nerve-stimulator-guided-injection-autologous-stem-cells-near-equine

Nerve Stimulator-guided Injection of Autologous Stem Cells Near the Equine Left Recurrent Laryngeal Nerve

Recurrent laryngeal neuropathy (RLN) commonly affects horses and is characterized by abnormal respiratory sounds and exercise intolerance. The recurrent ...

University of Liege

Mont-le-Soie Equine Research Centre

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.

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

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.

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

We investigated the oxygen (O(2)) uptake of equine articular chondrocytes to assess their reactions to anoxia/re-oxygenation. They were cultured under 5% or 21% gas phase O(2) and at glucose concentrations of 0, 1.0 or 4.5g/L in the culture medium (n=3). Afterwards, the O(2) consumption rate of the chondrocytes was monitored (oxymetry) before and after an anoxia period of 25min. The glucose consumption and lactate release were measured at the end of the re-oxygenation period. The chondrocytes showed a minimal O(2) consumption rate, which was hardly changed by anoxia. Independently from the O(2) tension, glucose uptake by the cells was about 30% of the available culture medium glucose, thus higher for cells at 4.5g/L glucose (n=3). Lactate release was also independent from O(2) tension, but lower for cells at 4.5g/L glucose (n=3). Our observations indicated that O(2) consumption by equine chondrocytes was very low despite a functional mitochondrial respiratory chain, and nearly insensitive to anoxia/re-oxygenation. But the chondrocytes metabolism was modified by an excess of O(2) and glucose.

Oxygen consumption of equine articular chondrocytes: Influence of applied oxygen tension and glucose concentration during culture.

Osteochondrosis (OC) is a developmental disease that affects growing horses and that severely affects their ability to perform. The genetic basis of its pathogenesis is poorly understood. The aim of the study was to analyze the transcript profile of leukocytes from horses affected with OC. Two transcriptome libraries were constructed from leukocytes of OC-affected and non-OC-affected horses using digital gene expression analysis (DGE) and real-time PCR. Statistical analysis allowed selection of 1,008 tags upregulated in the non-OC-affected group and 1,545 tags upregulated in the OC-affected group. Among these genes, 16 regulated genes and 5 housekeeping genes were selected. Metabolic pathways analysis showed an obvious dysregulation of several signaling pathways related to cartilage formation or cartilage repair, including Wnt, Indian hedgehog, and TGF-beta signaling. Other genes, including ISG, ApoB, MGAT4, and TBC1D9, showed a significantly different expression between groups. These genes may play a role in high carbohydrate diet, abnormal insulin metabolism, or inflammation, mechanisms suspected to be involved in OC. This DGE analysis of the transcript profile of leukocytes from OC-affected horses demonstrated significant differences in comparison to the control library. These results open new perspectives for the understanding of equine OC.

Gene expression profiling from leukocytes of horses affected by osteochondrosis.

In neutrophils (PMNs), superoxide anion (O2*-), the first reactive oxygen species (ROS) produced to kill pathogenic agents, is generated by NADPH oxidase, an enzymatic complex formed by the translocation of cytosolic subunits to the membrane flavocytochrome b558. In horses, excessive activation of PMNs is often associated with deadly pathologies and the modulation of their ROS production by acting on NADPH oxidase is a prime target to manage inflammation. We developed a cell-free assay to measure the activity of equine NADPH oxidase assembled in vitro, in order to test the effects of natural or synthetic compounds on the enzyme activity or assembly. The cell-free assay was validated with diphenyleneiodonium chloride and Gp91ds-tat, two inhibitors largely described for human NADPH oxidase. The anti-oxidant effects of curcumin and resveratrol at final concentration ranging from 10(-4) to 10(-6) M were studied on whole cells by chemiluminescence (CL) and by cell-free assay, in which the molecule was added before or after the enzyme assembly. The CL assay demonstrated that curcumin efficiently inhibited the O2(-) production and easily entered into PMNs or interacted with their membrane. Cell-free assay showed that curcumin acted on the reconstitution of NADPH oxidase even at 10(-5)M, while resveratrol appeared to be an O2*- scavenger rather than an inhibitor of NADPH oxidase activity, since it acted from outside the cell in CL and after the complex assembly in cell-free assay. By acting directly on NADPH oxidase, curcumin should be a good candidate for the treatment of acute or inflammatory diseases involving an excessive ROS production.

Curcumin and resveratrol act by different ways on NADPH oxidase activity and reactive oxygen species produced by equine neutrophils.

The aim of this work was to completely replace the egg yolk a classical diluent for freezing equine semen by a cyclodextrin-cholesterol complex. At the same time, the reduction in the glycerol content used for cryopreservation and the incubation time between sperm and the freezing media were evaluated. Horse ejaculates were frozen with four different freezing extenders: a frozen reference medium (IF) containing egg yolk and 2.5% glycerol and media without egg yolk but supplemented with 1.5 mg 2-hydroxypropyl-beta-cyclodextrin cholesterol (HPβCD-C) complex and containing either 1% (G1), 2% (G2) or 3% glycerol (G3). Three incubation times (90, 120 and 180 min) at 4 °C between the fresh semen and the different media were tested before freezing. Viability and motility analyses were performed with computer assisted semen analysis (CASA). Results showed that the freezing media containing the HPβCD-C complex with 1%, 2% and 3% glycerol significantly improve the 3 in vitro parameters of post thawing semen quality (viability, progressive and total mobilities) compared to IF. The best improvement of the parameters was obtained with G1 medium and the longest contact time. The substitution of egg yolk by HPβCD-C complex allows the decrease of protein charge of the medium while favouring the cholesterol supply to membrane spermatozoa offering it a better resistance to osmotic imbalance and a better tolerance to the glycerol toxicity. Our results highlight that the egg yolk of an extender for the freezing of horse semen can be completely substituted by HPβCD-C complex. 

Substitution of egg yolk by a cyclodextrin-cholesterol complex allows a reduction of the glycerol concentration into the freezing medium of equine sperm.

Osteochondrosis dissecans (OCD) is a developmental orthopedic disease caused by a failure of the endochondral ossification in epiphyseal plates and joint cartilage. This trouble may induce the presence of osteochondral fragments in the articulation, fissures or subchondral bone cysts in the growth cartilage. Occurrence of osteochondrosis is influenced by a complex interaction of different factors. Among these, the effect of the housing and the feeding of the foals during their first months of life, have been described as risk factors for the development of osteochondrosis. The aim of this study was to investigate the evolution of OCD lesions with a longitudinal study in 204 young foals from 6 to 18 months in comparison to the type of feeding and the type of housing conditions. These factors and OCD status were obtained by a questionnaire and radiological examination, respectively. This allowed dividing the foals into four groups according to the initial OCD status and the evolution of the condition. As a result, we found that foals fed with concentrates show a higher probability to develop OCD lesions (p=0.06), while foals not receiving concentrates, had a higher probability to heal from existing OCD lesions (p=0.001). This study supports the theory that management factors such as feeding or housing may influence the evolution of the osteochondrosis disease. 

Article	Year
Synoviocytes, not chondrocytes, release free radicals after cycles of anoxia/re-oxygenation. Biochemical and biophysical research communications\| PubMed ID: 16036131	2005
Histology of two rice bodies isolated from the stifle of an adult draught horse stallion. Journal of veterinary science\| PubMed ID: 16434856	2006
Oxygen consumption of equine articular chondrocytes: Influence of applied oxygen tension and glucose concentration during culture. Cell biology international\| PubMed ID: 17442596	2007
Gene expression profiling from leukocytes of horses affected by osteochondrosis. Journal of orthopaedic research : official publication of the Orthopaedic Research Society\| PubMed ID: 20108324	2010
Curcumin and resveratrol act by different ways on NADPH oxidase activity and reactive oxygen species produced by equine neutrophils. Chemico-biological interactions\| PubMed ID: 24060679	2013
Substitution of egg yolk by a cyclodextrin-cholesterol complex allows a reduction of the glycerol concentration into the freezing medium of equine sperm. Cryobiology\| PubMed ID: 26687387	2016
Impact of feeding and housing on the development of osteochondrosis in foals-A longitudinal study. Preventive veterinary medicine\| PubMed ID: 27094134	2016

Jean-Philippe Lejeune

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