Effect of different kinds of anoxia/reoxygenation on the mitochondrial function and the free radicals production of cultured primary equine skeletal myoblasts.

Horses are outstanding athletes, performing in many different disciplines involving different kinds of efforts and metabolic responses. Depending on exercise intensity, their skeletal muscle oxygenation decreases, and the reperfusion at cessation of the exercise can cause excessive production of free radicals. This study on cultured primary equine myoblasts investigated the effect of different kinds of anoxia/reoxygenation (A/R) on routine respiration, mitochondrial complex I specific activity and free radicals production. Our data revealed that short cycles of A/R caused a decrease of all the parameters, opposite to what a single long period of anoxia did. A preconditioning-like effect could explain our first pattern of results whereas mild uncoupling could be more appropriate for the second one. Anyway, it seems that mitochondrial complex I could play a major role in the regulation of the balance between metabolic and antioxidant protection of the muscular function of athletic horses.

Effect of myeloperoxidase and anoxia/reoxygenation on mitochondrial respiratory function of cultured primary equine skeletal myoblasts.

Horses are particularly sensitive to excessive inflammatory reaction where myeloperoxidase, a marker of inflammation, may contribute to mitochondrial dysfunctions. This study investigated the interaction between myeloperoxidase and cultured primary equine skeletal myoblasts, particularly its effect on mitochondrial respiration combined or not with anoxia followed by reoxygenation (AR). We showed that active myeloperoxidase entered into the cells, interacted with mitochondria and decreased routine and maximal respirations. When combined with AR, myeloperoxidase caused a further decrease of these respiratory parameters while the leak increased. Our results indicate that myeloperoxidase amplifies the mitochondrial damages initiated by AR phenomenon and alters the mitochondrial function.

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

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-α 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-α, LPS and LPS/TNF-α, induced a significant release of total MPO in whole blood but only the combinations CB/fMLP and LPS/TNF-α 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.

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.

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.

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.

Histology of two rice bodies isolated from the stifle of an adult draught horse stallion.

In the human and equine species, different kinds of free floating intra-articular particles are related to certain disorders. Osteochondral fragments formed during osteochondrosis dissecans are the most common finding in the equine species, whereas in humans rice bodies due to rheumatoid arthritis are more frequent. Herein we report a third type of floating body inside the stifle of an adult draught horse stallion, in macroscopic appearance similar to articular rice bodies known in humans. As revealed by histologic examination, the two particles consist of polypoid degenerated structures derived from synovial villi. Their formation was probably induced by ischemia.

Synoviocytes, not chondrocytes, release free radicals after cycles of anoxia/re-oxygenation.

By oxymetry and electron paramagnetic resonance (EPR), we investigated the effects of repeated anoxia/re-oxygenation (A/R) periods on the respiration and production of free radicals by synoviocytes (rabbit HIG-82 cell line and primary equine synoviocytes) and equine articular chondrocytes. Three periods of 20 min anoxia followed by re-oxygenation were applied to 10(7)cells; O(2) consumption was measured before anoxia and after each re-oxygenation. After the last A/R, cellular free radical formation was investigated by EPR spectroscopy with spin trapping technique (n=3 for each cell line). Both types of synoviocytes showed a high O(2) consumption, which was slowered after anoxia. By EPR with the spin trap POBN, we proved a free radical formation. Results were similar for equine and rabbit synoviocytes. For chondrocytes, we observed a low O(2) consumption, unchanged by anoxia, and no free radical production. These observations suggest an oxidant activity of synoviocytes, potentially important for the onset of osteoarthritis.