Evolution of blood myeloperoxidase in the perioperative period of horses undergoing emergency celiotomy.

Poster (2009, July)

Colic can cause an activation of neutrophils with release in the blood flow of myeloperoxidase (MPO), a specific enzyme with strong oxidative activity. The aim of this study was to describe the evolution ... [more ▼]

Colic can cause an activation of neutrophils with release in the blood flow of myeloperoxidase (MPO), a specific enzyme with strong oxidative activity. The aim of this study was to describe the evolution of plasma MPO after colic surgery. Materials included 13 adult horses that underwent an emergency celiotomy for acute intestinal obstruction. Venous blood samples were collected into EDTA anticoagulated tubes before surgery, during surgery after correction of the intestinal lesion and during the recovery of anaesthesia. In the postoperative period samples were taken every 4 hours during the first 4 days (from day 0 until day 4), every 12 hours during the days 4 and 5 and every 24 hours until day 10. MPO was assayed with a specific enzyme-linked immunosorbent assay. Statistical analysis was performed by one way Anova with student- Newman-Keuls post test on data obtained for each time point. Significance was set at p < 0.05. The horses were operated for an obstruction strangulated or not of the small or the large intestine. In six cases the postoperative period was uneventful, the 7 remaining developed one or 2 severe complications. Eight horses were discharged and 5 died during the hospitalization.The general aspect of the curve of mean plasmatic MPO can be described as follow: An increase was observed from the admission on until a peak of concentration occurring generally during the time of recovery from the anaesthesia with the highest mean value reaching 740.84 +/- 507.61ng/ml. This was followed by a progressive decrease until the lowest value, usually near to day 2 after the recovery from anaesthesia corresponding to 171.79 +/- 76.21 ng/ml of MPO. Afterwards, the mean concentrations increased slowly until postoperative day 10. In the majority of cases a stable and low MPO value (plateau) was observed during approximately 2 days (from day 1 to day 3 postoperatively).The initial peak of MPO after surgery could be associated to the neutrophil activation consequent to the intestinal disorder and the intense stimulation of the coeliotomy. The following significant reduction in concentration could be attributed to MPO infiltration into the tissues with a critical point at approximately 2 days after surgery. This study may contribute to a better understanding of the role of the MPO and neutrophils in the pathophysiology of horses in the postoperative period after colic surgery. [less ▲]

Evaluation of low-molecular-weight heparin for the prevention of equine laminitis after colic surgery

in Journal of Veterinary Emergency and Critical Care (2009), 19(1), 113-119

Objectives – The aim of this study is to describe the prevalence of postoperative laminitis in colic cases and to determine if low-molecular-weight heparin (LMWH) is effective in preventing this ... [more ▼]

Objectives – The aim of this study is to describe the prevalence of postoperative laminitis in colic cases and to determine if low-molecular-weight heparin (LMWH) is effective in preventing this complication. Design – Retrospective clinical study. Animals – Client-owned horses. Interventions – SC administration of enoxaparin during the postoperative period. Measurements and Main Results – Medical records of 360 horses undergoing surgery for colic and surviving at least 3 days were evaluated. Fifty-six horses admitted before 1995 did not receive LMWH (control group) and 304 admitted after 1995 received LMWH as a prophylaxis for laminitis (treatment group). Three grades of severity were defined for laminitis. Prevalence and severity of laminitis were compared between the 2 groups. Several parameters recorded on admission (sex, age, breed, site and nature of the disease, heart rate, PCV, gravity score, and shock score) and the administration of LMWH were tested as risk factors in the development of laminitis in a logistic regression procedure. Prevalence and grade of laminitis were significantly lower in the treatment group. Only the absence of LMWH was recognized as a significant risk factor in the logistic regression model. Conclusions – The administration of LMWH appears to be effective in the prophylaxis of laminitis following colic surgery and may be useful in the postoperative management of these horses. [less ▲]

Equine skeletal muscle cells submitted to anoxia/reoxygenation produce reactive oxygen species.

Poster (2009)

Activation of equine neutrophils by phorbol myristate acetate or N-formyl-methionyl-leucyl-phenylalanine induces a different response in reactive oxygen species production and release of active myeloperoxidase.

in Veterinary Immunology and Immunopathology (2009)

Neutrophil (PMN) contribution to the acute inflammatory processes may lead to an excessive generation of reactive oxygen metabolites species (ROS) and secretion of granule enzymes. We compared the effects ... [more ▼]

Neutrophil (PMN) contribution to the acute inflammatory processes may lead to an excessive generation of reactive oxygen metabolites species (ROS) and secretion of granule enzymes. We compared the effects of either phorbol myristate acetate (PMA) or N-formyl-methionyl-leucyl-phenylalanine (fMLP) in combination with a pre-treatment by cytochalasin B (CB) on the production of ROS and the release of total and active myeloperoxidase (MPO) by isolated equine PMNs. The ROS production was assessed by lucigenin dependent chemiluminescence (CL) and ethylene release by alpha-keto-gamma-methylthiobutyric acid (KMB) oxidation. In the supernatant of activated PMNs, total equine MPO was measured by ELISA and active MPO by the SIEFED (Specific Immunologic Extraction Followed by Enzymatic Detection) technique that allows for the study of the interaction of a compound directly with the enzyme. The stimulation of PMNs with CB-fMLP only modestly increased the release of MPO, but more than 70% of released MPO was active. PMA stimulation markedly increased the production of ROS and release of MPO, but more than 95% of released MPO was inactive. When PMNs were pre-incubated with superoxide dismutase (SOD) prior to PMA activation, the lucigenin enhanced CL, which is linked to the superoxide anion (O(2)(-)) production, was much more decreased than KMB oxidation, linked to the hydroxyl-like radical production. The addition of SOD prior to the activation of PMNs by PMA also limited the loss of the activity of released MPO. These results confirm the key role of O(2)(-) generation in the ROS cascade in PMN and reveal its critical role on MPO inactivation. [less ▲]

Method for co-purification of equine neutrophil elastase and myeloperoxidase from a limited blood volume.

in Research in Veterinary Science (2009)

Neutrophil myeloperoxidase (MPO) and elastase can be released in severe inflammatory diseases and cause tissue injuries. Equine enzymes have already been individually purified from large blood quantities ... [more ▼]

Neutrophil myeloperoxidase (MPO) and elastase can be released in severe inflammatory diseases and cause tissue injuries. Equine enzymes have already been individually purified from large blood quantities. We describe the isolation of both enzymes from a same limited blood volume. Both MPO and elastase were extracted by crushing PMN isolated by centrifugation on a percoll-gradient from a 460ml blood collection. MPO and elastase were separated by an ionic exchange chromatography phase and further purified by gel filtration chromatography on Superdex 200 and 75, respectively. Enzymes were identified in the collected fractions by specific enzymatic assays. The final purity was verified by electrophoresis. Specific activity was improved to 19.92 and 34.3x for elastase (final yield: 340mug) and MPO (final yield: 130mug), respectively, during the procedure. Results show the possibility of isolating both enzymes from the same blood sample with a sufficient yield and purity for future studies on their implication and interaction during inflammatory diseases. [less ▲]

Biphasic effect of acepromazine on the activity of equine myeloperoxidase in vitro

Poster (2008, October)

Rupture of small intestine into the mesentery without evidence of overload or obstruction in 4 horses

Poster (2008, January)

Datex-Ohmeda S/5 direct and indirect blood pressure monitoring during general anesthesia in the adult horse

(2008, January)

Effects of Unfractionated and Fractionated Heparins on Myeloperoxidase Activity and Interactions with Endothelial Cells: Possible Effects on the Pathophysiology of Equine Laminitis

in Veterinary Journal (2007), 178(1), 62-69

As heparins are sometimes used to prevent equine laminitis, the interactions between equine neutrophil myeloperoxidase (MPO), unfractionated (UFH) and fractionated low molecular weight (LMWH) heparins and ... [more ▼]

As heparins are sometimes used to prevent equine laminitis, the interactions between equine neutrophil myeloperoxidase (MPO), unfractionated (UFH) and fractionated low molecular weight (LMWH) heparins and digital endothelium have been investigated. The effects of the heparins on purified equine MPO activity were tested by immunocapture followed by enzymatic detection. Endothelium-MPO interactions were assessed by measuring total and active MPO uptake by arterial and venous digital endothelial cells in culture with or without the addition of heparins. A dose-dependent MPO inhibition by UFH and LMWH was seen, with the greatest reduction in MPO activity noted with the highest concentration of LMWH. The MPO capture was greater in arterial cells, but heparins better inhibited MPO capture in venous cells. The activity of cell-bound MPO was almost completely suppressed by the heparins, and no differences were observed between UFH and LMWH. The results confirm the anti-inflammatory properties of heparins and allow a better understanding of the potential role of MPO in laminitis. [less ▲]

Development of equine models of podal keratinocytes and endothelial cells cultures for the study of equine laminitis

Master of advanced studies dissertation (2006)

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 ... [more ▼]

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. [less ▲]