a rapid contraction time following stimulation and are used for brief-duration activity in carrying heavy loads and locomotion. These fibres are larger, have fewer (and smaller) mitochondria and contain less lipid and more glycogen than type I fibres. The gluteal muscles or semitendinosus muscle are commonly biopsied type II predominant muscles in the horse. Subgroups of type II fibres and fibres with overlap characteristics between type I and type II also exist. Information about changes in the myofiber types in a muscle biopsy often provides significant clues to the diagnosis, as different pathologic processes alter the ratio of the myofiber types and their distributions in the muscle. Diseases may selectively affect the size of one type or the other, for example equine motor neuron disease preferentially affect type I fibres whereas rhabdomyolysis syndrome is more pronounced in type II predominant muscles. Myopathology basics: In specialty myopathology laboratories muscle tissue is processed using a variety of histochemical and enzyme histochemical techniques to allow fibre typing and to expose changes in morphology, respiratory enzyme and mitochondrial function, abnormal glycogen and lipid storage, endomysial fibrosis and degenerating and regenerating fibres. Diseases of muscle are classified into two broad categories: 1. Neurogenic myopathies, in which the primary lesion affects lower motor neuron soma in the spinal cord or their axons in the peripheral nerves. 2. Myopathic myopathies, in which the muscle is primarily affected by various forms of congenital, inflammatory and degenerative disorders. Muscular dystrophies and mitochondrial myopathies are exceptionally rare in horses. Diseases in the horse causing neurogenic muscle changes include peripheral nerve and nerve root trauma, stringhalt and equine motor neuron disease. Denervation of skeletal muscle is followed by the progressive loss of tissue mass and impairment of its functional properties. Denervated myofibres classically become smaller and more angular and since a single motor unit supplies many fibres, denervation initially results in randomly scattered atrophic fibres. With further involvement of adjacent motor units, groups or whole fascicles of fibres become atrophic resulting in group atrophy. Type I fibres are targeted in equine motor neuron disease. If the denervated muscle fibres are in the vicinity of intact axons they may become reinnervated by collateral sprouting. Since the motor neuron determines the muscle fiber type, all of the reinnervated fibres are converted to a single histochemical fiber type with loss of the normal checkerboard pattern ( fibre type grouping. ). Fibres that are not ultimately reinnervated undergo cell death in a process resembling, but distinct from, classical apoptosis. End stage denervated muscle can show significant fibre necrosis and fibre loss results in endomysial fibrosis and fatty replacement
4 Myopathic diseases in horses include recurrent exertional rhabdomyolysis, muscle trauma, polysaccharide storage myopathy, white muscle disease and rarely malignant hyperthermia, inflammatory myopathies and myotonic dystrophy. Atypical myopathy is a devastating, enigmatic, pasture associated myodegenerative disease that has caused considerable morbidity and mortality in some areas of Europe. Necrosis followed by phagocystosis of muscle fibres is a common feature in many myopathic conditions especially in the early stages. This is largely responsible for elevated serum enzymes, notably creatine phosphokinase in many active myopathic conditions. Atrophy of fibres invariably results in hypertrophy of the remaining fibres and an abnormal fibre size variation. Type II fibres are selectively atrophic in a variety of unrelated myopathic conditions including metabolic myopathies and disuse atrophy. Nonspecific fibre changes seen in various myopathies also include split fibres and vacuolar changes. Horses can suffer from inflammatory myopathies but unlike dogs, horses do not have a specialised fibre type in masseter muscles and are not affected by from masticatory muscle myositis. Rhabdomyolysis in conjunction with Streptococcus equi infection, immune-mediated myositis and virus associated myopathies are exceptionally rare but have been reported. biopsy: The surgical procedure to obtain a muscle biopsy is relatively simple but it must be performed properly to optimize the information it can yield. A much broader range of procedures can be undertaken on cryostat frozen fresh samples rather than formalin fixed samples and careful sampling and shipping techniques, as detailed below, are important. Choice of muscle For generalised myopathies or focal disease it is advisable to biopsy an apparently affected muscle. Equine polysaccharide storage myopathy preferentially affects type II predominant ( fast twitch, locomotory) muscles such as the proximal semitendinosus, while Equine Motor Neuron Disease affects type I ( slow twitch, postural) muscles including the medial tail head muscle, the sacrocaudalis dorsalis medialis. Biopsy technique To obtain the biopsy, the horse should be sedated and administered local anaesthesia with either a caudal epidural or line block over the incision site, without actually injecting into the muscle to be sampled. Make a 3 cm longitudinal skin incision parallel to the muscle fibres. Repeat through the fascia. Obtain a strip of muscle approximately 3 cm in length by 1 cm in diameter. The muscle should be undermined first so it does not retract when one end is cut. Punch biopsies do not provide enough tissue for evaluation. [The techniques are well reviewed in Valentine et al. biopsy diagnosis of equine motor neuron disease and equine polysaccharide storage myopathy. Equine Vet Ed 10(1): 42-50, 1998]. Shipping 1. Most of the sample should be wrapped into saline moistened (ie not wet!) gauze, placed into a dry, water-tight container and shipped on ice, or better yet, cold packs. 2. A smaller portion of the biopsy should be smoothed onto a piece of cardboard, pinned in place, and then put in 10% formalin (in case there is a delay in arrival of the package)
5 Further reading: - Ledwith and Mcgowan. biopsy: a routine diagnostic procedure. Equine Veterinary Education 16(2)62-67, Votion D. M. et al. Atypical Myopathy (Atypical Myoglobinuria). Document No. R International Veterinary Information Service, Ithaca NY ( - Dickinson PJ and LeCouteur RA. and nerve biopsy. Veterinary Clinics of North America - Small Animal Practice 32:63-102,
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