Est-ce que la laparoscopie a une place dans la gestion des coliques chez le cheval ?

editorial reviewe

Clinical outcome after surgical correction of cleft palate by laryngeal tie-forward in 2 horses

Introduction: Cleft palate is a rare congenital defect in horses. Its description in literature is generally limited to cases of young foals referred for milk regurgitation from nostrils, coughing and dysphagia, with a guarded to poor prognosis if not treated. Surgical intervention consists mainly in palatoplasty that is considered a salvage procedure and complications are frequent. Only few cases of cleft palate in adult horses are described in literature. Laryngeal tie-forward, a surgical technique generally used to treat dorsal displacement of the soft palate, has been described only in one 4-year-old pony with cleft palate but clinical improvement after surgery was partial and temporary. Objectives: To describe laryngeal tie-forward as a valid option for the treatment of cleft palate in two adult horses not suitable for palatoplasty. Methods: Clinical records of two 8 y.o. jumping horses performing respectively at a low and intermediate intensity of exercise and competition. Both horses were referred for respiratory noise, exercise intolerance, cough and alimentary nasal discharge; they were diagnosed with a moderate soft palate defect and surgically treated by laryngeal tie-forward. A clinical examination and control endoscopy were realized at 4 months for one horse and at 3 years after surgery for the other. Results: In both horses postoperative endoscopy showed a visible reduction of the gap between soft palate and the larynx so that the epiglottis was covering the soft palate defect. One horse was examined 4 months after surgery, a residual respiratory noise during exercise was still present but lighter than prior to intervention, its performances had remarkably improved. The second horse was controlled 3 years after surgery, some cough was still observed but limited to the onset of exercise, performances were satisfying and the horse was working at the same intensity level. Globally, in both horses, initial symptoms had disappeared or were significantly reduced after surgery and owners were satisfied. Reduction of the soft palate defect was still effective, even several months after surgery. Conclusions: The description of these two clinical cases shows that some horses can grow up to adult age with moderate defects of the soft palate; they are likely to show clinical signs of variable severity. In such cases laryngeal tie-forward should be considered as an option if the defect is too large or asymmetrical to be corrected with palatoplasty

Generalized sarcoidosis and hypertrophic osteopathy in a Standardbred with poor-performance.

editorial reviewedBackground and objective: Equine sarcoidosis (ES) is a rare but emergent disease of unknown etiology. Body of literature on ES remains limited, especially on the generalized forms associated with hypertrophic osteopathy (HO). This case report describes clinical and imaging findings in a racehorse with exercise intolerance affected by generalized ES and subsequent HO. Material and methods: A 6-year-old Standardbred gelding was presented for sudden exercise intolerance, weight loss, pyrexia, skin lesions, facial and peripheral multifocal swelling. Results: The horse underwent diagnostic imaging of head and limbs. Radiography and ultrasonography of the head revealed periostitis and regional soft tissue swelling of the maxillary bone. Bilateral marked periostitis with palisade-like appearance involving diaphyseal or metaphyseal region was also identified on distal radius and metatarsal bones, suggestive of HO. Computed tomography excluded dental or sinus abnormalities. Due to the appearance (scaling, crusting and exfoliating) of skin lesions on the croup and lower hindlimbs during hospitalization, biopsies were performed revealing granulomatous dermatitis. This finding was consistent with ES, thus explaining HO. Rapidly declining conditions of the horse led to the decision of euthanasia. Post-mortem examination revealed splenomegaly and several pulmonary nodules. Histopathology on different organs confirmed severe granulomatous lesions; PCR on a pool of tissues identified EHV-5. Discussion and conclusions: ES, even in its generalized form, is an emergent disease that should be considered in case of unspecific clinical signs combining exercise intolerance, weight loss, facial/distal limb bone deformities and skin lesions; EHV-5 could play a role in the development of the disease

A case of true hermaphrodism in a horse

peer reviewedTrue hermaphroditism in horses, is a complex and poorly under- stood disorder of sexual development characterized by the presence of both male and female gonads as separate organs or in a combined structure called ovotestis. A 3-year-old Spanish nullipa- rous mare with a history of stallion behaviour and an abnormal reproductive exam was presented at the Equine Clinic of the University of Li ege. Externally, a small vulva with an enlarged clitoris was observed. Transrectal ultrasonography showed a thin (<1 cm) uterus, leading to a small heterogeneous structure in the normal place of the left ovary. No structure could be identified in the presumed location of the right ovary. Speculum exam was compatible with a vaginal agenesis, as the cervix opened craniad the urethral meatus. Oestradiol, progesterone and testosterone concen- trations were compatible with a stallion’s endocrinology. Gonads were laparoscopically found in the usual location of the ovaries in both flanks and excised. Histopathology of both gonads showed atrophic testicular tissue with hyperplastic Leydig cells. The left gonad also contained ovarian tissue with some scarce primordial follicles. Clitoral enlargement is the first symptom most commonly identified in animals with true hermaphroditism and can be explained by the production of testosterone by the gonads, however, the aetiology of the vaginal agenesis is unknown. True hermaph- roditism in horses is generally related to 64XX syndrome with or without SRY gene translocation (impending karyotyping analysis). After 5 days the mare was discharged from the clinic, and in absence of testosterone the stallion behaviour disappeared in a couple of months

Presence of neutrophil myeloperoxidase in lamellar tissue of horses with laminitis induced using a hyperinsulinemic model

peer reviewedLaminitis is a pathology of the equine digit resulting in failure of the dermo-epidermal interface. Inflammation is likely a central player in its pathophysiology. Neutrophil activation and the presence of myeloperoxidase have been observed in the black walnut heartwood extract model, used to investigate sepsis-related laminitis. However, less is known about the role of neutrophil activation in the prolonged euglycemic hyperinsulinemic clamp (pEHC) model. This model is used to investigate the pathophysiology of laminitis related to endocrine disturbances like hyperinsulinemia encountered in pituitary pars intermedia dysfunction and equine metabolic syndrome. Five horses were included in this study and randomly assigned to a control group (n = 2) and a pEHC group (n = 3). Histological sections of lamellar tissue from all horses were obtained and immunohistochemically stained for myeloperoxidase and counterstained with hematoxylin-eosin. The sections were examined for histopathological evidence of laminitis and myeloperoxidase. Histopathological changes that characterize insulin-induced laminitis and increased presence of myeloperoxidase, especially in the dermal lamellae, were observed in histologic sections of pEHC-treated horses compared to control horses. Neutrophil activation with myeloperoxidase release may play a role in the pathophysiology of endocrinopathic laminitis. The implications of this finding warrant further investigation

Gastrointestinal effects of general anaesthesia in horses undergoing non abdominal surgery: focus on the clinical parameters and ultrasonographic images

The ultrasonographic images of the gastrointestinal tract in horses can be influenced by fasting and sedation but the proper effect of general anaesthesia (GA) on them has not been determined yet. This study aimed to evaluate the effects of GA on ultrasonographic images of the gastrointestinal tract in horses and to compare these effects with a clinical evaluation. Twenty horses undergoing non-abdominal surgeries were evaluated by ultrasonography before and 4 times within 24 h after GA. Each ultrasonographic exam focused on the stomach, the duodenum and on 5 locations on the jejunum. The four-quadrant auscultation and the postoperative faecal output were also recorded. Pre and post anaesthetic values were compared using linear mixed effects models. None of the horses presented colic signs or reduced faecal output. During the first 2 post anaesthetic evaluations, the gut sounds were significantly decreased and, when taking all jejunal locations together, the jejunal diameter and visualisation frequency significantly increased. No intestinal loop appeared thickened and most of their diameters remained within the normal range. Our results suggest that the effects of GA on the ultrasonographic images of the small intestine are mild and of short duration and can therefore be differentiated from a pathological process

Effects of Juglone on Neutrophil Degranulation and Myeloperoxidase Activity Related to Equine Laminitis.

Experimental laminitis, characterized by a failure of the dermal-epidermal interface of the foot, can be induced in horses by the oral administration of a black walnut extract (BWE). In the early phase of this severe and painful disease, an activation of neutrophil occurs, with the release of myeloperoxidase (MPO), a pro-oxidant enzyme of neutrophils, in plasma, skin, and laminar tissue. Juglone, a naphthoquinone derivative endowed with redox properties, is found in walnuts and has been incriminated in this neutrophil activation. We report for the first time the inhibitory activity of juglone on the degranulation of neutrophils induced by cytochalasin B and formyl-methionyl-leucyl-phenylalanine as monitored by the MPO release (>90% inhibition for 25 and 50 μM). Moreover, it also acts on the peroxidase activity of MPO by interacting with the intermediate "π cation radical," as evidenced by the classical and specific immunological extraction followed by enzymatic detection (SIEFED) assays. These results are confirmed by a docking study showing the perfect positioning of juglone in the MPO enzyme active site and its interaction with one of the amino acids (Arg-239) of MPO apoprotein. By chemiluminescence and electron paramagnetic resonance techniques, we demonstrated that juglone inhibited reactive oxygen species (ROS) and superoxide anion free radical produced from phorbol myristate acetate (PMA)-activated polymorphonuclear neutrophils (PMNs). These results indicate that juglone is not the trigger for equine laminitis, at least if we focus on the modulation of neutrophil activation

Effect of non-sperm cells removal with single layer colloidal centrifugation on myeloperoxidase concentration in post-thaw equine semen

Myeloperoxidase (MPO) is a pro-oxidant enzyme contained in, and released by, neutrophils during degranulation or after lysis. Post-thaw semen contains MPO and its concentration is associated with decreased sperm motility. Recently, MPO concentration in post-thaw semen was shown to be associated with presence of non-sperm cells. The aim of this study was to describe the effect of centrifugal fractionation of semen prior to cryopreservation on post-thaw concentrations of non-sperm cells and MPO. The experimental design consisted in freezing semen with or without previous centrifugation through two concentrations of single layer colloid media. Non-sperm cells and MPO concentrations were assessed in pellet and upper layer at each step of the procedure and MPO was detected in cells by immunocytochemistry. Single layer colloid centrifugation decreased non-sperm cells and MPO concentrations in post-thaw semen. The MPO concentration was correlated with concentration of non-sperm cells in the upper layer of the supernatant. In post-thaw semen, with or without previous single layer colloid centrifugation, MPO concentration was correlated with concentration of non-sperm cells. Overall, neutrophils were rarely observed and non-sperm cells were mainly epithelial cells or cellular debris, as demonstrated by MPO immunocytochemistry. Following single layer colloid centrifugation, MPO concentration was decreased, and correlations observed in different samples of the experiments revealed an association between concentrations of MPO and non-sperm cells. At all steps of the semen processing and cryopreservation, MPO immunostaining was clearly identified only on non-sperm cells. Our results demonstrate that non-sperm cells present in fresh semen release MPO during freezing.SPERMP

Mise au point des modèles équins de culture des kératinocytes podaux et des cellules endothéliales pour l’étude de la fourbure équine

Laminitis is a painful systemic disease of which the principal manifestation is a diffuse aseptic pododermatitis. An ischaemia-reperfusion and/or an activation of matrix métalloproteinases following a proinflammatory status (mainly gastro-intestinal) or hormonal disorders involving metabolic dysfunctions related to the development of insulin resistance, are at the origin of laminitis. The presence of histological lesions of keratinocytes and endothelial cells, and the infiltration of polymorphonuclear neutrophils (PMNs) in laminar tissues, warranted the development of the two cellular culture models corresponding for the study of this disease. Therefore, keratinocytes and endothelial cells are isolated by enzymatic digestion and mechanical separation, respectively from coronary bands and carotids, and are put in culture under controlled atmosphere in DMEM supplemented with additives. Microscopic observations of phenotype, the immunocytochemistry, growth curves, and apoptosis stainings allowed to characterize the cells. The cells can be used, between passage 3 and 5 for the keratinocytes and 3 and 6 for the endothelial cells, in models of interaction with PMNs and their enzymes, in condition of anoxia/reoxygenation with variable glucose.concentrations

Interactions between neutrophils, endothelial cells, and keratinocytes in the pathogenesis of equine laminitis: role of myeloperoxidase and elastase

La pododermatite aseptique diffuse, connue sous le nom de fourbure, consiste en un état inflammatoire des tissus lamellaires du pied. Elle se traduit par un défaut d’attachement entre la phalange distale et la face interne de la muraille du pied et par l’apparition d’une boiterie caractéristique. Le coût émotionnel et économique de la fourbure, associé aux connaissances encore limitées de sa pathophysiologie et des moyens de traitement ou de prévention, rend cette maladie particulièrement frustrante pour les vétérinaires et les propriétaires. La fourbure reste une complication importante de nombreuses maladies médicales et chirurgicales du cheval. Elle est le plus souvent associée aux facteurs alimentaires (pâturage intensif, coliques, obésité), aux traumas de la région digitée ainsi qu’aux états hypovolémiques et septiques. Histologiquement, la fourbure se caractérise par la destruction de la membrane basale et le désengrènement subséquent du derme et de l’épiderme lamellaire.Deux grandes théories ont été avancées pour expliquer la fourbure. Dans la première, une chute de la perfusion sanguine du pied conduirait à une nécrose de l’interface lamellaire en réponse à un phénomène d’ischémie-reperfusion. Dans la seconde théorie, les facteurs déclencheurs de la fourbure seraient drainés par la voie vasculaire jusque dans le pied où ils induiraient la production et l’activation des métalloprotéinases matricielles (MMP) à l’origine de la destruction de la membrane basale. Plus récemment, les troubles endocriniens à l’origine de résistance à l’insuline ont aussi été reconnus comme une importante cause de fourbure.A partir des éléments connus de la pathophysiologie de la fourbure, il nous a semblé que les cellules endothéliales, les kératinocytes et les neutrophiles pourraient jouer un rôle prépondérant dans la pathophysiologie complexe de cette maladie. L’objectif général de ce travail était donc de préciser les rôles de la myéloperoxydase (MPO) et de l’élastase du neutrophile dans la fourbure et ce, particulièrement au niveau des interactions du neutrophile avec les cellules endothéliales et les kératinocytes. Les travaux de cette thèse ont conduit à la mise au point d’un système de dosage de l’élastase du neutrophile équin. En raison de la grande quantité de sang que demande la purification de la MPO et de l’élastase, nous avons d’abord élaboré une technique permettant la purification des 2 enzymes à la fois. Pour ce faire, la MPO et l’élastase ont été purifiées par une double étape de chromatographie en phase liquide à partir de neutrophiles isolés du même pool sanguin. L’élastase purifiée a ensuite été utilisée pour la production d’anticorps anti-élastase par immunisation chez le lapin et le cobaye. Les anticorps disponibles ont permis le développement et la validation d’un test ELISA pour la mesure de l’élastase équine, d’abord dans le plasma puis dans d’autres fluides biologiques. Les méthodes de dosage de la MPO et de l’élastase ont été appliquées dans un modèle expérimental de fourbure induite par administration orale d’extraits de noyer noir (BWHE). Les résultats indiquent une augmentation significative de la concentration plasmatique en MPO et en élastase pendant la phase prodromique de la fourbure, plus précoce pour la MPO que pour l’élastase. Dans les tissus lamellaires et dans la peau, une augmentation significative de la MPO et de l’élastase est aussi enregistrée dès 3h après l’administration de BWHE. L’activité enzymatique de la MPO mesurée par SIEFED (Specific Immuno-Extraction Followed by Enzymatic Detection) est, quant à elle, significativement plus élevée chez les animaux traités que chez les contrôles dès 1,5h après l’induction. L’immunohistochimie de la peau et des tissus lamellaires confirme l’infiltration de la MPO et de l’élastase pendant la phase prodromique de la fourbure et indique une forte affinité de ces enzymes pour le tissu épidermique.Notre étude préliminaire sur des fourbures naturelles hospitalisées dans notre clinique indique une concentration en élastase plasmatique significativement plus élevée chez les chevaux fourbus que chez les chevaux du groupe témoin et confirme les résultats obtenus dans le modèle expérimental. D’autre part, sur le plan clinique, nos travaux ont mis en évidence l’efficacité de l’administration d’une héparine de bas poids moléculaire (LMWH), l’énoxaparine, dans la prévention des fourbures postopératoires chez les chevaux en coliques. Nos modèles de culture cellulaire in vitro nous ont appris que les cellules endothéliales issues de l’extrémité distale du membre sont capables de fixer la MPO en solution et que la MPO associée aux cellules conserve une activité enzymatique. Par ailleurs, la présence d’héparine classique et de LMWH dans le milieu, réduit de manière significative la capture de MPO par les cellules endothéliales et l’activité enzymatique associée aux cellules. De plus, bien que les cellules artérielles semblent capter davantage la MPO que les cellules veineuses, l’inhibition de cette capture par les héparines semble plus efficace sur les cellules veineuses. En utilisant le SIEFED comme outil pharmacologique dans un modèle acellulaire, héparine classique et LMWH ont montré leur aptitude à inhiber l’activité de la MPO. Cette inhibition, meilleure à basse concentration pour l’héparine classique, est plus prononcée à plus forte concentration pour les LMWH. Une étude immunocytologique préliminaire menée sur des kératinocytes équins en culture (issus de la bande coronaire) confirme l’affinité de la MPO en solution pour ces cellules. Enfin, dans un modèle de culture de cellules endothéliales équines issues de carotides, nous avons montré par oxygraphie, chromatographie en phase gazeuse et résonance paramagnétique électronique, que l’anoxie-réoxygénation entraîne une altération de la fonction respiratoire de ces cellules et provoque une augmentation significative de leur production d’espèces réactives de l’oxygène, créant ainsi des conditions favorables au fonctionnement enzymatique de la MPO fixée sur les cellules endothéliales. Nos travaux permettent d’incriminer le recrutement et l’activation du neutrophile dans la pathophysiologie de la maladie. La MPO et l’élastase représentent respectivement le versant oxydant et protéolytique du neutrophile et leur présence dans la phase prodromique de la fourbure suggère un rôle de ces enzymes dans les évènements qui contribuent au développement de la maladie. L’effet modulateur de LMWH sur l’activité des neutrophiles et de leurs enzymes, ainsi que sur les interactions des neutrophiles avec l’endothélium, pourrait ainsi intervenir dans la prévention de la fourbure.Nos travaux, associés aux publications de la dernière décennie, suggèrent aussi que les théories vasculaire et enzymatique de la fourbure sont inextricablement liées l’une à l’autre comme les caractéristiques d’un syndrome de réponse inflammatoire systémique dont la défaillance organique ou multi-organique se traduit principalement, chez le cheval, par la séparation des tissus lamellaires. La réponse inflammatoire semble ainsi se situer au carrefour des fourbures liées au sepsis et de celles relatives aux désordres endocriniens. Dans le futur, le contrôle de l’activation et du recrutement des neutrophiles et la protection de l’endothélium devraient constituer des avancées stratégiques pour enfin traiter et prévenir la fourbure équine de manière efficace/Diffuse aseptic pododermatitis, known as laminitis, is an inflammatory condition of the hoof laminar tissues. It consists in default of attachment between the third phalanx and the inner hoof wall and results into a characteristic lameness. The emotional and economic cost of laminitis, coupled with our limited knowledge of its pathophysiology and the means of prevention or treatment, makes this disease particularly frustrating for veterinarians and owners.Laminitis is an important complication of many equine medical and surgical diseases. It is most often associated with dietary factors (intensive grazing, colic, obesity), trauma to the digital region and to the hypovolemic and septic states. Histologically, laminitis is characterized by the destruction of the basement membrane and the subsequent disengagement of the dermal and epidermal lamina.Two main theories have been advanced to explain laminitis. In the first one, a drop of blood perfusion of the foot would lead to necrosis of the lamellar interface in response to an ischemia-reperfusion phenomenon. In the second theory, the laminitis trigger factors would be drained into the foot through hematogenous pathway causing the production and activation of matrix metalloproteinases (MMP). This would result into the destruction of the basement membrane. More recently, endocrine disorders resulting in insulin resistance have also been recognized as an important cause of laminitis.From what is known about the pathophysiology of laminitis, it seemed that the endothelial cells, keratinocytes and neutrophils may play a role in the complex pathophysiology of this disease. The overall objective of this study was to clarify the roles of myeloperoxidase (MPO) and neutrophil elastase in laminitis, particularly at the level of neutrophil interactions with endothelial cells and keratinocytes. The works of this thesis have led to develop a mean of determination of neutrophil equine elastase. Due to the large amount of blood required for the purification of MPO and elastase, we first developed a technique for the purification of both enzymes. MPO and elastase were purified by a double step liquid chromatography from neutrophils isolated from the same pool of blood. The purified elastase was then used for the production of anti-elastase antibodies by immunizing rabbits and guinea pigs. The available antibodies have enabled the development and validation of an ELISA for measurement of equine elastase, first in the plasma and then in other biological fluids. The methods for MPO and elastase assay have been applied in an experimental model of laminitis induced by oral administration of black walnut heartwood extract (BWHE). The results indicate a significant increase in plasma concentration of MPO and elastase during the prodromal stage of laminitis, earlier for MPO than for elastase. In the skin and the lamellar tissues, a significant increase of MPO and elastase is also registered as early as 3h after administration of BWHE. The enzymatic activity of MPO measured by SIEFED (Specific Immuno-Extraction Followed by Enzymatic Detection) is, in turn, significantly higher in treated animals than in controls as early as 1.5 h after induction. Immunohistochemistry of the skin and lamellar tissues confirms the infiltration of MPO and elastase during the prodromal stage of laminitis, indicating a strong affinity of these enzymes for the skin tissue.Our preliminary study on naturally occurring laminitis in horses hospitalized in our clinic indicates a significantly higher plasma elastase concentration in foundered horses than in control horses and confirms the results obtained in the experimental model. Additionally, we clinically demonstrated the effectiveness of the administration of a low molecular weight heparin (LMWH), enoxaparin, in preventing postoperative laminitis in horses suffering from colic.Our in vitro cell culture models taught us that endothelial cells from the distal limb are able to bind MPO in solution and that cell-associated MPO retains enzymatic activity. Moreover, the presence of conventional heparin and LMWH in the medium significantly reduces the uptake of MPO by endothelial cells and the enzyme activity associated with cells. Furthermore, although the arterial cells seem to capture more MPO than venous ones, inhibition of this uptake by heparins appears to be more effective on venous cells. Using the SIEFED as pharmacological tool in a cell-free model, heparin and LMWH showed their ability to inhibit MPO activity. This inhibition is better at low-concentration for heparin and is conversely more pronounced at higher doses for LMWH. A preliminary immunocytochemical study on equine keratinocytes cultured from the coronary band cells confirms the affinity of MPO in solution for these cells. Finally, in a model of equine endothelial cells, cultured from the carotid artery, we showed by oxygraphy, gas chromatography and electron spin resonance, that anoxia-reoxygenation leads to an impaired respiratory function of the cells and causes their production of reactive oxygen species to significantly increase, thereby creating favorable conditions for the functioning of MPO enzyme attached to the endothelial cells. Our works incriminate the neutrophil recruitment and activation in the pathophysiology of laminitis. MPO and elastase respectively represent the oxidative and proteolytic side of neutrophils. Their presence in the prodromal phase of laminitis suggests a role for these enzymes in the events that contribute to the disease development. The modulating effect of LMWH on the activity of neutrophils, their enzymes and their interactions with the endothelium, could intervene in the prevention of laminitis. Our work, related to publications of the last decade, also suggest that the enzymatic and vascular theories of laminitis are inextricably linked to each other as they characterize a systemic inflammatory response syndrome associated with organ failure, which, in the horse, mainly translates into the separation of lamellar tissue. Inflammatory response seems to be at the crossroads of sepsis-related laminitis and those related to endocrine disorders. In the future, control of the neutrophil activation and recruitment and protection of the endothelium should become strategic steps to effectively treat and prevent equine laminitis