Creatine kinase (CK), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) activities in serum are used to assess muscle damage in large animals. Serum CK offers remarkable sensitivity as an indicator of myonecrosis and is predominantly located in skeletal and heart muscle. It is intimately involved in energy production within the cytoplasm and is readily liberated into the extracellular fluid when the muscle cell membrane is disrupted. Serum CK activity increases within hours of a muscle insult and peaks within 4 to 6 hours after injury (Fig. 42-2). Limited elevations in CK may accompany training, transport, and strenuous exercise. Elevations of CK to 400 or 500 IU/L may occur when training commences or in response to moderate exercise. Extreme fatiguing exercise (e.g., endurance rides or the cross-country phase of a 3-day event) may result in CK activities being increased to more than 1000 IU/L but usually less than 8000 IU/L. These elevations are not typically reflective of an extensive myopathy, and serum activities of CK rapidly return to baseline activities (i.e., <250 IU/L in 24 to 48 hours). Recumbent animals may also have slightly elevated CK activities that are usually less than 3000 IU/L. In contrast, more substantial elevations (from several thousand to hundreds of thousands of IU/L) in the activity of this enzyme may occur with rhabdomyolysis.

Serum AST has high activity in skeletal and cardiac muscle and also in liver, red blood cells, and other tissues. Elevations in AST are not specific for myonecrosis, and increases can be the result of hemolysis, muscle, liver, or other organ damage. Alterations in AST activity are an integral component of many serum biochemical “profiles.” AST activity rises more slowly in response to myonecrosis than does CK, often peaking 24 hours after the insult (see Fig. 42-2), and the half-life of AST is much longer than that of CK. By comparing serial activities of CK and AST in animals with suspected myopathy, information concerning the progression of myonecrosis may be derived. Thus (1) elevations in CK and AST reflect relatively recent or active myonecrosis; (2) if CK remains persistently elevated, myonecrosis is likely ongoing; and (3) elevated AST activities accompanied by decreasing or normal CK activities indicate that myonecrosis is not continuing. Elevations in serum LDH activity are not specific to skeletal muscle and can occur with rhabdomyolysis, myocardial necrosis, and/or hepatic necrosis.

Under ideal conditions samples for determination of serum CK, AST, and LDH should be harvested as soon as possible because anoxia and lysis of red blood cells allows the liberation of AST and LDH. They should be kept chilled (4° C, 39° F) if not taken immediately to a laboratory for analysis within 24 hours. When a delay of more than 12 hours is anticipated, freezing of the samples is desirable.

Elevations in plasma/serum myoglobin concentrations provide an indication of recent muscle damage. Unfortunately, few laboratories can routinely measure myoglobin. Normal concentrations in resting horses have been determined by nephelometry (range 0 to 9 µg/L),¹ with measured concentrations with rhabdomyolysis ranging from 10,000 to 800,000 µg/L.²

Urinalysis

Urine can be obtained free catch from horses placed in stalls with fresh bedding or via catheterization. The most accurate reflection of fractional excretion of electrolytes is obtained from samples obtained without tranquilization. Urinalysis is particularly important in horses with myoglobinuria, elevations in creatinine, or suspected electrolyte imbalances. Urine specific gravity, protein content, white blood cell (WBC) count, red blood cell (RBC) count, and evaluation of cast formation should be performed to assess the potential for concurrent renal disease. A positive Hemastix test (ortho-toluidine) in the absence of hemolysis or RBCs in urine is highly suggestive of myoglobinuria. Further differentiation of myoglobin from hemoglobin is sometimes warranted and, where available, electrophoresis, nephelometry, or spectroscopy may be used. Spectroscopy does not always reliably distinguish between myoglobin and hemoglobin.

Electrolyte, mineral, and creatinine concentrations in urine and blood have been used to determine electrolyte balance in horses with muscle cramping or exertional rhabdomyolysis.³ Problems often encountered, however, are the large fluctuations in daily electrolyte excretion that occur in the same horse and between horses, the interference of high urinary potassium concentrations with measures of sodium concentrations when an ion specific electrode is used, and the presence of calcium crystals in equine urine, which artifactually decrease the calcium and magnesium measured if urine samples are not acidified.⁴ Renal fractional excretions (FEs) can be calculated using the following formula:

FEx=SCr×Ux×100UCr×Sx

where U is urine, S is serum, x is measured electrolyte, and Cr is creatinine.

If urine calcium is to be determined, acidification of urine to dissolve all calcium oxalate crystals is recommended to provide exact calcium excretion. Determination of potassium, chloride, magnesium, and phosphorus concentrations can be performed using ion-specific electrodes or by inductively coupled plasma atomic absorption.

Normal values for FE of electrolytes depend on a horse’s diet. Normal values (%) for horses consuming grass, hay, and a sweet feed mix with available salt are FE_Na 0.04 to 0.08, FE_K 35 to 80, FE_Cl 0.4 to 1.2, FE_Ca 5.3 to 14.5, FE_P 0.05 to 4.1, and FE_Mg 14.2 to 21.4.^4,5

Exercise Testing

Evaluation of muscle disorders that are precipitated by exercise may require an exercise challenge test. Horses should be observed for evidence of exercise intolerance, as well as muscle stiffness. An exercise test should not be used in horses with overt signs of rhabdomyolysis but rather to determine if horses not currently showing clinical signs are prone to exertional rhabdomyolysis. The goal is to induce subclinical elevations in serum CK activity. Abnormal increases in CK are more likely to occur if slow trotting is performed rather than strenuous exercise.⁶ Often 15 minutes of exercise at a walk and trot in unfit horses or at a constant slow trot in fit horses will elucidate subclinical elevations on CK. If signs of stiffness develop before this, exercise should be concluded. CK activities in blood samples taken immediately after exercise do not reflect the amount of exercise-induced muscle damage. For best results, blood samples for CK activity should be taken before and 4 to 6 hours after exercise. In healthy horses, 15 to 30 minutes of light exercise rarely causes more than a threefold increase in CK activity.^7,8 Elevations greater than fivefold are indicative of exertional rhabdomyolysis. Standardized treadmill exercise testing can also be used to evaluate muscle responses and measure metabolic responses to exercise.

Electromyography

Electrodiagnostic studies detect spontaneous or evoked potentials of neurogenic or myogenic origin using electrodes positioned in the muscle. Electromyography (EMG) is particularly useful to evaluate large animals with altered muscle tone. EMG of normal skeletal muscle shows a brief burst of electrical activity when the needle is inserted (insertional activity) in muscle and then quiescence, unless motor units are recruited (motor unit action potentials), or the needle is close to a motor end plate (miniature end plate potentials). Normal muscle shows little spontaneous electrical activity unless the muscles contract or the horse moves. Horses with abnormalities in the electrical conduction system of muscle, or denervation of motor units, show abnormal spontaneous electrical activity in the form of fibrillation potentials, positive sharp waves, myotonic discharges, or complex repetitive discharges.^9–12 Fibrillation potentials and positive sharp waves represent spontaneous firing of muscle fibers. Myotonic discharges are bursts of complex high-frequency potentials, whereas complex repetitive discharges are similar but have fixed amplitude and frequency. They both represent abnormal simultaneous firing of groups of muscle fibers. Motor unit action potentials can be evaluated to assess their amplitude, duration, phase, and number of phases. Myopathic changes include a decrease in duration and amplitude of motor unit action potentials.^10,12 More information about the motor unit could be provided by nerve conduction velocities (NCVs); however, the inaccessibility of motor nerves makes measurement difficult in large animals. Both EMG and NCVs are used to classify the primary disease as neuropathic or myopathic, to determine the distribution of the disease, and to provide insight into the pathophysiologic mechanisms of the disease.^9,13 Equipment costs are relatively high, and expertise is required in operation and interpretation of results. Readers are advised to consult Chapter 35 before considering the use of EMG.

Nuclear Scintigraphy

Nuclear scintigraphy is useful for identification of some forms of muscle damage, particularly an area of deep muscle damage that was not suspected on the basis of clinical examination.¹⁴ Technetium 99 m methylene diphosphonate (MDP) is taken up in some damaged muscle in the horse and is best seen in the bone phase images (i.e., 3 hours after injection). Scintigraphy has been used in horses with a history of poor performance, with or without stiffness after exercise, to confirm a diagnosis of equine rhabdomyolysis.¹⁵ The mechanism of MDP binding is unknown, but the release of large amounts of calcium from damaged muscle or the exposure of calcium binding sites on protein macromolecules in the damaged muscle may be responsible. Scintigraphy may be helpful in some cases involving focal damage to either proximal forelimb or hindlimb muscles.¹⁴

Ultrasonography

Diagnostic ultrasonography is potentially useful for identification of muscle trauma, crepitus, fibrosis, and atrophy. Muscles have a rather typical striated echogenic pattern, but this varies according to the muscle group and careful comparisons must be made between similar sites in contralateral limbs, in both transverse and longitudinal images.¹⁴ The appearance of muscle is also sensitive to the way the animal is standing and whether the muscle is under tension, so it is important that the animal is standing squarely and bearing weight evenly. Muscle fascia appears as well-defined, relatively echodense bands.¹⁴ Care must be taken in identifying large vessels and artifacts created by them.

In an acute injury, muscle fiber disruption is seen as relatively hypoechoic areas within muscle, with loss of the normal muscle fiber striation. The jagged edge of the margin of the torn muscle may be increased in echogenicity.¹⁴ Tears in the muscle fascia may be identified. The defect in muscle may be filled by loculated hematoma that is hypoechoic. As the muscle repairs it becomes progressively more echogenic. Relatively hyperechoic regions may be a result of increased connective tissue or loss of muscle cell mass. Hyperechoic shadowing artifacts usually represent mineralization or gas pockets.¹⁴

Muscle Biopsy

Examination of muscle fibers, neuromuscular junctions, nerve branches, connective tissue, and blood vessels within a biopsy sample can provide additional information necessary to fully characterize a neuromuscular disorder.^16–18 Routine light and electron microscopic examinations, combined with histochemical evaluations, may provide insights into the particular manifestations of neuromuscular diseases and their rate of progression. A number of basic pathologic responses of muscle can be identified in paraffin-fixed sections. These include inflammatory infiltrates, muscle fiber necrosis, muscle fiber regeneration, increased number of central nuclei, variations in muscle fiber sizes and fiber shapes, vacuolar change, and proliferation of connective tissue. However, there are many pathologic alterations that cannot be detected in formalin-fixed tissue but can readily be seen in histochemical stains of fresh-frozen biopsy samples.^16–18 These include muscle fiber types and their pattern of distribution, differentiation of neurogenic atrophy from disuse atrophy or a primary myopathy, characterization of vacuolar storage material, characterization of inclusion bodies, assessment of mitochondrial density, and additional clues that may allow identification of a specific disorder or category of muscle disorders. Furthermore, formalin fixation results in artifactual cracking, fiber shrinkage, and leakage of substrates such as glycogen, which can impact proper interpretation of muscle pathology.

When considering collection of muscle biopsies, some general guidelines apply. Preferably, samples should be collected from what is considered abnormal or diseased muscle. A 6-mm-outer-diameter (Jørgen Kruuse A/S, Langeskov, Denmark) percutaneous needle biopsy technique can be used to obtain small muscle samples through a -inch skin incision using a local anesthetic subcutaneously. If this technique is used, enough muscle should be obtained to form a -inch square sample at a minimum. These samples do not, however, tolerate shipment to an outside laboratory. The optimum biopsy for shipment of histopathology tissues to a laboratory is collected using surgical or open techniques, performed under local anesthesia. Care must be exercised to infiltrate only the subcutaneous tissues, not the muscle, with the anesthetic agent. The objective is to obtain approximately a -inch cube of tissue; hence a suitably long skin incision is required. Two parallel incisions inch apart should be made longitudinal to the muscle fibers with a scalpel. The muscle should only be handled in one corner using forceps, and crushing should be avoided. The muscle sample is then excised by cross-secting incisions inch apart, and the tissue is fixed appropriately. Routine histopathology samples can be placed in formalin; fresh samples can be placed in a watertight hard container after being wrapped in gauze lightly moistened with saline and shipped chilled to laboratories for freezing. On arrival at specialized laboratories, fresh samples for histochemical analysis are fixed in isopentane (methylbutane) chilled in liquid nitrogen to ensure rapid freezing and minimization of freeze artifact. Samples that potentially may be used for biochemical analysis should be immediately frozen in liquid nitrogen. Other routine histopathologic techniques may also be of diagnostic value. A special fixative may be required if such practices are to be undertaken. Samples for electron microscopy (EM) require appropriate fixation in glutaraldehyde preparations. Ideally, thin sections of muscle for EM should be clamped in vivo to maintain fibers at a resting length before they are excised. However, if pathology other than the alignment of thick and thin myofilaments is to be investigated, small muscle pieces can be excised and placed directly in appropriate EM fixative.

Responses of strips of fresh muscle to stimuli such as caffeine, halothane, and a variety of other agents can also be performed by specialized laboratories to diagnose recurrent exertional rhabdomyolysis and susceptibility to malignant hyperthermia but are not routine diagnostic procedures.^19–21

Diagnosis

A tentative diagnosis frequently can be made on the basis of age; clinical signs (stiff gait, particularly at the onset of exercise); muscle bulging; and prolonged contractions following muscle stimulation.

Definitive diagnosis of myotonia is usually based on electromyographic examination. Affected muscle manifests pathognomonic, crescendo-decrescendo, high-frequency repetitive bursts with a characteristic “dive bomber” sound.^29,45,48 This sound is produced by the repetitive firing (after contractions) of affected muscle fibers. After a contraction diminishes, the excitability of muscle fibers is decreased and the action potentials recorded by the EMG reflect the diminution of electrical activity.⁴⁹

Examination of muscle biopsies from foals with myotonia congenita may be normal or may demonstrate extremely variable muscle fiber dimensions up to twice those of normal age-matched controls.⁴¹ Type I fiber hypertrophy or hypotrophy may be seen. The major changes noted with myotonic dystrophy are ringed fibers, alterations in the shape and position of myonuclei, sarcoplasmic masses, and an increase in endomysial and perimysial connective tissue.^29,30,45,50 Fiber type grouping and atrophy of both type I and type II muscle fibers may be present.

Treatment

Considering that the pathophysiologic basis of myotonia in horses has not been clearly identified, recommendations for specific, effective therapy are almost impossible. In affected humans and dogs some relief of signs has been provided by drugs such as quinine, procainamide, and phenytoin. However, responses vary among patients. Phenytoin has been reported to be efficacious in two Quarter Horses with hyperkalemic periodic paralysis and myotonic dystrophy.⁴⁸

Prognosis

Prognosis appears to be variable and dependent on the severity of clinical signs. Mildly affected animals with congenital myotonia may undergo some amelioration of clinical signs with increasing age over a period of months to years. The reason(s) for this regression of signs is unknown. Other, more severely affected horses, particularly those with dystrophic myotonia, may have progression of signs, including atrophy and fibrosis or pseudohypertrophy to the point at which the animal is no longer able to move without great pain and difficulty (Fig. 42-5). Euthanasia of such animals is often warranted. Although conclusive evidence regarding the genetic basis of dystrophic myotonia in horses is still not available, owners of affected horses should be cautioned as to the possibility of this disease being heritable.