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Abstract
Muscle samples were obtained at necropsy from 225 horses and ponies 1 year of age or older. Samples were processed in routine manner and were stained with hematoxylin and eosin and with periodic acid-Schiff for glycogen. Sections were examined for abnormal glycogen content and amylase-resistant complex polysaccharide and for chronic myopathic change (excessive fiber size variation, increase in number of internal nuclei). A total of 101 horses and ponies with lesions of polysaccharide storage myopathy were identified. Age of affected horses ranged from one to 30 years, with a mean of 14.7 years. Mean age of nonaffected horses was 12 years. Incidence of polysaccharide storage myopathy varied depending on breed; Thoroughbreds had the lowest (27%) and draft-related horses had the highest (86%) incidence. Chronic myopathic changes were more severe in polysaccharide storage myopathy-affected horses than in nonaffected horses. Results of this study indicate that polysaccharide storage myopathy is a common disorder of many breeds of horses and ponies.
Key words: Horses; muscular disorders; myopathy; polysaccharide.
Equine polysaccharide storage myopathy (EPSSM) is characterized by abnormal accumulation of glycogen and glycogen-related polysaccharide in skeletal muscle fibers.3–12 This disorder is believed to involve abnormal carbohydrate metabolism, but to the authors' knowledge, a specific defect has not been identified.7,10 A range of clinical signs of disease related to muscle dysfunction, including exertional rhabdomyolysis, muscular atrophy, abnormal gait, back pain, and postanesthetic myopathy have been described.3–9,11 Subclinical or unrecognized disease also is common,11,12 This disorder is thought to be inherited,3,7 and is most often reported in Quarter horse-related breeds, warmbloods, and draft-related breeds.3,4,6–11 Other breeds in which EPSSM has been reported include Thoroughbred crossbreds, Andalusian, Arabian, Morgan, Standardbred, and Welsh pony crossbreds.4,5,12 In a necropsy study of 37 draft-related horses and one draft mule, the incidence of pathologic changes indicative of EPSSM was 66%.11 To our knowledge, the incidence of this disorder in other breeds has not been reported. The purpose of the study reported here was to evaluate muscle samples from horses examined at necropsy to determine the incidence of EPSSM in various breeds.
A sample of semimembranosus or semitendinosus muscle was obtained from all horses and ponies 1 year of age or older that were examined after death at the Veterinary Diagnostic Laboratory at Oregon State University over a 2.5-year period. The sample obtained was a longitudinal strip of muscle approximately 5 cm long and 1 cm in diameter, similar to that obtained by biopsy.10 The samples were placed on a portion of a wooden tongue depressor and were immersed in neutral-buffered 10% formalin. Five to eight sections, one longitudinal section from the periphery of the sample, and the rest full-thickness transverse sections, were embedded in paraffin, sectioned at 4-µm thickness, and stained with hematoxylin and eosin (HE) and periodic acid-Schiff (PAS), with and without amylase digestion. Sections were examined in blinded manner by one of the authors (B. A. Valentine), and were evaluated for abnormal polysaccharide storage (subsarcolemmal aggregates of glycogen, central cytoplasmic bodies containing glycogen, inclusions of amylase-resistant material) and chronic myopathic change (excessive fiber size variation and increase in number of internal nuclei). Subsarcolemmal glycogen aggregates were graded as 0 (average, fewer than 1/10x field), 1+ (average, 1–3/10x; field), 2+ (average, 4–6/10x field), or 3+ (average, more than 6/10x field). Fiber size variation was subjectively judged to be absent (0), mild (1+), moderate (2+), or severe (3+). Internal nuclei were judged to be 0 (absent), 1+ (present, but average of fewer than 1/10x field), 2+ (average, 1–2/10x field or presence of fibers with two or more internal nuclei), or 3+ (average of more than 3/10x field). Central cytoplasmic bodies and amylase-resistant inclusions were recorded as present or absent.
A diagnosis of EPSSM was made for samples containing amylase-resistant inclusions (Fig. 1), 3+ subsarcolemmal aggregates of glycogen (Fig. 2), central cytoplasmic bodies containing glycogen (Fig. 3), or any combination of these findings. These criteria were based on findings of previous studies.3–12 Findings in PAS-stained sections of EPSSM-affected horses differed substantially from the glycogen staining pattern in muscle samples from horses considered normal (Fig. 4). Glycogen staining of all samples was extremely variable, and postmortem artifactual depletion of glycogen was common. Glycogen was often depleted in all but peripheral fibers within transverse sections (due to poor penetration of formalin into the interior of samples), and these areas were not included when evaluating samples for abnormal glycogen storage. Amylase-resistant inclusions persisted despite severe overall glycogen depletion, and the number present varied from one to many. In cases with numerous amylase-resistant inclusions, these could be seen as pale pink to blue-gray inclusions on HE-stained sections (Fig. 5). Subsarcolemmal vacuoles that were either clear or that contained pale pink hyaline material were also common in HE-stained sections from EPSSM-positive horses. Characteristic findings in many cases, however, were detected only after examination of PAS-stained sections. Myofiber necrosis or regeneration was uncommon. Chronic myopathic changes were best visualized on PAS-stained sections with amylase digestion.
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Medical records were obtained for all horses. The data were analyzed to determine the incidence of lesions of EPSSM in the major breeds represented. Crossbreds of Arabian, Morgan, draft, warmblood, Quarter horse, Paint, and Appaloosa were included in those breed headings. Other categories included only purebreds. The scores for fiber size variation and for internal nuclei were added to obtain a chronic myopathic score for each horse. The mean chronic myopathic score (± SE), mean score for subsarcolemmal glycogen aggregates (± SE), and mean age (± SE) were calculated for all EPSSM-positive horses and for all EPSSM-negative horses. Mean ages, mean glycogen scores, and mean chronic myopathic scores of EPSSM-positive and EPSSM-negative horses were analyzed for significant differences (Student's t-test).
Data from breeds with six or more horses are presented in Table 1. Data from breeds with fewer than six horses are presented in Table 2. Pathologic findings characteristic of EPSSM were detected in horses and ponies of many breeds (Tables 1, 2), with incidence ranging from 27% to 86% (Table 1). A sex predisposition was not apparent (no analysis performed). The mean chronic myopathic score for EPSSM-positive horses (2.66 ± 0.14) was significantly higher than that for EPSSM-negative horses (1.33 ± 0.14; P < 0.001). The mean score for subsarcolemmal glycogen aggregates was significantly higher in EPSSM-positive horses (2.63 ± 0.06) than in EPSSM-negative horses (0.92 ± 0.07; P < 0.001). Affected horses ranged from 1 year to 30 years of age. Mean age of affected horses (14.7 ± 0.82 years) was significantly different from the mean age of nonaffected horses (12 ± 0.66 years, P < 0.01).
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To date and to our knowledge, an immunohistochemical procedure has not been developed for diagnosis of EPSSM. Although severe cases contained obvious inclusions on HE-stained sections (Fig. 5), detection of EPSSM often required examination of PAS-stained sections. Cases in which amylase-resistant inclusions and internal glycogen inclusions involved few fibers indicate that examination of small samples or of only one or two sections from a sample may not be sufficient to detect these changes. When present, chronic myopathic change was a diffuse lesion that was readily detected in all sections. Formation of subsarcolemmal glycogen aggregates also was a widespread lesion, but detection required adequate preservation of glycogen; severe postmortem glycogen depletion will make it difficult or impossible to diagnose EPSSM in such cases.
Central glycogen-associated cytoplasmic bodies were considered to be a finding related to EPSSM, and have been associated with this myopathy.9 Similar structures have been reported in clinically normal horses as well as horses with neuromuscular disease, leading to the suggestion that they are a "non-pathological degenerative structure."1 Most of the horses of this study with central cytoplasmic bodies also had other findings indicative of EPSSM, suggesting that these structures are a feature of this myopathy. Given that muscle spasm and abnormal muscle tension are frequent clinical findings in EPSSM-positive horses,3,5,9,10 it is interesting that similar myofiber inclusions have developed in rat muscle following induction of localized tetanus.2 Formation of cytoplasmic bodies may be secondary to increased muscle tension. Further studies of cytoplasmic bodies in equine muscle are clearly needed.
Chronic myopathic change is a feature of many neuromuscular diseases.10 The finding of a significantly higher chronic myopathic change score in horses of this study that were diagnosed with EPSSM is evidence that the pathologic criteria used for diagnosis of this disorder are valid. It is also possible that increased degree of myopathic change is age related, as the mean age of EPSSM-positive horses was slightly but significantly higher than the mean age of nonaffected horses. However, the difference in mean age between EPSSM-affected and nonaffected horses does not appear to be sufficiently large to account for the highly significant difference in myopathic score. The characteristic pathologic changes in EPSSM are progressive3; therefore, it is not surprising that the mean age of affected horses in this study was slightly higher than that of unaffected horses.
Most horses studied did not have a recent history of clinical signs of muscular dysfunction, nor had they been evaluated for evidence of muscle disease. Therefore, the clinical relevance of finding evidence of EPSSM in these horses cannot be determined. As an attempt was not made to determine whether clinical signs of muscular dysfunction had been evident prior to death or euthanasia, it is possible that such clinical signs had been recognized but were not recorded in the medical history. However, the presence of this underlying myopathy could result in clinical signs of disease (e.g., weakness, pain, muscle atrophy, difficult recovery from general anesthesia) and clinicopathologic findings (i.e., increased serum activity of creatine kinase and/or aspartate aminotransferase) in affected horses that develop other disorders.
Acknowledgements
We thank Dr. Jerry Heidel, Dr. Christiane Löhr, Dr. Rob Bildfell, Mehmet Kupeli, and Jermey Becker for collection of samples, and Patricia DeVaughn for assistance with medical record data collection.
This study was funded by a donation from Tom and Mindy Wolfe.
References
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