Contracture test and histologic and histochemical analyses of muscle biopsy specimens from horses with exertional rhabdomyolysis

Biopsy specimens of the cutaneous omobrachialis muscle were obtained from 10 horses with a problem of myositis from mild exercise. One horse had been evaluated previously and malignant hyperthermia-like contractures developed in its muscle biopsy specimen during the contracture test. In this study, the halothane-caffeine contracture test and histologic and histochemical evaluations were performed on muscle biopsy specimens. In the contracture test, no muscle biopsy specimen developed contracture in the presence of 2 or 4% halothane alone. The mean (+/- SEM) caffeine-specific concentration in the presence of halothane was 5.23 +/- 0.5 mM for 2% halothane, and 4.46 +/- 0.6 mM for 4% halothane. The caffeine-specific concentration values were not significantly different. Contracture response for any muscle specimen did not resemble contracture associated with malignant hyperthermia. The cutaneous omobrachialis muscle was composed of type-II fibers, with type-I fibers seldom seen. For 9 of the 10 horses, overall fiber morphology was normal; 1 horse had necrotic fibers. Of the 10 muscle specimens, 9 had fibers that had positive reaction for alkaline phosphatase activity; 3 muscle specimens contained ringed myofibers. Three horses of this study were administered general anesthesia; 2 were research horses, anesthetized with halothane and succinylcholine, and 1 was a clinical case given halothane anesthesia plus a non-depolarizing muscle relaxant. One research horse developed a malignant hyperthermia-like reaction to anesthesia, with severe rhabdomyolysis evident after anesthesia, and an episode of muscle cramping in its stall 2 days after anesthesia. The other 2 horses had unremarkable postanesthetic periods.     

Intramuscular Distribution of Fibre Types in the Gluteus Medius of the Horse: A Histochemical Analysis

The results from a recent study by Bruce and Schurg (1990) indicate that the gluteus medius (Gm) of the horse is a complex muscle subdivided by an internal tendinous sheet into two regions (dorsal and ventral) which may subserve different functions. In the present study, both regions were found to contain a similar proportion of histochemically-determined muscle fibre types. Fibre type composition did not generally vary over the length of the muscle at the same relative depth. In contrast, there was a significant tendency for the percentage of type I fibres to increase gradually in accordance with depth. These histochemical results do not suggest that the two regions of the Gm assume distinct functional roles; they do, however, indicate that at a given relative depth the muscle fibre type distribution is relatively uniform over the length of the muscle. Nevertheless, care should be exercised in sampling and interpreting data obtained from limited biopsy of equine Gm, due to the substantial histochemical variation of fibre type compositon as a function of sampling depth.     

Histochemical staining characteristics of normal horse skeletal muscle

The histologic and histochemical staining characteristics of the triceps brachii (long head), extensor carpi radialis, gluteus medius, vastus lateralis, biceps femoris, semimembranosus, semitendinosus, and extensor digitorum longus muscles of 8 Thoroughbreds, 2 Quarter Horses, 1 Arabian, 1 Paso Fino, and 1 Shetland Pony are described. Muscle fiber morphology, staining distribution and intensity, amount of IM connective tissue, number of IM blood vessels and IM nerves, calcium-activated adenosine triphosphatase activity (CaATPase), percentage of fibertype population, percentage of relative fibertype area, mean fiber diameter, nonspecific esterase activity, alkaline phosphatase activity, and acid phosphatase activity were evaluated, using 10 common histochemical and histologic stains. Two fiber types (I, II) and 3 subtypes (IIA, IIB, IIC) were observed, using CaATPase-, nicotinamide-adenine dinucleotide-tetrazolium reductase-, periodic acid-Schiff hematoxylin-, and nonspecific esterase-stained frozen serial muscle sections. Type I muscle fibers in general had low CaATPase activity, high oxidative capacity, low glycogen capacity, and low esterase activity. Type IIA muscle fibers had high CaATPase activity, intermediate oxidative capacity, high glycogen concentration, and high esterase activity. Type IIB fibers had high CaATPase activity, low oxidative capacity, high glycogen concentration, and a high esterase activity. Type IIC muscle fibers had high CaATPase activity, high oxidative capacity, variable glycogen concentration, and high esterase activity. Type II (IIA and IIB) muscle fibers predominated in the muscles. The percentage of muscle fiber population, mean minimal muscle fiber diameter, and percentage of relative muscle fiber area were determined for each sampled muscle. Type IIA and IIB muscle fibers predominated in the percentage of muscle fiber population and percentage of relative muscle fiber area. Type IIB muscle fibers had the greatest minimal fiber diameter, type IIA muscle fibers had intermediate minimal fiber diameter, and type I muscle fibers had the smallest minimal fiber diameter. The percentage of relative muscle fiber area was less variable (P less than or equal to 0.05) than the percentage of muscle fiber population. Mean muscle fiber diameter did not significantly differ between breeds. Alkaline and acid phosphatase activities were at low levels in all muscles biopsied and were limited to the IM connective tissue fibrocytes, macrophages, and capillaries.(ABSTRACT TRUNCATED AT 400 WORDS)     

Acute rhabdomyolysis ("tying-up") in standardbred horses. A morphological and biochemical study

LINDHOLM, A., H.-E. JOHANSSON & P. KJÆRSGAARD: Acute rhabdomyolysis (“tying-up”) in standardbred horses. A morphological and biochemical study. Acta vet. scand. 1974, 15, 325–339. — Morphological, biochemical and histochemical changes were studied in muscle needle biopsy specimens (gluteus medius) from 59 standardbred trotters with acute clinical symptoms of the “tying-up” disease. All horses had increased levels of serum enzymes SGOT and SCPK. The biopsy specimens were taken at various intervals after onset of clinical symptoms (1–4 hrs., 18–24 hrs. and 2–20 days). Ry light microscopy it was shown that the muscular alterations had a focal distribution and were of the hyalin degeneration type with insignificant inflammatory reaction and slight calcification. The ultrastructural changes apparently commenced with myofibrillar waving, mitochondrial and sarcotubular alterations and terminated with myofibrillar degeneration and necrosis with invasion of inflammatory cells. The inflammatory cells were ultrastructurally similar to monocytes and macrophages. The degenerative changes mainly comprised fast twitch (FT and FTH) fibres as histochemically evidenced by myofibrillar ATPase and alkaline phosphatase staining.Riopsies from diseased muscle 1–4 hrs. after the onset of “tyingup” contained a low muscle concentration of glycogen, ATP and CP and a high concentration of lactate and glucose. Hence it is suggested that the described muscular alterations may be caused by a deranged carbohydrate metabolism caused by a local hypoxia.It was found that the “tying-up” disease resembled idiopathic rhabdomyolysis in man and was thus designated “equine rhabdomyolysis”.histochemistry; horse; rhabdomyolysis; skeletal muscle; “tying-up”; ultrastructure.     

Myotonic dystrophy-like disease in a dog

A mature female Rhodesian Ridgeback was determined to have a progressive, degenerative myopathy associated with myotonia, dysphagia, and marked muscle wasting. Clinical findings revealed a diffuse muscular disease with percussion dimpling, dysphagia, and creatine kinase elevation. A paroxysmal atrial tachycardia was found. Electromyography revealed a diffuse myopathy with high-frequency bizarre waves, myotonic discharges especially in the masticatory, laryngeal, and pharyngeal muscles. A few positive sharp waves were found in some of the appendicular muscles. Histopathologic and histochemical stains on skeletal muscle biopsy specimens demonstrated moderate fiber-size variation, myofiber architectural changes, muscle-fiber splitting, focal necrosis and phagocytosis, high percentage of internal nuclei, and atrophy of type-2 muscle fibers. A review of myotonic myopathies in the dog is presented. The clinical, electrophysiologic, and histochemical findings are similar to those for myotonic muscular dystrophy in man.     

Neuromuscular pedicle graft for restoration of arytenoid abductor function in dogs with experimentally induced laryngeal hemiplegia

Left laryngeal hemiplegia was induced by resection of the left recurrent laryngeal nerve in 12 dogs. A neuromuscular pedicle graft formed from the first cervical nerve and sternothyroideus muscle was transplanted after 1 week to the denervated cricoarytenoideus dorsalis muscle in 8 dogs. The remaining 4 dogs served as controls. Left arytenoid abduction was blindly evaluated by laryngoscopy with video photography at time 0, at 1 week, and at 19 weeks in all dogs. At 19 weeks, biopsy specimens of the left cricoarytenoideus dorsalis muscle and the neuromuscular pedicle were taken from 4 of the treatment dogs, and biopsy specimens of the left cricoarytenoideus dorsalis muscle were taken from the 4 control dogs. All biopsy specimens were blindly evaluated by histologic and histochemical examination. At 36 to 44 weeks, the remaining 4 treatment dogs, from which biopsy specimens had not been taken, were reevaluated by use of laryngoscopy with video photography. Complications and difficulties encountered during surgery included hemorrhage in the area of the cricoarytenoideus dorsalis muscle, location of a branch of the first cervical nerve that was long enough to prevent tension at the graft site, orientation of the muscle pedicle in the cricoarytenoideus dorsalis muscle without the use of an operating microscope, and preservation of the terminal portion of the first cervical nerve while forming the neuromuscular pedicle. Results of the arytenoid movement evaluations revealed improvement in arytenoid abductor function in the treatment group, compared with that in the control group at 19 weeks. Arytenoid abduction in the treatment group at this time, however, was still significantly decreased (P less than 0.05), compared with presurgical movement evaluations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enzyme histochemistry on muscle biopsies as an aid in the diagnosis of diseases of the equine neuromuscular system: a study of six cases

Muscle biopsies from six horses with clinical histories of muscle atrophy, muscle tremors, myopathic symptoms, unsteadiness of pelvic limbs and progressive ataxia were examined. Muscle biopsies were studied with enzyme histochemical techniques to evaluate the diagnostic values of these methods in cases suspected of suffering from neuromuscular disorders. Hypertrophy, atrophy, fibre splitting, waxy degeneration, phagocytosis and necrosis were seen in haematoxylin eosin stained sections of the different cases. Fibre type predominance and fibre type grouping were seen in the calcium ion stimulated myosine ATP-ase (Ca-ATP-ase) stained sections of some cases. 'Moth-eaten fibres' were demonstrated in three cases by staining with NADH: nitro blue tetrazolium oxidoreductase (NADH-TR), succinate dehydrogenase (SDH), NADH dependent malate dehydrogenase, cytochrome c oxidase and by lactate dehydrogenase. The catabolic enzymes, acid phosphatase (ACP) and 5'-nucleotidase were active in cases with fibre phagocytosis. The oxidative part of the pentose phosphate pathway in myopathic tissue seemed to be important in three cases, demonstrated by the increased activity of glucose-6-phosphate dehydrogenase (GPDH) and 6-phosphogluconate dehydrogenase (PGDH). The important feature of diseased horse muscle was that the pathohistochemical changes were exactly the same as in diseased skeletal muscles of humans. The application of tissue saving enzyme histochemical techniques can be recommended in the study of muscle tissue from horses suffering from suspected neuromuscular disorders.     

Primary culture of fibroblasts and cementoblasts of the equine periodontium

Fibroblasts and cementoblasts in the periodontal ligament (PDL) of equine cheek teeth were harvested, and monocultures were obtained by means of a "selective detachment" procedure. Cells were characterized by morphological criteria and by immunostaining for vimentin, FVIII, pan-cytokeratin, smooth muscle actin, and pro-collagen. Cementogenic potential of the cells was determined by immunostaining for osteopontin and by histochemical detection of alkaline phosphatase. Equine periodontal fibroblasts (EPF) were spindle-shaped and polygonal. Equine dental cementoblasts (EDC) grew in cobblestone-like clusters. Both EPF and EDC stained positive for vimentin. Only EPF contained smooth muscle actin, pro-collagen, and alkaline phosphatase. Few EDC stained positive for osteopontin. The phenotypes of EPF and EDC and their specific expression of proteins corresponded to PDL fibroblasts and dental cementoblasts of other species. These results indicate the potential use of EPF and EDC in an adequate in vitro model of equine cementogenesis and equine periodontal remodeling.     

Electron microscopic and histochemical studies of the transverse striated musculature of birds after long-term hypokinesia]

Electromicroscopical and histochemical studies of the gastrocnemius muscle has been carried out in 4 months old cockerels of the laying hybrid after hypokinesis lasting 15 and 30 days. It was found that restricted movement resulted in dystrophic changes of myofibrils, enlargement of the sarcoplasmic reticulum and oedem of interfibrillar spaces. Histochemical studies revealed focuses of increased activity of non-specific esterase decreased activity of dehydrogenase of lactic acid and a positive reaction of acid phosphatase.     

Equine muscle fiber types: A histological and histochemical analysis of select thoroughbred yearlings

Equine skeletal muscle was histologically and histochemically analyzed in orderto measure fiber composition. Well-sampled open muscle biopsies were obtained from the semitendinosus muscle in select yearling Thoroughbreds. Each biopsy was sectioned and stained for the Harris Hematoxylin and Eosin and the modified Gomori Trichrome in order to demonstrate the basic muscle fiber morphology delineating intra and extracellular elements. A thirdstain, CA++ activated myosin Adenosine Triphosphatase at pH 9.4 was used to differentiate between specific types on the basis of enzyme activities. Subsequent differentiation of Type I slow twitch and Type II fast twitch muscle fibers was assessed on the basisof staining intensity. The high predominance of Type II fast twitch fibers ranged from 85.1 to 100 % with a mean of 90.5 %. Racing records, as two- and three-year-olds, were monitored in order to qualitatively compare histochemical results with performance achievements. While caution must be exercised, results of this investigation suggested that it may be possible, at an early age to partially predict the potential of each horse to adapt to training and to compete successfully in races of varying distances.     

Centronuclear myopathy in a Border collie dog

A two‐year old, male entire Border collie was presented with a one‐year history of exercise‐induced collapsing on the pelvic limbs. Physical examination revealed generalised muscle atrophy. Neurological examination supported a generalised neuromuscular disorder. Electromyography revealed spontaneous electrical activity in almost all muscles. Unfixed and formaldehyde‐fixed biopsy samples were collected from the triceps brachii, longissimus and vastus lateralis muscles. Histopathological, histochemical and ultrastructural examinations of biopsy specimens were consistent with either centronuclear or myotubular myopathy. The dog clinically improved with supportive treatment with L‐carnitine, co‐enzyme Q10 and vitamin B compound. To the authors’ knowledge, this is the first report of centronuclear/myotubular myopathy in a Border collie.     

Reduced Phagocytotic Activity of Macrophages in the Bovine Retained Placenta

This study was carried out to investigate the distribution of immune cells in the bovine placenta during the postpartum period and to compare these cells between normal and retained placenta. Within 1 h after normal calving, biopsy samples of placentomes were collected from 10 cows. The occurrence of retention of fetal membranes was monitored for more than 8 h post-calving, and the samples obtained were divided into two groups: normally discharged and retained placenta (n = 5 each). Immunohistochemical procedures were utilized to detect macrophages and T lymphocytes. Numerous CD14-positive macrophages were found in the stroma of both normal placenta and retained placenta whereas only a few CD3-positive T lymphocytes were found in both cases. However, histochemical staining for acid phosphatase, a predominant lysozomal enzyme, revealed that almost all macrophages showed strong enzyme activity in the normally discharged placentas, whereas in retained placenta the activity of acid phosphatase was conspicuously decreased in intensity. These results indicate that there are functional differences in placental macrophages between normal and retained placenta.     

Mucometra with Persistent Corpus Luteum in Goats

Ovaries of two goats with persistent corpus luteum were studied by histochemical methods. Lutein cells showed moderate alkaline phosphatase and acid phosphatase activity associated with strong delta 5-3β-hydroxysteroid dehydrogenase and succinic tetrazolium reductase activity. The results are interpreted as an indication that the corpus luteum was functioning.     

Effects of a submaximal treadmill training programme on histochemical properties, enzyme activities and glycogen utilisation of skeletal muscle in the horse

The effects of training on skeletal muscle composition were studied in four Standardbred geldings given a seven week submaximal treadmill training programme. Before the start of training, muscle biopsies were collected from the left middle gluteal muscle for the determination of muscle fibre types, oxidative capacity and capillary numbers using histochemical techniques. The concentrations of citrate synthase, 3-hydroxyacyl-CoA dehydrogenase (HAD), lactate dehydrogenase and total muscle glycogen were measured using fluorometric methods. Muscle biopsy samples were repeated after one, three, five and seven weeks of training and the same measurements performed. No significant changes were found in muscle fibre types or capillary numbers as a result of training, although there was an increase in the oxidative capacity of the Type IIB fibres after seven weeks training. After seven weeks of training there were also increases in the concentrations of citrate synthase and HAD and a decrease in lactate dehydrogenase. Before commencement of training the horses underwent a standardised submaximal exercise test and muscle glycogen concentrations were measured before and immediately after the exercise. This procedure was repeated after one, three, five and seven weeks of training. A progressive decrease in the rate of glycogen utilisation occurred throughout the period of training, which by seven weeks was 36 per cent lower than that before training.     

A comparative enzyme histochemical and histological study of the effect of ischaemia and post mortem change on rat kidneys.

A histochemical study of the effect of ischaemia on rat kidneys showed that changes were demonstrable in adenosine triphosphatase, alkaline phosphatase and succinic dehydrogenase within 2 h. Further changes occurred with increasing time. The activity of acid phosphatase was little affected up to 24 h although at this time there was marked tubular disruption. Paraffin embedded H and E sections also showed marked changes within 2 h. Enzyme histochemical and histological changes in kidneys taken at varying periods after the death of the animal showed very similar changes to those in ischaemic kidneys. Differences were mainly in the rate and extent of the changes.     

Preliminary Study on Age- and Sex-Dependent Alterations in the Composition of Skeletal Muscle Fibers of Brasileiro de Hipismo Horses

The aim of this work was to characterize the distribution of myofibers in the gluteus medius muscle of inactive horses of the Brasileiro de Hipismo (BH) breed at different ages by means of histochemical analyses, according to sex and depth of the biopsy. A total of 78 inactive horses (9 castrated males, 35 stallions, and 34 females) of the BH breed, aged 1 to 4 years, were used. A percutaneous muscle biopsy was obtained with a 6.0-mm Bergström-type needle, which allowed the removal of muscle fragments at depths of 20 and 60 mm. Myofiber types were determined based on myofibrillar adenosine triphosphatase (mATPase) and nicotinamide dinucleotide tetrazolium reductase (NADH-TR) techniques. Morphometry of the fibers was determined based on cross-sectional area (CSA), mean frequency (F), and relative cross-sectional area (RCSA). The current study demonstrated that BH horses 3 and 4 years of age show a greater percentage of, and area occupied by, type IIA fibers and lower percentage of type IIX fibers in the gluteus medius muscle compared with horses 1 and 2 years of age. No difference was found between sexes in the frequency of and area occupied by the different fiber types at any of the depths and ages studied. In this study, females showed a greater CSA for all fibers in comparison with males, at 1 year of age. The results of the current study indicate that the gluteus medius muscle of inactive BH horses shows modifications in its structural and biochemical composition during the growth of the animals, leading to a better oxidative capacity.     

Experimental studies on the ketogenic effect of high doses of proteins in dairy cows]

A 14-month feeding experiment was conducted with 16 high-yielding cows, each given a daily supplement of 2 kg soya meal regardless of milk yield or the stage of lactation, in comparison with 8 normally-fed cows. At monthly intervals various parameters of carbohydrate, fat and protein metabolism and of liver function were determined. At the end of the trial, samples of liver were obtained by biopsy for morphological and histochemical examination. Supplementation had a pronounced effect on milk yield and fat content, averaging over 900 kg of milk and 0.2% fat content more than during the preceding year. The parameters of metabolism revealed clear signs of subclinical ketosis, particularly during the first third of the trial period. Changes were greatest during the 3rd and 4th months of the trial, when the protein content of the ration was particularly high (over 3 kg) and when the energy supply ratio was particularly unfavourable (PEQ 300). At the end of the trial some abnormalities were detected in individual animals in the biochemical properties of blood and urine, and the morphological, histochemical and ultrastructural appearance of the liver. These changes included increases in KK, BTS and bilirubin concentrations in the blood; ketonuria, proteinuria; perilobular fat infiltration with glycogen depletion; changes in succinate dehydrogenase and acid phosphatase activities. In most animals the liver seemed to have become adapted to a high protein intake.     

Nemaline Rods in Canine Myopathies: 4 Case Reports and Literature Review

The diagnosis of nemaline rod myopathy (NM) is based on the presence of numerous pathognomonic rods within a fresh frozen muscle biopsy specimen. Three forms of congenital NM have been described in humans, and rods have been found to occur in various other conditions. A similar myopathy was described in 1986 in a family of cats. In this report, we describe a case of congenital NM in a 10-month-old Border Collie, an adult-onset NM in an 11-year-old Schipperke, and 2 acquired myopathies with nemaline rods in adult dogs associated with hypothyroidism and Cushing's syndrome. Common clinical features included exercise intolerance, abnormal electromyography, and the presence of nemaline rods in fresh, frozen, and glutaraldehyde-fixed biopsies from proximal appendicular limb muscles. Staining of cryostat sections of muscle biopsy specimens by the modified Gomori trichrome technique disclosed numerous rod bodies that were localized to type 1 fibers by the histochemical adenosine triphosphatase reaction. Accumulation of rods also was demonstrated by electron microscopy in 2 of the cases with localized enlargement and streaming of Z lines. Documentation of NM in a young Border Collie and the adult-onset form in the Schipperke alerts clinicians to the existence of this disorder in these breeds.     

Histochemical differentiation of Dirofilaria immitis, Dirofilaria repens and Acanthocheilonema dracunculoides microfilariae by staining with a commercial kit, Leucognost-SP.

The diagnosis of canine heartworm infection is based upon the presence of circulating Dirofilaria immitis microfilariae or on techniques for the detection of serum antibodies or antigens. In the first of these, discrimination between D. immitis, D. repens and Acanthocheilonema dracunculoides microfilariae is based upon the acid phosphatase histochemical stain. In this paper, we propose an alternative technique for histochemical staining using a commercial kit test of naphthol-AS-OL (Leucognost-SP). This offers the advantages of speed and simplicity as compared to the standard Barka procedure.