An in vitro whole blood model to test the effects of different stimuli conditions on the release of myeloperoxidase and elastase by equine neutrophils.

in Veterinary Immunology and Immunopathology (2012), 150(3-4), 221-7

Horses are particularly sensitive and exposed to excessive inflammatory responses evolving toward an important stimulation of polymorphonuclear neutrophils (PMNs). The aim of this work was to stimulate ... [more ▼]

Horses are particularly sensitive and exposed to excessive inflammatory responses evolving toward an important stimulation of polymorphonuclear neutrophils (PMNs). The aim of this work was to stimulate equine neutrophils in whole blood and to evaluate their response by measuring the release of total and active myeloperoxidase (MPO) and total elastase, considered as markers of neutrophil stimulation and degranulation. Because of the critical importance of the concomitant presence of LPS and TNF-alpha in equine pathological situations, we combined these two natural mediators to stimulate PMN and compared the response with those obtained after the PMN stimulation with each mediator used alone and well-known artificial stimulation systems such as 12-phorbol 13-myristate acetate (PMA) and the combination of cytochalasin B (CB) and N-formyl-methionyl-leucyl-phenylalanine (fMLP). All the activation systems, PMA, CB/fMLP, TNF-alpha, LPS and LPS/TNF-alpha, induced a significant release of total MPO in whole blood but only the combinations CB/fMLP and LPS/TNF-alpha significantly favored the release of active MPO. Regarding the total elastase, we did not observe a significant release in all the stimulated conditions except with PMA. It appears clearly that the choice of the neutrophil stimulation model is fundamental for the selection of potentially active pharmacological agents, especially on MPO activity. [less ▲]

Les kératomes: lésions et traitements chez 19 chevaux

Conference (2012, October 12)

Introduction: Les kératomes, masses hyperplasiques de kératine, croissent entre la phalange distale (P3) et la paroi du sabot et provoquent des boiteries chez le cheval. La littérature comporte de ... [more ▼]

Introduction: Les kératomes, masses hyperplasiques de kératine, croissent entre la phalange distale (P3) et la paroi du sabot et provoquent des boiteries chez le cheval. La littérature comporte de nombreux rapports de cas isolés mais peu de séries cliniques. Matériel et méthodes: Les dossiers cliniques des cas de kératomes présentés depuis 2004 à la faculté de médecine vétérinaire de Liège ont été revus. Résultats: Vingt-trois kératomes ont été diagnostiqués sur 19 chevaux boiteux. Un cheval présentait 2 kératomes sur le même pied et 3 chevaux présentaient 2 kératomes sur des pieds différents. Sur les 22 pieds atteints, les lésions observées étaient: un abcès, souvent récidivant (18 cas), une déformation de la muraille (6 cas), une déviation de la ligne blanche (6 cas) et une seime (3 cas). Un kératome était situé sous la sole et 22 sous la muraille. La durée des symptômes variait de 2 semaines à 15 ans (plus d'un an sur 12 pieds). Sur les radiographies, une lyse par compression de P3 était visible en regard de 20 kératomes. Le cheval présentant 2 kératomes sur le même pied a été euthanasié. Trois kératomes ont été traités de manière conservative. Dix-huit kératomes (chez 16 chevaux) ont été excisés chirurgicalement dont 16 par avulsion complète de muraille, 1 par avulsion partielle de muraille et 1 par curetage d'une portion de sole. Aucune complication postopératoire n'a été observée dans 7/18 cas (39%). Les complications rencontrées étaient: une granulation excessive (10 cas), une douleur importante pendant plusieurs jours (5 cas), une légère infection (4 cas), un enfoncement de P3 en regard de la zone avulsée (2 cas) et une récidive (2 cas, dont 1 a été réopéré avec succès ultérieurement). Un cheval est encore en convalescence au moment de cette étude. Le suivi à long terme d’un cheval a été perdu. Des 14 chevaux repris dans l'évaluation postopératoire, 1 a été euthanasié pour une autre raison que le kératome, 3 chevaux ont gardé une boiterie résiduelle au trot et 10 chevaux ont récupéré leur niveau d'activité. Discussion: La littérature décrit les kératomes comme rares1. Or, 21% des chevaux de cette étude (4 chevaux sur 19) présentaient plusieurs kératomes. De plus, dans 54 % des cas, les symptômes duraient depuis plus d’un an. Dès lors, il apparait que les kératomes sont sous diagnostiqués ou le sont souvent tardivement. La chronicité et l’étendue des kératomes de cette étude n’a que très rarement permis leur résection par avulsion partielle de muraille, technique de choix dont les complications postopératoires sont moindres². Un diagnostic précoce par inspection minutieuse des sabots, particulièrement en cas d’abcès de pied récidivant permettrait une excision par une technique moins invasive, qui diminuerait la convalescence et les complications postopératoires. Bibliographie: 1. Sundberg, J.P. et al. Neoplasms of Equidae. Journal of American veterinary medical association, 1977, 170: 150-152. 2. Boys Smith SJ. et al. Complete and partial hoof wall resection for keratoma removal: postoperative complications and final outcome in 26 horses (1994-2004). Equine Veterinary Journal, 2006, 38 (2): 127-133. [less ▲]

Sevoflurane inhibits equine myeloperoxidase release and activity in vitro.

in Veterinary Anaesthesia & Analgesia (2012)

Objective To investigate the effects of the volatile anaesthetic sevoflurane on the release of total and active myeloperoxidase (MPO) by non-stimulated and stimulated polymorphonuclear neutrophils (PMNs ... [more ▼]

Objective To investigate the effects of the volatile anaesthetic sevoflurane on the release of total and active myeloperoxidase (MPO) by non-stimulated and stimulated polymorphonuclear neutrophils (PMNs) in whole blood from healthy horses. Study design In vitro experimental study. Animals Adult healthy horses. Methods Samples of whole venous blood were collected and incubated in air or in air plus 2.3% or 4.6% sevoflurane for 1 hour. PMNs were stimulated with N-formyl-methionyl-leucyl-phenylalanine (fMLP), with a combination of cytochalasin B (CB) and fMLP or with phorbol myristate acetate (PMA). Total and active MPO contents released by PMNs in blood were measured by enzyme-linked immunosorbent assay (ELISA) and specific immunological extraction followed by enzymatic detection (SIEFED) respectively. Additional experiments were performed to assess the effect of sevoflurane on the peroxidase and chlorination cycles of purified equine MPO using Amplex Red and 3'-(p-aminophenyl) fluorescein as fluorogenic substrates respectively. Results As compared with air alone, 1 hour exposure of whole blood to 4.6% sevoflurane in air significantly inhibited the release of total and active MPO by unstimulated and both fMLP- and CB + fMLP-stimulated PMNs but not by PMA-stimulated PMNs. Although 2.3% sevoflurane had no effect on total MPO release by unstimulated and stimulated PMNs, it significantly reduced the release of active MPO by unstimulated and fMLP-stimulated PMNs. Additionally, sevoflurane reversibly inhibited the activity of MPO, especially the peroxidase cycle of the enzyme. Conclusions and clinical relevance Although our experimental study was not designed to assess the effects of sevoflurane in vivo, this inhibition of MPO release and activity may have relevance for anaesthetized horses and deserves further studies to examine the clinical importance of these findings. [less ▲]

Assessment of reactive oxygen species production in cultured equine skeletal myoblasts in response to conditions of anoxia followed by reoxygenation with or without exposure to peroxidases.

in American Journal of Veterinary Research (2012), 73(3), 426-434

Objective—To culture equine myoblasts from muscle microbiopsy specimens, examine myoblast production of reactive oxygen species (ROS) in conditions of anoxia followed by reoxygenation, and assess the ... [more ▼]

Objective—To culture equine myoblasts from muscle microbiopsy specimens, examine myoblast production of reactive oxygen species (ROS) in conditions of anoxia followed by reoxygenation, and assess the effects of horseradish peroxidase (HRP) and myeloperoxidase (MPO) on ROS production. Animals—5 healthy horses (5 to 15 years old). Procedures—Equine skeletal myoblast cultures were derived from 1 or 2 microbiopsy specimens obtained from a triceps brachii muscle of each horse. Cultured myoblasts were exposed to conditions of anoxia followed by reoxygenation or to conditions of normoxia (control cells). Cell production of ROS in the presence or absence of HRP or MPO was assessed by use of a gas chromatography method, after which cells were treated with a 3,3′-diaminobenzidine chromogen solution to detect peroxidase binding. Results—Equine skeletal myoblasts were successfully cultured from microbiopsy specimens. In response to anoxia and reoxygenation, ROS production of myoblasts increased by 71%, compared with that of control cells. When experiments were performed in the presence of HRP or MPO, ROS production in myoblasts exposed to anoxia and reoxygenation was increased by 228% and 183%, respectively, compared with findings for control cells. Chromogen reaction revealed a close adherence of peroxidases to cells, even after several washes. Conclusions and Clinical Relevance—Results indicated that equine skeletal myoblast cultures can be generated from muscle microbiopsy specimens. Anoxia-reoxygenation– treated myoblasts produced ROS, and production was enhanced in the presence of peroxidases. This experimental model could be used to study the damaging effect of exercise on muscles in athletic horses. [less ▲]

Time trends of blood leucocytes, neutrophils and plasmatic myeloperoxidase in the perioperative period of horses undergoing colic surgery.

Poster (2011, July)

Background: Despite the recent advances in this area, colic remains a major cause of morbidity and death in horses. Neutrophilic activation and degranulation may play a key role in the postoperative ... [more ▼]

Background: Despite the recent advances in this area, colic remains a major cause of morbidity and death in horses. Neutrophilic activation and degranulation may play a key role in the postoperative complications. Activated neutrophils release enzymes like proteases and myeloperoxidase (MPO). MPO concentrations in plasma and tissue are considered as a marker of neutrophil activation. (McConnico et al. 1999; Hoy et al. 2002). When freed in the tissue, active MPO is able to oxidize, nitrate and chlorate most organic molecules (Klebanoff 2005). Objectives: The aim of this study was 1) to determine the time trends of blood leukocyte and neutrophil counts as well as of plasmatic MPO concentrations in the perioperative period of horses undergoing colic surgery and 2) to relate these time trends to the location of the pathology, the severity of postoperative complications and to the outcome of the patients. Methods: Fifty two horses undergoing colic surgery at the Equine Teaching Hospital of the University of Liege were included in this study. The location of the predominant lesion of the intestine, the severity of the postoperative complications and the outcome were recorded for each horse. Total leukocyte and neutrophil counts were performed in all of the horses while plasmatic myeloperoxidase levels were determined in 16 of them. The blood samplings were realized before and during the surgery (after correction of the intestinal lesion), during the recovery and every 4 hours during the first 4 days (from day 0 until day 4) and then every 12 hours until day 6 (150th hour after the first blood sampling) or until euthanasia. Hematologic analyses were performed at the time of sampling by use of the Medonic CA 530 (Menarini, Zaventem, Belgium). The blood was then centrifuged and the plasma was aliquoted and frozen at -20° C until assayed. MPO was assayed with a specific enzyme-linked immunosorbent assay (Equine MPO-ELISA kit, BiopTis, Liège, Belgium). A mixed model was used to analyze the time trends of leukocytes, neutrophils and MPO. All computations were done with the SAS (Statistical Analysis System) procedure Proc Mixed, with Satterthwaite degrees of freedom. The significance level was set at p=0.05. Results: The main pathology was found in the large intestine in 30 horses (58%) and the small intestine in 22 horses (42%). Forty horses (77%) survived to discharge from the clinic. Twelve horses were euthanized during the postoperative period. Their survival time varied from 0.5 day to 20 days with a mean of 7.8 days. Twenty-two horses (42%) showed none or mild complications, 12 horses (23%) showed moderate complications and 18 horses (35%) suffered from severe complications. Time trends for leukocytes and neutrophils were similar to each other (p= 0.7205) and significantly different (p< 0.0001) from the MPO time trend, which increased during the first hours, while the neutrophil time trend decreased immediately after the admission. The time trend of neutrophils was higher in large intestinal than in small intestinal pathologies and the time trend of MPO was lower in large intestinal than in small intestinal pathologies. The time trends of neutrophils were significantly different between the degrees of complications (no/mild vs moderate vs severe). For the first part of the curve, the more severe the complication, the lower is the time trend. The time trend of MPO was lower in survivors. The time trend of neutrophils in survivors was higher during the first 4 days thereafter it becomes lower than in non survivors. Conclusions: These results confirm that neutrophil counts and MPO levels undergo timely changes and that they are related to the severity of the inflammatory reaction in surgical colic cases. Knowing the kinetics of these parameters is an essential step to further determine cut-off values (with a larger group of horses) for the prognosis of horses after colic surgery. References Hoy, A., Leininger-Muller, B., Kutter, D., Siest, G. and Visvikis, S. (2002) Growing significance of myeloperoxidase in non-infectious diseases. Clin. Chem. Lab. Med. 40, 2-8. Klebanoff, S.J. (2005) Myeloperoxidase: friend and foe. J. Leukoc. Biol. 77, 598-625. McConnico, R.S., Weinstock, D., Poston, M.E. and Roberts, M.C. (1999) Myeloperoxidase activity of the large intestine in an equine model of acute colitis. Am. J. Vet. Res. 60, 807-813. [less ▲]

Production of free radicals and oxygen consumption by primary equine endothelial cells during anoxia-reoxygenation.

in Open Biochemistry Journal (The) (2011), 5

The endothelium plays an active role in ischemia/reperfusion injuries. Herein, we report the effect of a single or successive cycles of anoxia/reoxygenation (A/R) on the mitochondrial respiratory function ... [more ▼]

The endothelium plays an active role in ischemia/reperfusion injuries. Herein, we report the effect of a single or successive cycles of anoxia/reoxygenation (A/R) on the mitochondrial respiratory function of equine endothelial cells (cultured from carotids) monitored by high resolution oxymetry, and on their production of reactive oxygen species (ROS). ROS were measured by electron paramagnetic resonance (ESR) using POBN and DMPO spin traps, and by gas chromatography (GC) of ethylene released by ROS-induced alpha-keto-gamma-(methylthio)butyric acid (KMB) oxidation. The oxygen consumption significantly decreased with the number of A/R cycles, and POBN-ESR spectra were specific of adducts formed in the cells from superoxide anion. After a one-hour A/R cycle, high intensity DMPO-ESR spectra were observed and assigned to superoxide anion trapping; the GC results confirmed an important production of ROS compared to normoxic cells. These results show that A/R induces mitochondrial alterations in endothelial cells, and strongly stimulates their oxidative activity as demonstrated by ESR and GC methods. [less ▲]

Modulating effects of acepromazine on the reactive oxygen species production by stimulated equine neutrophils

in Veterinary Anaesthesia & Analgesia (2011), 38

To investigate the effect of acepromazine (ACP) on reactive oxygen species (ROS) production by stimulated equine neutrophils.

Is neutrophil elastase associated with myeloperoxidase concentration and post-thawing parameters in equine frozen semen?

in Animal Reproduction Science (2010), 121S

Non sperm cells concentration is higher in unfreezable semen. A relation between non-sperm cells in fresh semen and MPO and post thawing quality has been observed. Neutrophil Elastase seems to have no ... [more ▼]

Non sperm cells concentration is higher in unfreezable semen. A relation between non-sperm cells in fresh semen and MPO and post thawing quality has been observed. Neutrophil Elastase seems to have no effect on semen characteristics and to be not associated with on-sperm cells and freezability. [less ▲]

Specific immuno-extraction followed by enzymatic detection (SIEFED) of myeloperoxidase and mitochondrial complex I in muscular microbiopsies: preliminary results in endurance horses

in Equine Veterinary Journal. Supplement (2010), 42(Suppl. 38), 296-302

Effect of intensive exercise on plasmatic neutrophil elastase level in eventing and endurance horses

in Equine Veterinary Journal. Supplement (2010), 48

Reasons for performing the study – Intensive exercise induces a systemic inflammatory response characterized by an increase of blood neutrophil count and myeloperoxidase (MPO) release. Neutrophil elastase ... [more ▼]

Reasons for performing the study – Intensive exercise induces a systemic inflammatory response characterized by an increase of blood neutrophil count and myeloperoxidase (MPO) release. Neutrophil elastase (NE) could also contribute to tissues lesions by their proteinase activities. Objective – To compare plasmatic NE concentrations before and after different forms of intensive exercise. Materials and Methods – EDTA blood samples were taken from 51 eventing horses (EvH) and 32 endurance horses (EndH) were sampled before the race (T0). Blood sampling was performed 2 h (T1) after completing either phase D of a one or two star eventing competition (n=51) or a 120 or 160 km endurance race (n=32). Plasmatic NE and MPO were measured by a specific equine ELISA. Neutrophil counts and creatine kinase (CK) levels were also measured. A Wilcoxon test for paired samples was used to compare mean values of neutrophils, CK, MPO and NE at T0 and T1 in EvH and in EndH. Correlations were calculated between all the 4 parameters in EvH and EndH. Results – At T0, mean NE levels were 14.43 ± 3.63 ng/ml for EvH and 11.7 ± 2.11 ng/ml for EndH. The competition induced a significant increase of NE levels in (58.57 ± 24.06 ng/mL) EvH and (95.74 ± 22.70 ng/mL) EndH (p < 0,05). NE was significantly (p < 0,0001) correlated to MPO in EvH (r = 0.293) and EndH (0.594) and to CK (r = 0.297) in EndH (p<0.0001). Neutrophils, CK and MPO were significantly increased between T0 and T1 in both types of horses. Conclusions – Significant increase of NE was observed after intense exercise with a significant correlation between NE and MPO. The huge variability in MPO and ELT, indicates, that not all horses show the same intensity of systemic inflammatory response. [less ▲]