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Background Horses are affected by various peripheral nerve disorders but defining their aetiology and pathophysiology is hampered by limited understanding of associated morphological and pathological changes and involvement of specific axonal types. Objectives To investigate the hypothesis that selected antibody markers, used in conjunction with various tissue processing methods, would enable identification of axons with different functional modalities within a range of equine peripheral nerves. Study design Optimisation and validation study METHODS: A range of antibodies were evaluated immunohistochemically via fluorescence confocal microscopy in cadaver equine nerve samples of primary motor, mixed or primary sensory functions (recurrent laryngeal, phrenic, plantar digital) within formalin-fixed paraffin-embedded (FFPE) and formalin-fixed frozen (FFF) tissues subjected to different antigen retrieval protocols. Results Immunohistochemistry of FFPE-derived nerve samples with selected antibodies and specific antigen retrieval methods, enabled identification of myelinated and unmyelinated axons, cholinergic, sympathetic and peptidergic axons. The recurrent laryngeal and phrenic nerves are composed of myelinated cholinergic (motor), myelinated sensory fibres, unmyelinated adrenergic (sympathetic) axons and unmyelinated peptidergic (sensory) axons. In contrast, as expected, the plantar digital nerve had no myelinated motor fibres being mainly composed of myelinated sensory fibres, unmyelinated sympathetic and unmyelinated peptidergic sensory axons. Main limitation Attempts specifically to label parasympathetic fibres were unsuccessful in any nerve examined in both FFPE and FFF tissues. Conclusions A panel of antibody markers can be used to reveal morphological and functional properties of equine nerves. Future work should enable better characterisation of morphological changes in equine neuropathies at various stages of disease development.

INTRODUCTION Equine recurrent laryngeal neuropathy (RLN) is a naturally occurring model of length-dependent axonopathy characterized by asymmetrical degeneration of recurrent laryngeal nerve axons (RLn). Distal RLn degeneration is marked, but it is unclear whether degeneration extends to include cell bodies (consistent with a neuronopathy). METHODS With examiners blinded to RLN severity, brainstem location, and side, we examined correlations between RLN severity (assessed using left distal RLn myelinated axon count) and histopathological features (including chromatolysis and glial responses) in the nucleus ambiguus cell bodies, and myelinated axon count of the right distal RLn of 16 horses. RESULTS RLN severity was not associated with RLn cell body number (P > .05), or degeneration. A positive correlation between the left and right distal RLn myelinated axon counts was identified (R2 = 0.57, P

Horse racing is a popular and financially important industry worldwide and researchers and horse owners are interested in genetic and training influences that maximise athletic performance. An association has been found between the presence of a short interspersed nuclear element (SINE) mutation in the myostatin (MSTN) gene promoter and optimal race distance in Thoroughbred horses. There is previous laboratory evidence that this mutation reduces MSTN expression in a cell culture model and influences skeletal muscle fibre type proportions in horses. Manipulating MSTN expression has been proposed for illicit gene doping in human and equine athletes and already, researchers have generated homozygous and heterozygous MSTN-null horse embryos following CRISPR/Cas9 editing at the equine MSTN locus and nuclear transfer, aiming artificially to enhance performance. To date however, the role of the naturally-occurring equine MSTN SINE mutation in vivo has remained unclear; here we hypothesised that it reduces, but does not ablate circulating myostatin expression. Following validation of an ELISA for detection of myostatin in equine serum and using residual whole blood and serum samples from 176 Thoroughbred racehorses under identical management, horses were genotyped for the SINE mutation by PCR and their serum myostatin concentrations measured. In our population, the proportions of SINE homozygotes, heterozygotes and normal horses were 27%, 46% and 27% respectively. Results indicated that horses that are homozygous for the SINE mutation have detectable, but significantly lower (pMSTN gene associated with exercise performance. Determining the reason for variation in expression of myostatin within SINE-genotyped groups might identify additional performance-associated environmental or genetic influences in Thoroughbreds. Understanding the mechanism by which altered myostatin expression influences skeletal muscle fibre type remains to be determined.

Highlights • Normalized muscle volumes distinguish between wild type and DE50-MD dogs. • Global muscle T2 signal intensities discriminate between wild type and DE50-MD dogs. • Musculoskeletal changes detected by MRI reflect those seen in human DMD patients. • Musculoskeletal MRI in this model will be useful to assess treatment efficacy., The DE50-MD canine model of Duchenne muscular dystrophy (DMD) has a dystrophin gene splice site mutation causing deletion of exon 50, an out-of-frame transcript and absence of dystrophin expression in striated muscles. We hypothesized that the musculoskeletal phenotype of DE50-MD dogs could be detected using Magnetic Resonance Imaging (MRI), that it would progress with age and that it would reflect those in other canine models and DMD patients. 15 DE50-MD and 10 age-matched littermate wild type (WT) male dogs underwent MRI every 3 months from 3 to 18 months of age. Normalized muscle volumes, global muscle T2 and ratio of post- to pre-gadolinium T1-weighted SI were evaluated in 7 pelvic limb and 4 lumbar muscles bilaterally. DE50-MD dogs, compared to WT, had smaller volumes in all muscles, except the cranial sartorius; global muscle T2 was significantly higher in DE50-MD dogs compared to WT. Muscle volumes plateaued and global muscle T2 decreased with age. Normalized muscle volumes and global muscle T2 revealed significant differences between groups longitudinally and should be useful to determine efficacy of therapeutics in this model with suitable power and low sample sizes. Musculoskeletal changes reflect those of DMD patients and other dog models.

Gene editing and muscular dystrophy Duchenne muscular dystrophy (DMD) is characterized by progressive muscle weakness and a shortened life span. The disease is caused by mutations that reduce or prevent expression of dystrophin, an essential structural protein in skeletal and heart muscle. The gene editing technology CRISPR-Cas9 can correct disease-causing mutations and has yielded promising results in mouse models of DMD. In a small, short-term study, Amoasii et al. tested this strategy in a dog model of DMD that exhibits many features of the human disease. Intramuscular or systemic delivery of the gene editing components resulted in a substantial increase in dystrophin protein levels in skeletal and heart muscle. Restoration of dystrophin expression was accompanied by improved muscle histology. Science , this issue p. 86

SummaryBackground Agreement amongst experienced clinicians is poor when assessing presence and severity of ataxia, especially when signs are mild. Consequently, objective gait measurements might be beneficial for assessment of horses with neurological diseases. Objectives To assess diagnostic criteria using motion capture to measure variability of spatial gait-characteristics and swing duration derived from ataxic and non-ataxic horses and to assess if variability increases with blindfolding. Study design Cross-sectional. Methods Twenty-one horses underwent measurements in a gait laboratory and live neurological grading by multiple raters. In the gait laboratory the horses were walked across a runway surrounded by a 12-camera motion capture system with a sample frequency of 240 Hz. They were walked normally and with a blindfold in at least 3 trials each. Displacements of reflective markers on head, fetlock, hoof, 4th lumbar vertebra, tuber coxae and sacrum derived from 3-4 consecutive strides were processed and descriptive statistics, receiver operator characteristics (ROC) to determine the diagnostic sensitivity, specificity and area under the curve (AUC) and correlation between median ataxia grade and gait parameters were determined. Results For horses with a median ataxia grade ≥2, Coefficient of Variation for the location of maximum vertical displacement of pelvic and thoracic distal limbs generated good diagnostic yield. The hoofs of the thoracic limbs yielded an AUC of 0.81 with 64% sensitivity and 90% specificity. Blindfolding exacerbated the variation for ataxic horses compared to non-ataxic horses with the hoof marker having an AUC of 0.89 with 82% sensitivity and 90% specificity. Main limitations The low number of consecutive strides per horse obtained with motion capture could decrease diagnostic utility. Conclusions Motion capture can objectively aid the assessment of horses with ataxia. Furthermore blindfolding increases variation of distal pelvic limb kinematics making it a useful clinical tool. This article is protected by copyright. All rights reserved.

Mutations in hydroxyacyl-coA dehydratase 1 (HACD1/PTPLA) cause recessive congenital myopathies in humans and dogs. Hydroxyacyl-coA dehydratases are required for elongation of very long chain fatty acids and HACD1 has a role in early myogenesis but the functions of this striated muscle-specific enzyme in more differentiated skeletal muscle remain unknown. Canine HACD1-deficiency is histopathologically classified as a centronuclear myopathy (CNM): we investigated the hypothesis that muscle from HACD1-deficient dogs has membrane abnormalities in common with CNMs with different genetic causes. We demonstrate progressive changes in tubuloreticular and sarcolemmal membranes and mislocalized triads and mitochondria in skeletal muscle from animals deficient in HACD1. Further, comparable membranous abnormalities in cultured HACD1-deficient myotubes provide additional evidence that these defects are a primary consequence of altered HACD1 expression. Our novel findings, including t-tubule dilatation and disorganization, associated with defects in this additional CNM-associated gene provide a definitive pathophysiological link with these disorders, confirm that dogs deficient in HACD1 are relevant models and strengthen the evidence for a unifying pathogenesis in CNMs via defective membrane trafficking and excitation-contraction coupling in muscle. These results build on previous work by determining further functional roles of HACD1 in muscle and provide new insight into the pathology and pathogenetic mechanisms of HACD1-CNM. Consequently, alterations in membrane properties associated with HACD1 mutations should be investigated in humans with related phenotypes.

Summary Although horses are affected by cranial nerve disease, our understanding of these structures' imaging anatomy is limited, and the optimal modality for imaging of each of these nerves is unclear. The aim of this study was to describe the imaging appearance of the equine cranial nerves on high-resolution 1.5T magnetic resonance imaging (MRI) and computed tomography (CT) scans of a cadaver head, and with these as standards, examine the utility of MRI and CT performed in clinical cases. High-resolution MRI and CT images were prospectively acquired of the head of a normal Thoroughbred gelding following euthanasia. Ten clinical cases undergoing high-field MRI under general anaesthesia and 10 clinical cases undergoing CT in the standing horse under sedation were retrospectively evaluated by three reviewers to assess cranial nerve visibility. On high-resolution, thin-slice, MRI scans of the normal cadaver head, each of the 12 cranial nerves and their topographic location could be appreciated. On high-resolution cadaver CT, cranial nerves II, V and VII were clearly visible, but others were less easily identified; osseous structures were clearly visualised. Clinical MRI and CT allowed for variable visualisation of the cranial nerves, dependent on the sequence and the orientation of scan planes. High-field MRI allowed excellent visualisation of equine cranial nerves, whereas CT allowed for more detailed visualisation of the osseous canals and foramina. In live horses, the ability to identify all 12 nerves is challenging with either MRI or CT; however, high-field MRI enables better visualisation of the nerve bundles than CT.

Introduction A unilateral neurectomy model was used to study the relationship between histologic and ultrasonographic tissue characteristics during muscle atrophy over time. Methods This investigation was an in vivo experimental study in an equine model (n = 28). Mean pixel intensity of ultrasonographic images was measured, a muscle appearance grade was assigned weekly, and muscles were harvested from 4 to 32 weeks. Minimum fiber diameter, fiber density per unit area, percent collagen, percent fat, and fiber type profile were measured from muscle cryosections and correlated with the ultrasonographic parameters. Results A significant relationship was identified between collagen content, minimum fiber diameter, and ultrasonographic muscle appearance by as early as 8 weeks. There was no apparent association between fat content of muscle and the ultrasonographic appearance of atrophy before 28 weeks. Conclusions Early muscle atrophy before fatty infiltration is detectable with ultrasound. The effect of muscle collagen content on echointensity may be mediated by reduced fiber diameter.

Recurrent Laryngeal Neuropathy (RLN) is a highly prevalent and predominantly left-sided, degenerative disorder of the recurrent laryngeal nerves (RLn) of tall horses, that causes inspiratory stridor at exercise because of intrinsic laryngeal muscle paresis. The associated laryngeal dysfunction and exercise intolerance in athletic horses commonly leads to surgical intervention, retirement or euthanasia with associated financial and welfare implications. Despite speculation, there is a lack of consensus and conflicting evidence supporting the primary classification of RLN, as either a distal ("dying back") axonopathy or as a primary myelinopathy and as either a (bilateral) mononeuropathy or a polyneuropathy; this uncertainty hinders etiological and pathophysiological research. In this review, we discuss the neuropathological changes and electrophysiological deficits reported in the RLn of affected horses, and the evidence for correct classification of the disorder. In so doing, we summarize and reveal the limitations of much historical research on RLN and propose future directions that might best help identify the etiology and pathophysiology of this enigmatic disorder.

The veterinary profession needs to become more successful in producing the next generation of clinician scientists, say Richard Mellanby and others, who set out a roadmap for future academic postgraduate clinical training.

Summary Reasons for performing study The pathogenesis of bilateral dynamic laryngeal collapse associated with poll flexion (DLC) of horses is unknown but might be associated with intrinsic laryngeal muscle weakness. Objectives To investigate histopathological characteristics of the cricoarytenoideus dorsalis, the cricothyroid (CT) and the cricoarytenoideus lateralis muscles in DLC-affected horses and compare these with unaffected controls. Our hypotheses were that evidence of neurogenic atrophy of the CT or cricoarytenoideus dorsalis muscles would be found in DLC-affected horses and that observed changes would be symmetrically (left/right) distributed, or that muscle fibre diameter would be significantly reduced in DLC-affected horses compared to unaffected controls, reflecting an underlying paresis. Study design Case–control study. Methods Five DLC horses and 8 controls were included. Muscle samples were harvested immediately following euthanasia. Fibre type proportions and size were evaluated by multiple immunofluorescence labelling of cryosections, and compared between sides (left/right) and groups (DLC-affected cases/ unaffected controls). Subjective and objective assessments of fibre type grouping were compared between sides and groups. Results Fibre type proportions, fibre size and the subjective assessment of fibre type grouping did not reveal any statistically significant differences between the groups. Objective assessment of fibre type grouping revealed significantly more large clusters of T1 fibres within the left cricoarytenoideus lateralis muscle of DLC-affected cases versus controls, and within the right CT muscle of control horses compared to the DLC-cases. Conclusions The absence of bilateral symmetric fibre type grouping, fibre type loss and fibre atrophy in the DLC-affected cases do not support a neuromuscular component within the pathogenesis of DLC. The objective assessment of fibre type grouping revealed some statistical differences between the DLC-affected cases and the unaffected controls; however, these findings were inconsistent with regard to DLC. An alternative aetiology of DLC seems likely.

Histopathological interpretation of semimembranosus muscle samples from an adult Warmblood mare with clinical signs suggestive of exertional rhabdomyolysis and intermittent mild elevations in muscle enzyme activities revealed abundant sarcoplasmic vacuoles in all fibre-types containing fine, apparently proteinaceous debris. Vacuolar contents stained lightly with PAS, but did not appear to contain amylopectate, lipid or acid phosphatase and their periphery was unstained with dystrophin immunohistochemistry. Electron microscopy revealed that vacuoles were not membrane bound. No vacuoles were detected in muscle samples evaluated at post mortem following 4 months of rest. To our knowledge, this is the first report of a presumed primary vacuolar myopathy in a horse.

SummaryBackground Equine atypical myopathy (AM) is a toxic rhabdomyolysis associated with ingestion of hypoglycin A, derived typically in Europe, from Acer pseudoplatanus tree. Despite the wide distribution of this tree species in the UK, the number of cases reported annually varies, and there has been an apparent increase in prevalence in recent years. Although AM was first recognised in the UK, epidemiological studies have never been conducted focused solely on this country. Objectives To describe the spatiotemporal distribution, presentation, treatment and outcome of AM cases reported in the UK. Study design Retrospective case series. Methods British AM cases reported to the atypical myopathy alert website, between 2011 and 2015 were included (n = 224). Data were obtained via standardised epidemiological questionnaires from owners and veterinarians. Factors associated with survival were assessed using logistic regression. Results Most cases reported were from England (87.9%). Survival was 38.6% (n = 73/189). Clinical factors associated with reduced odds of survival included, hypothermia (odds ratio (OR) 0.18; CI 0.06-0.57; p = 0.01), bladder distension (OR 0.11; CI 0.02-0.59; p = 0.01), tachycardia (OR 0.97; CI 0.94-0.99; p = 0.04) and serum creatine kinase activity >100,000 IU/L (OR 0.17; CI 0.04-0.68; p = 0.01) in the univariable analysis as well as recumbency. The latter was the only sign retained in multivariable analysis (OR = 0.19; CI 0.06-0.62; p = 0.006). Administration of vitamins during the disease was associated with survival (OR 3.75; CI 1.21-11.57; p = 0.02). Main limitations Reporting cases to the atypical myopathy alert group is voluntary; therefore, under-reporting will result in underestimation of AM cases; furthermore, direct owner-reporting could have introduced misdiagnosis bias. Conclusion Some areas of the UK reported AM cases more commonly. Clinical signs such as recumbency, rectal temperature, distended bladder and serum CK activity might be useful prognostic indicators though should be considered in the context of the clinical picture. Treatment with vitamins increases survival. This article is protected by copyright. All rights reserved.

Background Type 1 polysaccharide storage myopathy (PSSM1), an equine glycogen storage disorder caused by a gain of function mutation (R309H) in the gene encoding glycogen synthase (GYS1), is associated with the accumulation of amylase-resistant alpha-crystalline polysaccharide inclusions within skeletal muscle. Several glycogenoses in humans have a cardiac phenotype, and reports exist of horses with PSSM and polysaccharide inclusions in cardiac muscle. Hypothesis/Objectives To investigate the hypothesis that horses with PSSM1 display a cardiac phenotype. Our objectives were to compare plasma cardiac troponin I (cTnI) concentration and the incidence of cardiac arrhythmias in PSSM1 homozygotes, heterozygotes, and control horses. Methods One hundred and twenty-five Belgian and Percheron horses under the same management were genotyped for the R309H GYS1 mutation. From these, 8 age-, breed-, and sex-matched cohorts of each genotype were identified. Plasma cTnI concentration and incidence of cardiac arrhythmias (determined by 24-hour Holter ECG) were compared between the groups. Results Although some PSSM1-affected horses had mildly increased plasma cTnI concentrations, there was no significant difference in cTnI concentrations between groups. There were no significant differences in the incidence of ectopic beats, cardiac conduction intervals or mean heart rate between groups. Conclusions and clinical importance We found no evidence of clinically relevant cardiac myocyte injury or arrhythmias in horses with PSSM1. Additional study is required to determine whether myocardial function may be compromised in this disorder.

Summary Reasons for performing study: Improved understanding of the epidemiology of atypical myopathy (AM) will help to define the environmental factors that permit or support the causal agent(s) to exert toxicity. Objectives: This European survey of AM aimed to describe spatiotemporal distribution, survival, clinical signs, circumstances in which AM develops and its different expressions between countries and over time. Methods: The spatiotemporal distribution, history and clinical features of AM cases reported to the Atypical Myopathy Alert Group from 2006 to 2009 were described. Comparisons of data from the most severely affected countries and from the large outbreaks were made with Fisher's exact and Welch's tests with Bonferroni correction. Results: Of 600 suspected cases, 354 met the diagnostic criteria for confirmed or highly probable AM. The largest outbreaks occurred during the autumns of 2006 and 2009 in Belgium, France and Germany. For the first time, donkeys, zebras and old horses were affected, and clinical signs such as gastrointestinal impaction, diarrhoea, penile prolapse, buccal ulceration and renal dysfunction were observed. Affected horses spent >6 h/day on pastures that almost always contained or were surrounded by trees. The latency period was estimated at up to 4 days. Overall survival rate was 26%. Although differences between countries in affected breeds, body condition, horse management and pasture characteristics were recognised, the common presenting clinical signs and mortality were similar between countries. Conclusions and potential relevance: This study describes new data on case details, history and clinical course of AM that is of preventive, diagnostic and therapeutic value. However, the true impact of the findings of this study on the development of or severity of AM should be tested with case–control studies.

Reasons for performing study: Appropriate management of atypical myopathy (AM) requires the establishment of an accurate diagnosis and prognosis. Furthermore, preventive measures to avoid AM need to be refined. Objectives: The aims of the study were as follows: 1) to improve the diagnosis of AM; 2) to identify prognostic predictors; and 3) to refine recommended preventive measures based on indicators of risk factors. Methods: An exploratory analysis of cases in Europe between 2006 and 2009 reported to the Atypical Myopathy Alert Group was conducted. Based on clinical data, reported cases were allocated into 2 groups: confirmed or highly probable AM (AM group; further divided into survivors and nonsurvivors); and cases with a low probability of having AM or with another final diagnosis (non-AM group). Using Welch's test and odds ratios corrected for multiple comparisons, the AM vs. non-AM groups were compared to identify indicators for diagnosis and risk factors, and survivors vs. nonsurvivors in the AM group were compared to identify prognostic factors. Sensitivity, specificity and positive and negative predictive values were calculated for specific clinical signs related to final diagnosis and outcome. Results: From 600 reported cases, 354 AM cases (survival rate of 26%) and 69 non-AM cases were identified, while there were insufficient data to categorise the remainder. Variables valuable for diagnosing AM compared with similar diseases were as follows: presence of dead leaves and wood and/or trees on pastures; sloping pastures; full-time pasture access; no food supplementation; normal body condition; pigmenturia; normothermia; and congested mucous membranes. Nonsurvival was associated with recumbency, sweating, anorexia, dyspnoea, tachypnoea and/or tachycardia. Survival was associated with remaining standing most of the time, normothermia, normal mucous membranes, defaecation and vitamin and antioxidant therapy. Conclusions and potential relevance: This study refines the list of risk factors for AM. Clinical signs valuable for diagnosis and prognosis have been identified, enabling clinicians to improve management of AM cases.

The aim of this study was to describe and report the prevalence of conditions found at necropsy examination of UK donkeys. Records from 1,444 donkeys over a 7-year period were included in the analysis. Sixty-one categories of post-mortem finding were identified from 9,744 observations. The four most prevalent conditions noted were dental disorder (78.7%), vascular disease other than aneurysm (60.9%), arthritis (55.4%) and foot disorder (44.8%). Gastric ulceration was found in 42% of the donkeys and gastrointestinal impaction in 18.6%. The most frequent combination of two post-mortem findings in the same animal was arthritis and dental disorder. The most common disorders were associated with age, body weight and/or body condition post mortem and, for some disorders, gender. For many of the post-mortem findings, crude associations were found between the presence of one finding and the odds of also having certain other post-mortem findings. This study is the first to summarize all conditions noted at necropsy examination for a large group of donkeys. The findings increase knowledge of diseases and conditions of this species and may be useful when investigating the relevance of various pathological conditions in the live animal.

Summary Hydranencephaly is well recognised in several domestic animals, especially ruminants, but is virtually unknown in the horse. This case report describes a premature filly foal that on the day of delivery was found with a severe neurological disorder that initially improved but then progressively worsened. The foal was subjected to euthanasia on humane grounds and post mortem examination revealed somewhat asymmetric but bilateral destruction of the telencephalon identified as hydranencephaly. The possible causes of hydranencephaly in foals are discussed.

Summary Reasons for performing study: The aetiology of temporohyoid osteoarthropathy (THO) is unknown; both primary infectious and degenerative causes have been suggested. Hypothesis: There is a significant association between increasing age and severity of temporohyoid joint degeneration. To examine the histopathology of the temporohyoid articulation in aged horses and to compare the appearance of the joint with computed tomography (CT) and peripheral quantitative CT (pQCT). Methods: pQCT scans of the temporohyoid articulations were obtained bilaterally from 31 horses (range age 1–44 years) post mortem and images were graded by 2 blinded observers on 2 occasions for the presence of osteophytes, irregularity of the joint surface and mineralisation. Eight heads had been examined previously by CT, with the images similarly graded for the shape and density of the proximal stylohyoid bones, bone proliferation surrounding the joint, mineralisation of the tympanohyoid cartilage and the relationship of the petrous temporal bone to the stylohyoid bone. Sixteen temporohyoid joints were then evaluated histologically. Results: There was significant association between the mean pQCT degeneration score and age (rho = 0.75; P

EDMD (Emery–Dreifuss muscular dystrophy) is caused by mutations in either the gene encoding for lamin A/C (LMNA) located at 1q21.2–q21.3 or emerin (EMD) located at Xq28. Autosomal dominant EDMD caused by LMNA mutations is more common than the X-linked form and often more severe, with an earlier onset. At the histological and histochemical levels, both X-linked and autosomal dominant EDMD appear similar. However, individuals with the same genetic disorder often show remarkable differences in clinical severity, a finding generally attributed to the genetic background. The clinical and pathological findings in EDMD patients found to have mutations in more than one gene are also discussed. There is now much interest in the phenotype of several animal models for EDMD which should lead to an increased insight into the pathogenesis of this disorder, particularly that relating to the heart phenotype.

Mutations in fukutin related protein (FKRP) are responsible for a common group of muscular dystrophies ranging from adult onset limb girdle muscular dystrophies to severe congenital forms with associated structural brain involvement, including Muscle Eye Brain disease. A common feature of these disorders is the variable reduction in the glycosylation of skeletal muscle alpha-dystroglycan. In order to gain insight into the pathogenesis and clinical variability, we have generated two lines of mice, the first containing a missense mutation and a neomycin cassette, FKRP-Neo(Tyr307Asn) and the second containing the FKRP(Tyr307Asn) mutation alone. We have previously associated this missense mutation with a severe muscle-eye-brain phenotype in several families. Homozygote Fkrp-Neo(Tyr307Asn) mice die soon after birth and show a reduction in the laminin-binding epitope of alpha-dystroglycan in muscle, eye and brain, and have reduced levels of FKRP transcript. Homozygous Fkrp(Tyr307Asn) mice showed no discernible phenotype up to 6 months of age, contrary to the severe clinical course observed in patients with the same mutation. These results suggest the generation of a mouse model for FKRP related muscular dystrophy requires a knock-down rather than a knock-in strategy in order to give rise to a disease phenotype.

Objective—To determine the effects of racing and nontraining on plasma thyroxine (T4), free thyroxine (fT4), thyroid-stimulating hormone (TSH), and thyroglobulin autoantibody (TgAA) concentrations in sled dogs and compare results with reference ranges established for dogs of other breeds. Design—Cross-sectional study. Animals—122 sled dogs. Procedure—Plasma thyroid hormone concentrations were measured before dogs began and after they finished or were removed from the Iditarod Trail Sled Dog Race in Alaska and approximately 3 months after the race. Results—Concentrations of T4 and fT4 before the race were less than the reference range for nonsled dogs in 26% and 18% of sled dogs, respectively. Immediately after racing, 92% of sled dogs had plasma T4 concentrations less than the reference range. Three months after the race, 25% of sled dogs had plasma T4 concentrations less than the reference range. For T4, fT4, TSH, and TgAA, significant differences were not detected in samples collected before the race versus 3 months later. Conclusions and Clinical Relevance—Plasma T4, fT4, and TSH concentrations decreased in dogs that complete a long distance sled dog race. Many clinically normal sled dogs have plasma T4 and fT4 values that are lower than the reference range for nonsled dogs. We suggest that the reference ranges for sled dogs are 5.3 to 40.3 nmol/L and 3.0 to 24.0 pmol/L for plasma T4 and fT4 concentrations, respectively, and 8.0 to 37.0 mU/L for TSH. (J Am Vet Med Assoc 2004;224:226–231)

Dysfunctional skeletal muscle calcium homeostasis plays a central role in the pathophysiology of several human and animal skeletal muscle disorders, in particular, genetic disorders associated with ryanodine receptor 1 (RYR1) mutations, such as malignant hyperthermia, central core disease, multiminicore disease and certain centronuclear myopathies. In addition, aberrant skeletal muscle calcium handling is believed to play a pivotal role in the highly prevalent disorder of Thoroughbred racehorses, known as Recurrent Exertional Rhabdomyolysis. Traditionally, such defects were studied in human and equine subjects by examining the contractile responses of biopsied muscle strips exposed to caffeine, a potent RYR1 agonist. However, this test is not widely available and, due to its invasive nature, is potentially less suitable for valuable animals in training or in the human paediatric setting. Furthermore, increasingly, RYR1 gene polymorphisms (of unknown pathogenicity and significance) are being identified through next generation sequencing projects. Consequently, we have investigated a less invasive test that can be used to study calcium homeostasis in cultured, skin-derived fibroblasts that are converted to the muscle lineage by viral transduction with a MyoD (myogenic differentiation 1) transgene. Similar models have been utilised to examine calcium homeostasis in human patient cells, however, to date, there has been no detailed assessment of the cells’ calcium homeostasis, and in particular, the responses to agonists and antagonists of RYR1. Here we describe experiments conducted to assess calcium handling of the cells and examine responses to treatment with dantrolene, a drug commonly used for prophylaxis of recurrent exertional rhabdomyolysis in horses and malignant hyperthermia in humans.

Background Reproducible and accurate recognition of presence and severity of ataxia in horses with neurologic disease is important when establishing a diagnosis, assessing response to treatment, and making recommendations that might influence rider safety or a decision for euthanasia. Objectives To determine the reproducibility and validity of the gait assessment component in the neurologic examination of horses. Animals Twenty-five horses referred to the Royal Veterinary College Equine Referral Hospital for neurological assessment (n = 15), purchased (without a history of gait abnormalities) for an unrelated study (n = 5), or donated because of perceived ataxia (n = 5). Methods Utilizing a prospective study design; a group of board-certified medicine (n = 2) and surgery (n = 2) clinicians and residents (n = 2) assessed components of the equine neurologic examination (live and video recorded) and assigned individual and overall neurologic gait deficit grades (0–4). Inter-rater agreement and assessment-reassessment reliability were quantified using intraclass correlation coefficients (ICC). Results The ICCs of the selected components of the neurologic examination ranged from 0 to 0.69. “Backing up” and “recognition of mistakes over obstacle” were the only components with an ICC > 0.6. Assessment-reassessment agreement was poor to fair. The agreement on gait grading was good overall (ICC = 0.74), but poor for grades ≤ 1 (ICC = 0.08) and fair for ataxia grades ≥ 2 (ICC = 0.43). Clinicians with prior knowledge of a possible gait abnormality were more likely to assign a grade higher than the median grade. Conclusion and Clinical Importance Clinicians should be aware of poor agreement even between skilled observers of equine gait abnormalities, especially when the clinical signs are subtle.

Several human and animal myopathies, such as malignant hyperthermia (MH), central core disease and equine recurrent exertional rhabdomyolysis (RER) are confirmed or thought to be associated with dysfunction of skeletal muscle calcium regulation. For some patients in whom the genetic cause is unknown, or when mutational analysis reveals genetic variants with unclear pathogenicity, defects are further studied through use of muscle histopathology and in vitro contraction tests, the latter in particular, when assessing responses to ryanodine receptor agonists, such as caffeine. However, since muscle biopsy is not always suitable, researchers have used cultured cells to model these diseases, by examining calcium regulation in myotubes derived from skin, following forced expression of muscle-specific transcription factors. Here we describe a novel adenoviral vector that we used to express equine MyoD in dermal fibroblasts. In permissive conditions, transduced equine and human fibroblasts differentiated into multinucleated myotubes. We demonstrate that these cells have a functional excitation-calcium release mechanism and, similarly to primary muscle-derived myotubes, respond in a dose-dependent manner to increasing concentrations of caffeine. MyoD-induced conversion of equine skin-derived fibroblasts offers an attractive method for evaluating calcium homeostasis defects in vitro without the need for invasive muscle biopsy.

Equine type 1 polysaccharide storage myopathy (PSSM1), a common glycogenosis associated with an R309H founder mutation in the glycogen synthase 1 gene (GYS1), shares pathological features with several human myopathies. In common with related human disorders, the pathogenesis remains unclear in particular, the marked phenotypic variability between affected animals. Given that affected animals accumulate glycogen and alpha-crystalline polysaccharide within their muscles, it is possible that physical disruption associated with the presence of this material could exacerbate the phenotype. The aim of this study was to compare the histopathological changes in horses with PSSM1, and specifically, to investigate the hypothesis that the severity of underlying pathology, (e.g. vacuolation and inclusion formation) would (1) be higher in homozygotes than heterozygotes and (2) correlate with clinical severity. Resting and post-exercise plasma creatine kinase (CK) and aspartate aminotransferase (AST) enzyme activity measurements and muscle pathology were assessed in matched cohorts of PSSM1 homozygotes, heterozygotes or control horses. Median (interquartile range (IR)) resting CK activities were 364 (332-764) U/L for homozygotes, 301 (222-377) U/L for heterozygotes and 260 (216-320) U/L for controls, and mean (+/- SD) AST activity for homozygotes were 502 (+/116) U/L, for heterozygotes, 357 (+/-92) U/L and for controls, 311 (+/-64) U/L and were significantly different between groups (P = 0.04 and P = 0.01 respectively). Resting plasma AST activity was significantly associated with the severity of subsarcolemmal vacuolation (rho = 0.816; P = 0.01) and cytoplasmic inclusions (rho = 0.766; P = 0.01). There were fewer type 2× and more type 2a muscle fibres in PSSM1-affected horses. Our results indicate that PSSM1 has incomplete dominance. Furthermore, the association between plasma muscle enzyme activity and severity of underlying pathology suggests that physical disruption of myofibres may contribute to the myopathic phenotype. This work provides insight into PSSM1 pathogenesis and has implications for related human glycogenoses.

Reasons for performing study Equine cervical osteoarthritis is a common disease known to contribute to both neck pain and cervical vertebral compressive myelopathy. There are no published studies demonstrating the usefulness of latero-oblique radiography as a diagnostic tool for cervical osteoarthritis. Objectives To determine the sensitivity, specificity and positive and negative predictive values of latero-oblique radiography as a diagnostic tool for cervical articular process joint osteoarthritis. Study design Prospective cadaver study. Methods Latero-oblique radiographs and CT images were collected, and post mortem examinations performed, on 27 cadaver necks from Thoroughbred-type horses of various ages and sexes. Two equine clinicians independently reviewed each set of images for the presence of osteoarthritis. The authors, under the guidance of a veterinary pathologist, reviewed all articular process joints for bony changes indicative of osteoarthritis. Results The prevalence of osteoarthritis identified on CT images was 57.9% (Assessor A) and 23% (Assessor B). The prevalence of lesions identified on post mortem examination was 25.6%. Latero-oblique radiography showed a low sensitivity for identifying osteoarthritis when compared to both CT imaging (6.3–16.1%) and post mortem examination (2.2–4.4%). However, it showed a high specificity when compared to both CT imaging (92.1–97.8%) and post mortem examination (87.2–95.0%). The positive predictive value for identifying osteoarthritis was moderate (40.9–80.0%) when compared to CT imaging and poor (4.2–18.2%) when compared to post mortem examination. The negative predictive value was moderate when compared to both CT imaging (42.9–76.3%) and post mortem examination (78.0–79.8%). Conclusions Latero-oblique radiography has low sensitivity, but high specificity for the detection of cervical osteoarthritis when compared to both CT imaging and post mortem examination. Further investigation comparing the sensitivity, specificity and positive and negative predictive values of latero-oblique radiography vs. laterolateral radiography would determine whether taking latero-oblique radiographs, which can be more difficult to obtain and interpret, is necessary for the diagnosis of cervical osteoarthritis in practice. Ethical animal research: Ethical committee oversight not currently required by this congress: The study was performed on material obtained from an abattoir. Sources of funding: None. Competing interests: None.

SPINAL ataxia is a common presenting sign in horses with neurological disease in the UK and in most cases the signs are attributed to spinal cord compression. This report describes an unusual, but perhpas overlooked degenerative cause of spinal ataxia in horses in which diagnosis was confirmed at

HERNIATION through the inguinal ring is an uncommon complication of castration. Herniation has been reported to occur up to 12 days after castration but usually occurs within hours ([Hutchins and Rawlinson 1972][1], [Boussauw and Wilderjans 1996][2]). Draught breeds and Andalusian horses may be more

Reasons for performing study: The extent to which variability affects endoscopic grading of arytenoid cartilage movement is uncertain. Objective: To determine the observer and within horse variability of grading arytenoid cartilage movement in horses during resting endoscopic examination, using a 7-grade system. Methods: Endoscopic recordings of the upper respiratory tract made at rest in 270 draught horses were reviewed independently by 2 veterinarians to assess interobserver variability when scoring horses' laryngeal function with a 7-grade system. Grading was repeated by both examiners in 80 randomly selected recordings in order to assess intraobserver variability. In 120 horses, endoscopy was repeated after 24-48 h, with videos graded by both veterinarians to assess intrahorse variability. Results: The mean weighted K statistic for concordance within examiners was 0.867, with a mean intraobserver agreement of 76.3%. The weighted kappa statistic for concordance between the 2 examiners was 0.765, with an interobserver agreement of 63.1%. Of the horses receiving 2 endoscopic examinations, the same grade was assigned to 57.1% of horses at the second examination, when effects resulting from interobserver variability were removed. The mean weighted K statistic for concordance between the grade assigned at first vs. second examinations was 0.588, indicating only moderate agreement. Conclusions and potential relevance: Intra- and interobserver reliability of resting endoscopic grading of arytenoid cartilage movement using a 7-grade system was high when examinations were conducted by experienced veterinarians. However, there was moderate daily intrahorse variability, suggesting that results of resting endoscopic examinations performed on a single day should be interpreted with caution.

REASONS FOR PERFORMING STUDY Muscle biopsy is increasingly used in equine veterinary practice for investigating exertional, inflammatory or immune mediated myopathies and unexplained muscle atrophy. Although formalin-fixed samples are often used, for complete evaluation, fresh-frozen tissue is required. Freezing muscle in veterinary practice is impractical: samples sent to specialist laboratories for processing are therefore susceptible to delays, potentially leading to artefact and compromising histological interpretation. HYPOTHESIS Altered temperature, duration and hydration status influence the severity of storage-induced artefact in equine muscle. METHODS Skeletal muscle obtained immediately post euthanasia was divided into 6 independent samples from each of 8 horses. One sample per horse was frozen immediately in isopentane precooled in liquid nitrogen. Additional samples were stored in conditions designed to mimic possible situations encountered in practice, including increased storage times, temperature and hydration status. Following storage, stored samples were frozen as before. Cryosections were stained using haematoxylin and eosin and ranked for artefact on 2 occasions by 2 blinded observers. The best samples were processed subsequently with a panel of routine stains and immunolabelled for collagen V to enable the measurement of minimum fibre diameters. RESULTS Both prolonged storage and increased hydration resulted in more storage-associated artefact. Samples stored for 24 h chilled on dry gauze were ranked higher than those stored on damp gauze; however, a panel of routinely-used histochemical staining techniques was unaffected by chilled 24 h storage. There was no significant effect of storage on mean fibre diameter; however, both chilled dry and damp storage for 24 h caused a significant increase in fibre-size variability. CONCLUSION AND POTENTIAL RELEVANCE Caution should be exercised when interpreting fibre size profiles in shipped samples. Equine muscle biopsy samples are optimally shipped in dry gauze, sealed in plastic containers and shipped on ice packs to be processed within 24 h and can thus be interpreted by the receiving laboratory with minimal artefact.

Autosomal dominant Emery-Dreifuss muscular dystrophy (AD-EDMD) is one of a number of allelic disorders caused by mutations in the nuclear lamina proteins, lamins A and C. The disorder is characterised by the early onset of skeletal muscle weakness and joint contractures and later, by dilated cardiomyopathy and cardiac arrythmias. Although the pathophysiology is not understood, one theory suggests that disordered structural organisation at weakened nuclei in contractile cells may underlie the disease. Previous work shows that mice deficient in lamin A/C develop similar skeletal and cardiac muscle signs to patients with AD-EDMD and ultrastructural examination of muscle from these mice shows abnormal localisation of desmin. We hypothesised therefore that desmin localisation may be abnormal in muscle or cells from patients with AD-EDMD and/or in cells expressing mutant lamins. In order to evaluate this, desmin immunolocalisation was determined in skeletal muscle biopsy sections from patients with AD-EDMD and cell lines including MyoD-transfected fibroblast-derived myotubes from AD-EDMD patients and murine embryonic stem cell-derived cardiomyocytes stably transfected with mutant human lamin A. Ultrastructural examination of patient muscle was also performed. Desmin was expressed and localised normally in patient muscle and cell lines and ultrastructural examination was similar to controls. These results fail to provide any evidence that dominant mutations in lamin A/C lead to a disorganisation of the desmin associated cytoskeleton.

Mutations of the LMNA gene, encoding the nuclear envelope proteins lamins A and C, give rise to Emery-Dreifuss muscular dystrophy and to limb-girdle muscular dystrophy 1B (EDMD and LGMD1B). With one exception, all the reported EDMD and LGMD1B mutations are confined to the first 10 exons of the gene. We report four separate cases, with mutations in the same codon of LMNA exon 11, characterized by remarkable variability of clinical findings, in addition to features not previously reported. One patient had congenital weakness and died in early childhood. In two other patients, severe cardiac problems arose early and, in one of these, cardiac signs preceded by many years the onset of skeletal muscle weakness. The fourth case had a mild and late-onset LGMD1B phenotype. Our cases further expand the clinical spectrum associated with mutations in the LMNA gene and provide new evidence of the role played by the C-terminal domain of lamin A.

Reasons for performing study Recurrent laryngeal neuropathy (RLN) is a common equine distal axonopathy associated with neurogenic atrophy of intrinsic laryngeal muscles (particularly the left) causing laryngeal paresis and poor performance. An objective, reliable method of assessing changes in structure that correlates with laryngeal muscle function in vivo is necessary to determine response to novel treatments, including functional electrical stimulation. This study aimed to determine whether laryngeal muscle and nerve morphology (determined by CT and histopathology) correlated with laryngeal function. Study design Prospective cohort study. Methods Resting (grades 1–4) and exercising (grades A–C) laryngeal function was graded in adult Thoroughbred horses. Standing CT of the larynx was performed to compare volume, cross-sectional area (XSA) and tissue density between left and right cricoarytenoideus dorsalis (CAD) muscles in 22 horses. CAD muscles and recurrent laryngeal nerves were collected in a subgroup of 10 horses and analysed for fibrosis, fat infiltration and nerve fibre density (axons/μm2). Results Horses with grade C RLN on exercising endoscopy had significantly (P

Reasons for performing study Advanced imaging modalities enable assessment of the equine skull and brain. Horses are susceptible to neurological dysfunction of the cranial nerves; however, our understanding of these structures’ imaging anatomy is limited. Hypothesis Magnetic resonance imaging (MRI) will be superior to computed tomography (CT) for the identification of cranial nerves, but optimal assessment may depend on both modalities. Objectives Identify cranial nerves and compare and contrast the utility of MRI and CT images of cadaver and clinical material. Interpret images from both modalities and determine the cranial nerve imaging anatomy. Study design Prospective cadaver anatomical study combined with retrospective clinical case study. Methods The head of a neurologically normal 9-year-old Thoroughbred gelding was scanned immediately following euthanasia (performed for reasons unrelated to this study). High resolution MRI (1.5 Tesla) and CT examinations were conducted over a 12 h interval following euthanasia. Images obtained were compared with selected clinical cases which were scanned during anaesthesia (MRI; approximately 30–60 min) or standing sedation (CT; approximately 30 s). Results On a high resolution MRI scan of a cadaver equine skull, each of the 12 cranial nerves and their topographic location was readily appreciated. Cranial nerves 1, 2, 3, 5, 7 and 8 were more easily identified in clinically relevant MRI scans. CT allowed visualisation of the stylomastoid foramen, the inner and middle ear and cranial nerves 2, 3, 5 and 7. Conclusions High field MRI allows for excellent visualisation of equine cranial nerves. CT allows for detailed visualisation of the osseous canals and foramina. This study advances anatomical knowledge of the normal equine cranial nerves to aid interpretation in horses that display neurological dysfunction localising to the brain and brainstem. Ethical animal research: Horse owners gave their consent for their animals to be included in the study. Sources of funding: Institutional. Competing interests: None.