Neutrophil extracellular traps and active myeloperoxidase concentrate in lamellar tissue of equids with naturally occurring laminitis.

Laminitis is a pathology of the equine digit ultimately leading to a failure of the dermo-epidermal interface. Neutrophil activation is recognized as a major factor in SIRS-associated laminitis and has recently been described in induced endocrinopathic laminitis evidenced by the presence of myeloperoxidase (MPO). Neutrophil extracellular traps (NET) are released with neutrophil activation. This study aimed to investigate the presence and activity of MPO and NET in the lamellar tissue of equids presented with naturally occurring laminitis. Samples of lamellar tissue of five horses and five donkeys presented with laminitis, as well as eight control horses without laminitis, were collected. Lamellar tissue extracts were submitted to ELISA and specific immuno-extraction followed by enzymatic detection (SIEFED) assays to confirm the presence and activity of both MPO and NET. Lamellar sections were also immunohistopathologically stained for MPO and NET. Analysis of lamellar tissue extracts revealed that laminitis cases had significantly higher levels of total MPO concentration, MPO activity, and NET-bound MPO activity in comparison to control horses. Moreover, a strong correlation was identified between the activity of NET-bound MPO and the total MPO activity, which suggests that MPO activity partly originates from NET-bound MPO. Immunohistochemical staining showed that MPO and NET labelling in laminitis cases was moderate to marked, primarily in the epidermis and in inflammatory infiltrates containing neutrophils, while labelling in control horses was minimal. This article constitutes the first indication of the presence and activity of NET-bound MPO in the lamellar tissue of horses and donkeys with naturally occurring laminitis. Targeting these substances may provide new treatment possibilities for this debilitating disease.

Presence of Myeloperoxidase in Lamellar Tissue of Horses Induced by an Euglycemic Hyperinsulinemic Clamp.

Laminitis is a pathology of the equine digit leading to a failure of the dermo-epidermal interface. Neutrophil activation is recognized as a major factor in SIRS-associated laminitis. Less is known about the role of neutrophil activation in laminitis associated with metabolic disorders. The aim of this descriptive study was to observe whether myeloperoxidase is increased in the laminae during early stage laminitis in three horses subjected to a prolonged euglycemic hyperinsulinemic clamp (pEHC). After 48 h of pEHC-treatment, horses were subjected to euthanasia. Two healthy horses are used as control. Histological sections of lamellar tissue from all horses were immunohistochemically stained for myeloperoxidase and counterstained with hematoxylin-eosin. Histopathological changes that characterize insulin-induced laminitis and increased presence of myeloperoxidase, especially in the dermal lamellae, were increased in histologic sections of pEHC-treated horses. Neutrophil myeloperoxidase release may contribute to the pathophysiology of endocrinopathic laminitis.

Endoscopy Guided Photoablation of Endometrial Cysts using a 980 nm Laser with a Contact Fiber in Mares.

In mares, endometrial cysts are associated with endometriosis and can cause maternal recognition failure or compromise and delay pregnancy diagnoses. Historical treatments were invasive and had adverse effects on the endometrium. Hysteroscopically guided laser therapy is easy and effective for endometrial cysts resection, with no deleterious effects for the endometrium. A 110 cm long and 1.0 cm wide endoscope is sterilely introduced in the uterus through the open cervix of an estrous mare after vulvar cleaning. The uterus is slowly infused with less than 1 L of physiologic solution and the laser fiber is inserted in the biopsy canal of the endoscope. Cysts are then cauterized with the 980 nm diode laser with a contact fiber set at 20‒2 5W in continuous mode. Each cyst is punctured until complete voiding of the cyst and shrinking of the cyst wall around the fiber. Uterine lavages with sterile saline solution are performed directly after the surgery and for one or two days as non-inflammatory fluid can be observed. This procedure is easy and quickly performed, with no obvious deleterious effects. Cysts resection makes ultrasound pregnancy diagnosis easier and, in some cases, could restore proper embryo migration in the uterine horns between day 6.5 and 17. However, this treatment does not improve the underlying histological lesions related to endometriosis. These considerations should be clearly expressed to the breeder before this procedure.

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 the presence of non-sperm cells (NSC). The objective of this study was to evaluate the effect of a single-layer colloidal centrifugation before cryopreservation on NSC and MPO concentrations in equine semen. The experimental design consisted of 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 NSC and MPO concentrations in post-thaw semen. The MPO concentration was correlated with concentration of NSC 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 NSC. Overall, neutrophils were rarely observed and NSC were mainly epithelial cells or cellular debris, as demonstrated by MPO immunocytochemistry. At all steps of the semen processing and cryopreservation, MPO immunostaining was clearly identified only on NSC. In conclusion, our study shows that NSC present in fresh semen release MPO during freezing.

Sevoflurane inhibits equine myeloperoxidase release and activity in vitro.

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.

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

OBJECTIVE: To investigate the effect of acepromazine (ACP) on reactive oxygen species (ROS) production by stimulated equine neutrophils. STUDY DESIGN: Ex vivo biochemical experiments. ANIMALS: Isolated neutrophils from healthy untreated horses. METHODS: Neutrophils were incubated with ACP at concentrations of 10(-4), 10(-5) or 10(-6) M and then stimulated with phorbol-myristate-acetate (PMA) before measurement of lucigenin-enhanced chemiluminescence (CL). In a second experiment neutrophils were incubated in the presence of α-keto-γ methylthiobutyric acid (KMB) and treated with ACP at concentrations of 10(-4), 10(-5) or 10(-6) M. Subsequent PMA stimulation lead to neutrophilic ROS production and decomposition of KMB to ethylene, which is measured by gas chromatography. Electron paramagnetic resonance-spin trapping (EPR) analysis was performed with PMA-stimulated neutrophils in the presence of ACP (10(-4), 10(-5) or 10(-6) M) directly added to the cell suspension. In the second experiment, the same concentrations of ACP were pre-incubated with neutrophils, then centrifuged to eliminate the excess of ACP and re-suspended in phosphate buffer before stimulation with PMA. In all experiments, the results of ACP-treated and ACP-untreated stimulated neutrophils were compared. RESULTS: Overall, results obtained with lucigenin-enhanced CL and KMB oxidation were in agreement with those seen in electron paramagnetic resonance spectroscopy. Acepromazine induced a dose-dependent inhibitory effect on neutrophilic ROS production. Electron paramagnetic resonance also showed, at high ACP concentration, the appearance of a cation radical derived from ACP. In contrast, electron paramagnetic resonance study performed with pre-incubated neutrophils showed an important dose-dependent inhibitory effect of ACP. CONCLUSION: The results indicate that ACP can neutralize O.-2 or its by-products during the stimulation of neutrophils. CLINICAL RELEVANCE: These findings may have a therapeutic relevance when phenothiazines are used in horses suffering from inflammatory diseases in which neutrophil activation and ROS production are implicated.

Neutrophil myeloperoxidase measurements in plasma, laminar tissue, and skin of horses given black walnut extract.

OBJECTIVE: To compare measurements of myeloperoxidase (MPO) in plasma, laminar tissues, and skin obtained from control horses and horses given black walnut heartwood extract (BWHE). ANIMALS: 22 healthy 5- to 15-year-old horses. PROCEDURES: Horses were randomly assigned to 4 groups as follows: a control group given water (n = 5) and 3 experimental groups given BWHE (17) via nasogastric intubation. Experimental groups consisted of 5, 6, and 6 horses that received BWHE and were euthanatized at 1.5, 3, and 12 hours after intubation, respectively. Control horses were euthanatized at 12 hours after intubation. Plasma samples were obtained hourly for all horses. Laminar tissue and skin from the middle region of the neck were harvested at the time of euthanasia. Plasma and tissue MPO concentrations were determined via an ELISA; tissue MPO activity was measured by use of specific immunologic extraction followed by enzymatic detection. RESULTS: Tissues and plasma of horses receiving BWHE contained significantly higher concentrations of MPO beginning at hour 3. Laminar tissue and skin from horses in experimental groups contained significantly higher MPO activity than tissues from control horses. Concentrations and activities of MPO in skin and laminar tissues were similar over time. CONCLUSIONS AND CLINICAL RELEVANCE: In horses, BWHE administration causes increases in MPO concentration and activity in laminar tissue and skin and the time of increased MPO concentration correlates with emigration of WBCs from the vasculature. These findings support the hypothesis that activation of peripheral WBCs is an early step in the pathogenesis of acute laminitis.