ÉTICO Y CONEXOS

Histology of skeletal muscle and liver samples was performed in 20 dogs. In all cases, the muscles sampled and the liver appeared grossly normal. Additional tissues were collected and examined histologically where possible, including heart (15 dogs), gastrointestinal tract (9), lung (9), kidney (7), spleen (6), brain (4), lymph node (3) and spinal cord (1). Samples for histology were collected at varying time points in relation

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to the onset of clinical signs, with the earliest samples collected on day 2 (four cases). Other cases were sampled on day 3 (three dogs), day 5 - 6 (three dogs), day 10 - 20 (four dogs) and day 45 – 430 (six dogs). The most significant lesions were present within sections of skeletal muscle, with similar histological changes seen in the triceps, biceps femoris and other muscles sampled. In severe acute cases (7 days or fewer duration), there was scattered segmental degeneration of individual myofibres, characterised by fragmentation, vacuolation or hyalinisation of the sarcoplasm and loss of cross-striations (Figure 3.10A-B). Within the sarcoplasm of some degenerate myofibres there were small numbers of mixed inflammatory cells, predominantly macrophages, with rare neutrophils, lymphocytes and plasma cells (Figure 3.10C-D), but in most cases inflammation was minimal to absent. In muscle samples collected within 2 days of the initial onset of clinical signs, there was no evidence of muscle regeneration. In dogs sampled from day 3 onwards, regeneration of myofibres was a consistent feature, and acute myofibre degeneration and regeneration were seen within the same muscle bundles (Figure 3.10E-G). Regenerative fibres were smaller than surrounding normal myofibres, with lightly basophilic sarcoplasm and no clearly visible cross striations. These fibres had multiple, centrally located large nuclei, each with 1 -2 large nucleoli. In cases with mild clinical signs and small increases in serum creatine kinase activities, no overt myodegeneration or necrosis was seen. Instead, the predominant finding in these cases was the presence of scattered small (10 – 50% of the diameter of normal myofibres), hypereosinophilic triangular fibres in transverse sections (Figure 3.10H). There were also occasional thin bands of connective tissue running through the middle of larger myofibres in cross section, dividing them into 2 smaller fibres (fibre splitting). PAS staining of skeletal muscle sections was performed in four of the cases recruited early in the study (duration of clinical signs ranging from 2 days to 4 weeks), as well as one control dog. There were no appreciable differences in the pattern of fibre staining with PAS in these dogs.

In chronic cases (greater than 7 days’ duration), there were scattered small triangular myofibres and fibre splitting as described in acute cases, resulting in variation in myofibre diameter in transverse sections. There were also occasional individual hypereosinophilic myofibres, separated from surrounding normal fibres by a thin rim

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of clear space (Figure 3.11A) These myofibres had small, dark peripheral nuclei (pyknosis) and a homogenous appearance to the sarcoplasm, with a loss of cross striations. Variable numbers of degenerate and regenerative myofibres were also seen in chronic cases, with no significant inflammatory response, and in some cases the lesions were very subtle. In three dogs, there were multifocal areas of mildly increased fibrous connective tissue around myofibres in the triceps and biceps femoris, and some fibres in these areas had increased numbers of peripheral satellite nuclei. Scattered adipocytes were present in the centre of muscle bundles in two cases, alongside myofibre regeneration (Figure 3.11B), suggestive of myofibre loss and replacement with adipose tissue. In all cases, perimysial connective tissue, blood vessels and intramuscular sections of peripheral nerves were histologically normal. There was no evidence of Trichinella spiralis or other parasites in any of the muscle sections examined. No microscopic abnormalities were observed in the cardiac muscle samples from affected dogs (n = 15).

In the livers of 18 out of the 20 dogs sampled, the cytoplasm of centrilobular and mid- zonal hepatocytes was expanded by numerous indistinct, small, clear vacuoles

(vacuolar hepatopathy) without peripheral displacement of the nucleus (Figure 3.11C- D). This is most consistent with microvesicular steatosis (accumulation of lipid in hepatocytes), although accumulation of glycogen within hepatocytes can also be associated with a similar vacuolar change. Special stains for lipid were not performed as all samples were formalin-fixed and paraffin embedded, and any lipid present is usually dissolved during processing. In three cases, the Periodic acid–Schiff (PAS) stain, which detects glycogen, was applied to liver sections and the clear material did not take up the PAS stain. In addition to the microvesicular change, there were also scattered foci of large, discrete clear vacuoles that displaced and compressed hepatocyte nuclei (macrovesicular steatosis) in one acute case (2 days’ duration). In the two cases where hepatocytes were not vacuolated, there was marked congestion of sinusoids in centrilobular areas resulting in attenuation of hepatic cords. Periportal lymphatic vessels were occasionally dilated in affected dogs, but the portal areas were otherwise normal. There was no significant inflammation or hepatocyte necrosis.

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Histologic changes were seen in the kidneys of two dogs that were euthanised due to clinical deterioration despite intensive symptomatic and supportive care. In these two dogs, numerous large, variably sized, brightly eosinophilic round droplets (interpreted as myoglobin) were present within the lumen of both proximal and distal convoluted tubules (Figure 3.11E-F). There was no degeneration of renal tubular epithelial cells associated with this, and glomeruli were normal. One of these dogs had increased serum urea, creatinine and phosphate concentrations on serum biochemistry, but urine specific gravity was not measured to allow differentiation between pre-renal and renal azotaemia. The kidneys were normal in the remaining five cases where samples were collected, and none of these dogs showed clinical signs or had biochemical changes suggestive of renal dysfunction.

Of the nine cases where fixed samples of the gastrointestinal tract were collected, six were histologically normal. In the remaining three dogs, there were multifocal areas of mild to moderate haemorrhage in the lamina propria and along the mucosal surface of the ileum and colon (Figure 3.11G-H). This was associated with mild blunting and fusion of villi, but there was no erosion of the mucosal surface or accompanying inflammation. The tunica muscularis was normal. All three of these dogs were

reported to have haemorrhagic diarrhoea, and samples were collected within 5 days of the initial onset of clinical signs. Other tissues examined from affected dogs were within normal limits, including the brain, spinal cord and peripheral nerves.

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A (2 days) B (2 days)

C (3 days) D (3 days)

E (6 days) F (6 days)

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Figure 3.10 (opposite page): Photomicrographs of longitudinal and transverse sections of

skeletal muscle from dogs with ‘Go Slow’ myopathy. The number of days refers to the time between the initial onset of clinical signs and sample collection. A-B (2 days): Scattered degenerate myofibres, characterised by disruption of the sarcoplasm and loss of cross striations. There are also several shrunken, hypereosinophilic fibres in the transverse section (B). C-D (3 days): Myofibre degeneration as in A, with small numbers of mononuclear inflammatory cells present within the sarcoplasm of degenerate fibres. Small numbers of regenerative fibres, characterised by basophilia of the sarcoplasm, are also seen in D. E-F (6

days): Acute degeneration of myofibres, degeneration with inflammation, and myofibre

regeneration are seen within the same section. Regenerative fibres have sarcoplasmic basophilia and multiple, large nuclei that can be located centrally within the cell. G (10 days): Regeneration of myofibres; no degeneration is seen in this section. H (19 days): Small, angular, hypereosinophilic myofibres are scattered amongst normal myofibres.

78 A (240 days) B (chronic) C (19 days) D (19 days) E (2 days) F (2 days) G (5 days) H (2 days)

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Figure 3.11 (opposite page): Photomicrographs of sections of skeletal muscle, liver, kidneys

and intestine from dogs with ‘Go Slow’ myopathy. The number of days refers to the time between the initial onset of clinical signs and sample collection. A (240 days): Transverse section of skeletal muscle hypereosinophilic myofibres separated from surrounding normal fibres by a thin rim of clear space; nuclei are pyknotic. B (chronic case, unknown duration): Transverse section of skeletal muscle showing numerous fibres with centrally located nuclei, indicative of myocyte regeneration. Multifocally, myofibres are replaced by adipocytes with a large, clear cytoplasm. C-D (19 days): Section of liver at 100X (C) and 400X magnification. In centrilobular to mid-zonal areas there is vacuolation of hepatocyte cytoplasm. Periportal hepatocytes are normal. Lymphatic vessels adjacent to the portal area are dilated. E-F (2 days): Sections of kidney at 100x and 400x showing the presence of variably sized, brightly

eosinophilic droplets within the lumen of renal tubules. Glomeruli are normal and there is no evidence of tubular degeneration. G (5 days): Section of ileum showing mild haemorrhage along the mucosal surface. H (2 days): Section of ileum showing haemorrhage within the lamina propria, with mild blunting and fusion of villi.