John Keen

Biochemical indices of vascular function, glucose metabolism and oxidative stress in horses with equine Cushing's disease

REASONS FOR PERFORMING STUDY The mechanisms underlying the increased risk of laminitis in horses with equine Cushings disease (ECD) are poorly understood. HYPOTHESIS That abnormalities in glucose homeostasis, similar to those which cause microvascular dysfunction in human diabetics, contribute to development of laminitis in horses with ECD. METHODS Thirty-one aged horses were divided into 3 groups based on clinical signs and dexamethasone suppression testing (DST). Group 1 (n = 12) had clinical ECD as evidenced by hirsutism. Group 2 (n = 10) had a positive DST but no hirsutism. Group 3 (n = 9) were controls without ECD, with a negative DST and no clinical evidence of ECD. Biochemical indices of glucose metabolism, vascular function and oxidative stress were determined in single morning blood samples. RESULTS Group 1 had abnormalities in glucose homeostasis, including increased levels of glucose and insulin, compared to Groups 2 and 3. Groups 1 and 2 had significantly lower plasma thiol (PSH) levels and nonsignificantly lower albumin-corrected PSH levels than Group 3, consistent with oxidative stress. CONCLUSIONS AND POTENTIAL RELEVANCE The observed abnormalities in glucose metabolism and oxidative stress could potentially contribute to development of laminitis in horses with ECD, by similar mechanisms to those that cause microvascular dysfunction in human diabetics. The absence of inter-group differences in the biochemical indices of vascular function precludes their use as preclinical diagnostic indicators of vascular dysfunction. The study also highlighted limitations in the premortem diagnosis of ECD.

European outbreaks of atypical myopathy in grazing equids (2006-2009). Spatiotemporal distribution, history and clinical features

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 Fishers exact and Welchs 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.

European outbreaks of atypical myopathy in grazing horses (2006–2009): determination of indicators for risk and prognostic factors

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 Welchs 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.

Study of the short- and long-term outcomes of 65 horses with peritonitis

The records of 65 horses with peritonitis examined at two uk referral centres over a period of 12 years were reviewed. Peritonitis was defined in terms of the horses peritoneal fluid containing more than 5 × 109 nucleated cells/l. Horses that had developed peritonitis after abdominal surgery or a rupture of the gastrointestinal tract were excluded. Of the 65 horses, 56 (86 per cent) survived to be discharged. Follow-up information was obtained from practice records and telephone calls to the owners for 38 of the horses. Of these, 32 (84 per cent) had survived for at least 12 months and were considered to be long-term survivors; the others six were euthanased within 12 months. Thirteen (34 per cent) of the horses discharged had experienced complications that could have been sequelae to peritonitis and eight of the 13 were euthanased. The cause of the peritonitis was identified in 15 cases; survival rates were lowest in horses with peritonitis secondary to urinary tract involvement or intra-abdominal masses. Of the other 50 cases, 47 (94 per cent) survived to discharge, but two were euthanased owing to recurrent colic.

European outbreak of atypical myopathy in the autumn 2009.

BACKGROUND Atypical myopathy is an acute, severe rhabdomyolysis occurring in grazing horses. In the beginning of October 2009, a new outbreak occurred in several European countries. Geographic, demographic and clinical data of the reported cases in the month October 2009 are described. KEY FINDINGS The survival rate in this outbreak was 25%. The most frequently observed clinical signs were congested mucous membranes, dyspnea, tachycardia, depression, weakness, stiffness, recumbency, trembling, sweating, and myoglobinuria. Nonsurvivors were significantly more likely to be recumbent than survivors. Prognostic factors, symptomatic treatment, and preventive measures are discussed. SIGNIFICANCE Differences were encountered during the described outbreak of atypical myopathy in October 2009 compared with previous outbreaks reported. Equine practitioners should be aware that previous epidemiological studies have shown that after a high prevalence in the autumn, new cases are likely to occur in the following spring.

Repeatability and Intra‐ and Inter‐observer Agreement of Cervical Vertebral Sagittal Diameter Ratios in Horses with Neurological Disease

Background Sagittal ratio values (SRVs) of cervical vertebrae are used for ante‐mortem diagnosis of cervical vertebral stenotic myelopathy, but intraobserver and interobserver variability in measurement may influence radiographic interpretation of vertebral stenosis in horses with neurological disease. Objectives To determine intraobserver repeatability in SRVs, intra‐ and interobserver agreement in SRVs and whether or not agreement was influenced by animal age. Animals Forty‐two horses (>1 year old) with neurological disease from which laterolateral computed radiographic images of C2–C7 were obtained. Methods Four observers made measurements from C2 to C7 for each horse and interobserver agreement for intra‐ and intervertebral SRVs was determined using Bland–Altman analysis (acceptable agreement: limits of agreement [LOA] ≤ 0.05) on all horses and those ≤3 (n = 25) and >3 (n = 17) years old. Each observer also made repeated measurements for 10 horses and intraobserver repeatability and agreement were determined. Results Adequate intraobserver repeatability was achieved for 6 sites. Within observers, paired measurements had a median difference ≤5.7%, but a large range in differences often occurred, most frequently at intervertebral sites. For C5, C6, C7, and C3–4, LOA ≤ 0.05 were achieved by at least 1 observer. With the exception of C5 for 1 pair, LOA were >0.05 for interobserver agreement, regardless of animal age. LOA were largest at intervertebral sites. Conclusions and Clinical Importance Within and between observers, measurement error may limit the diagnostic accuracy of SRVs and result in discrepancies of diagnosis and treatment and warrants consideration when used clinically in horses with neurological disease.

Diagnosis and management of equine rhabdomyolysis

Our knowledge of rhabdomyolysis has increased greatly in the past 10 years. Detailed clinical and molecular investigations are starting to uncover and identify more diverse disorders that lead to a clinical end-point that represents signs of muscle disease. In equine practice, despite the fact that cases of acute sporadic rhabdomyolysis (ie, ‘tying up’) are quite common, the pathophysiology of this disease is still poorly understood. The astute clinician can, however, recognise chronic cases that may have an underlying metabolic defect that predisposes to muscle disease even when management practices are optimal. This article describes the most commonly seen types of rhabdomyolysis in horses, discusses the approach to diagnosis and highlights the options for treatment in affected cases.

Proteomic Profiling of Cranial (Superior) Cervical Ganglia Reveals Beta-Amyloid and Ubiquitin Proteasome System Perturbations in an Equine Multiple System Neuropathy

Equine grass sickness (EGS) is an acute, predominantly fatal, multiple system neuropathy of grazing horses with reported incidence rates of ∼2%. An apparently identical disease occurs in multiple species, including but not limited to cats, dogs, and rabbits. Although the precise etiology remains unclear, ultrastructural findings have suggested that the primary lesion lies in the glycoprotein biosynthetic pathway of specific neuronal populations. The goal of this study was therefore to identify the molecular processes underpinning neurodegeneration in EGS. Here, we use a bottom-up approach beginning with the application of modern proteomic tools to the analysis of cranial (superior) cervical ganglion (CCG, a consistently affected tissue) from EGS-affected patients and appropriate control cases postmortem. In what appears to be the proteomic application of modern proteomic tools to equine neuronal tissues and/or to an inherent neurodegenerative disease of large animals (not a model of human disease), we identified 2,311 proteins in CCG extracts, with 320 proteins increased and 186 decreased by greater than 20% relative to controls. Further examination of selected proteomic candidates by quantitative fluorescent Western blotting (QFWB) and subcellular expression profiling by immunohistochemistry highlighted a previously unreported dysregulation in proteins commonly associated with protein misfolding/aggregation responses seen in a myriad of human neurodegenerative conditions, including but not limited to amyloid precursor protein (APP), microtubule associated protein (Tau), and multiple components of the ubiquitin proteasome system (UPS). Differentially expressed proteins eligible for in silico pathway analysis clustered predominantly into the following biofunctions: (1) diseases and disorders, including; neurological disease and skeletal and muscular disorders and (2) molecular and cellular functions, including cellular assembly and organization, cell-to-cell signaling and interaction (including epinephrine, dopamine, and adrenergic signaling and receptor function), and small molecule biochemistry. Interestingly, while the biofunctions identified in this study may represent pathways underpinning EGS-induced neurodegeneration, this is also the first demonstration of potential molecular conservation (including previously unreported dysregulation of the UPS and APP) spanning the degenerative cascades from an apparently unrelated condition of large animals, to small animal models with altered neuronal vulnerability, and human neurological conditions. Importantly, this study highlights the feasibility and benefits of applying modern proteomic techniques to veterinary investigations of neurodegenerative processes in diseases of large animals.

Expression of putative markers of pluripotency in equine embryonic and adult tissues.

Expression of several putative markers of pluripotency (OCT4, SOX2, NANOG, LIN28A, REX1, DNMT3B and TERT) was examined in a range of equine tissues, including early embryos, induced pluripotent stem cells (iPSCs), testis, adipose- and bone marrow-derived mesenchymal stromal cells (MSCs), and keratinocytes. Transcript levels of all markers were highest in embryos and iPSCs and, except for SOX2, were very low or undetectable in keratinocytes. Mean expression levels of all markers were lower in testis than in embryos or iPSCs and, except for DNMT3B, were higher in testis than in MSCs. Expression of OCT4, NANOG and DNMT3B, but not the other markers, was detected in MSCs. Of all markers analysed, only LIN28A, REX1 and TERT were associated exclusively with pluripotent cells in the horse.

Carbonyl reductase 1 catalyzes 20β-reduction of glucocorticoids, modulating receptor activation and metabolic complications of obesity

Carbonyl Reductase 1 (CBR1) is a ubiquitously expressed cytosolic enzyme important in exogenous drug metabolism but the physiological function of which is unknown. Here, we describe a role for CBR1 in metabolism of glucocorticoids. CBR1 catalyzes the NADPH- dependent production of 20β-dihydrocortisol (20β-DHF) from cortisol. CBR1 provides the major route of cortisol metabolism in horses and is up-regulated in adipose tissue in obesity in horses, humans and mice. We demonstrate that 20β-DHF is a weak endogenous agonist of the human glucocorticoid receptor (GR). Pharmacological inhibition of CBR1 in diet-induced obesity in mice results in more marked glucose intolerance with evidence for enhanced hepatic GR signaling. These findings suggest that CBR1 generating 20β-dihydrocortisol is a novel pathway modulating GR activation and providing enzymatic protection against excessive GR activation in obesity.