Mammalian muscle-derived stem cells
Serteyn, Didier ; Ceusters, Justine
The present invention provides a new method of obtaining muscle-derived mesenchymal stem cells from microbiopsies of mammalian origin. The invention provides for a minimally invasive methodology yielding ... [more ▼]
The present invention provides a new method of obtaining muscle-derived mesenchymal stem cells from microbiopsies of mammalian origin. The invention provides for a minimally invasive methodology yielding high amounts of MSCs that can differentiate into different cell lineages. [less ▲]Detailed reference viewed: 36 (3 ULg)
EquiNox2: a new method to measure NADPH oxidase activity and to study effect of inhibitors and their interactions with the enzyme
Derochette, Sandrine ; Serteyn, Didier ; Mouithys-Mickalad, Ange et al
in Talanta (2015)
Excessive neutrophil stimulation and reactive oxygen species (ROS) production are involved in numerous human or horse pathologies. The modulation of the neutrophil NADPH oxidase (NOX) has a great ... [more ▼]
Excessive neutrophil stimulation and reactive oxygen species (ROS) production are involved in numerous human or horse pathologies. The modulation of the neutrophil NADPH oxidase (NOX) has a great therapeutic potential since this enzyme produces superoxide anion whose most of the other ROS derive. The measurement of NOX activity by cell-free systems is often used to test potential inhibitors of the enzyme. A major drawback of this technique is the possible interferences between inhibitors and the probe, ferricytochrome c, used to measure the activity. We designed the "EquiNox2", a new pharmacological tool, to determine the direct interaction of potential inhibitors with equine phagocytic NOX and their effect on the enzyme activity or assembly. This method consists in binding the membrane fractions of neutrophils containing flavocytochrome b558 or the entire complex, reconstituted in vitro from membrane and cytosolic fractions of PMNs, onto the wells of a microplate followed by incubation with potential inhibitors or drugs. After incubation, the excess of the drug is simply eliminated or washed prior measuring the activity of the reconstituted complex. This latter step avoid the risk of interference between the inhibitor and the revelation solution and can distinguish if inhibitors, strongly bound or not, could interfere with the assembly of the enzymatic complex or with its activity. The EquiNox2 was validated using diphenyliodonium chloride and Gp91ds-tat, two well-known inhibitors largely described for human NADPH oxidase. The present technique was used to study and understand better the effect of curcumin and its water-soluble derivative, NDS27, on the assembly and activity of NOX. We demonstrated that curcumin and NDS27 can strongly bind to the enzyme and prevents its assembly making these molecules good candidates for the treatment of horse or human pathologies implying an excessive activation of neutrophils. [less ▲]Detailed reference viewed: 23 (4 ULg)
Effects of isoflurane and sevoflurane on the neutrophil myeloperoxidase system of horses
MINGUET, Grégory ; Franck, Thierry ; JORIS, Jean et al
in Veterinary Immunology and Immunopathology (2015)Detailed reference viewed: 27 (10 ULg)
Muscle Mitochondrial Dysfunction in Horses Affected by Acute Laminitis
Serteyn, Didier ; de la Rebière de Pouyade, Geoffroy ; Sandersen, Charlotte et al
in Bioenergetics (2014)
Laminitis is a common and debilitating disease affecting horses and ponies. It often leads to the demise of the animal. Energy deficiency is suspected to entrain the disruption of the hemidesmosomes ... [more ▼]
Laminitis is a common and debilitating disease affecting horses and ponies. It often leads to the demise of the animal. Energy deficiency is suspected to entrain the disruption of the hemidesmosomes leading to the failure of the dermal-epidermal interface. The aim of this study was to measure the muscle mitochondrial function by high resolution respirometry. Muscle micro-biopsies were obtained from 11 horses affected by acute metabolic laminitis, 6 horses affected by acute laminitis resulting from a systemic inflammation response syndrome and 28 healthy horses distributed in 2 control groups: 17 horses with a body condition score [BSC, ranging from 0 (emaciated) to 5 (obese)] of 2 to 3 and 11 horses with a BSC of 4 to 5. During the acute phase of laminitis, a significant reduction of the muscle mitochondrial respiration was observed. The muscle mitochondrial dysfunction occurred independently of the etiology (metabolic disorder or systemic inflammation) leading to laminitis. The reduction of the oxidative phosphorylation and of the maximal respiratory capacity (after uncoupling) may induce depletion of the cell’s ATP content. If the same mitochondrial alteration occurs in the foot lamina, mitochondria targeting should be considered for the future, not only to better understand the physiopathology of the disease but also to maintain and to support the mitochondrial function before reaching the « mitochondrial dysfunction threshold » that may lead to the failure of the dermal-epidermal interface. [less ▲]Detailed reference viewed: 85 (23 ULg)
NDS27 combines the effect of curcumin lysinate and hydroxypropyl-β-cyclodextrin to inhibit equine PKCδ and NADPH oxidase involved in the oxidative burst of neutrophils
Derochette, Sandrine ; Mouithys-Mickalad, Ange ; Franck, Thierry et al
in FEBS Open Bio (2014), 4(0), 1021-1029
Polymorphonuclear neutrophils (PMNs) are involved in host defence against infections by the production of reactive oxygen species (ROS), but excessive PMN stimulation is associated with the development of ... [more ▼]
Polymorphonuclear neutrophils (PMNs) are involved in host defence against infections by the production of reactive oxygen species (ROS), but excessive PMN stimulation is associated with the development of inflammatory diseases. After appropriate stimuli, protein kinase C (PKC) triggers the assembly of NADPH oxidase (Nox2) which produces superoxide anion (O2●-), from which ROS derive. The therapeutic use of polyphenols is proposed to lower ROS production by limiting Nox2 and PKC activities. The purpose of this study was to compare the antioxidant effect of NDS27 and NDS28, two water-soluble forms of curcumin lysinate respectively complexed with hydroxypropyl-β-cyclodextrin (HPβCD) and γ-cyclodextrin (γ-CD), on the activity of Nox2 and PKCδ, involved in the Nox2 activation pathway. Our results, showed that NDS27 is the best inhibitor for Nox2 and PKCδ. This was illustrated by the combined effect of HPβCD and curcumin lysinate: HPβCD, but not γ-CD, improved the release of curcumin lysinate and its exchange against lipid or cholesterol as demonstrated by the lipid coloration with Oil red O, the extraction of radical lipophilic probes recorded by ESR and the HPLC measurements of curcumin. HPβCD not only solubilised and transported curcumin, but also indirectly enhanced its action on both PKC and Nox2 activities. The modulatory effect of NDS27 on the Nox2 activation pathway of neutrophils may open therapeutic perspectives for the control of pathologies with excessive inflammatory reactions. [less ▲]Detailed reference viewed: 84 (15 ULg)
Effect of different kinds of anoxia/reoxygenation on the mitochondrial function and the free radicals production of cultured primary equine skeletal myoblasts.
Ceusters, Justine ; Mouithys-Mickalad, Ange ; Franck, Thierry et al
in Research in Veterinary Science (2013), 95
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 ... [more ▼]
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. [less ▲]Detailed reference viewed: 50 (13 ULg)
New 5-Aryl-1H-imidazoles display in vitro antitumor activity against apoptosis-resistant cancer models, including melanomas, through mitochondrial targeting.
; ; Ceusters, Justine et al
in Journal of Medicinal Chemistry (2013), 56(17), 6626-6637Detailed reference viewed: 12 (0 ULg)
The challenge of understanding myopathies in horses using permeabilized muscle cells
Votion, Dominique ; Mouithys-Mickalad, Ange ; Ceusters, Justine et al
in In proceedings 9th Conference on Mitochondrial Physiology (2013, September)Detailed reference viewed: 52 (22 ULg)
Curcumin and resveratrol act by different ways on NADPH oxidase activity and reactive oxygen species produced by equine neutrophils
Derochette, Sandrine ; Franck, Thierry ; Mouithys-Mickalad, Ange et al
in Chemico-Biological Interactions (2013), 206
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 ... [more ▼]
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. [less ▲]Detailed reference viewed: 57 (13 ULg)
Effect of myeloperoxidase and anoxia/reoxygenation on mitochondrial respiratory function of cultured primary equine skeletal myoblasts.
Ceusters, Justine ; Mouithys-Mickalad, Ange ; Franck, Thierry et al
in Mitochondrion (2013), 13(5),
Horses are particularly sensitive to excessive inflammatory reaction where myeloperoxidase, a marker of inflammation, may contribute to mitochondrial dysfunctions. This study investigated the interaction ... [more ▼]
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. [less ▲]Detailed reference viewed: 58 (18 ULg)
Effects of diazoxide, benzothiadiazine and benzopyrane derivatives on mitochondrial proton and electron leaks of cardiomyocytes (H9C2 cell line).
Mouithys-Mickalad, Ange ; Ceusters, Justine ; et al
Background: Mitochondria are double membrane- organelles that play a central role in cellular metabolism, calcium homeostasis and redox signaling. They have been also considered as main producers of ... [more ▼]
Background: Mitochondria are double membrane- organelles that play a central role in cellular metabolism, calcium homeostasis and redox signaling. They have been also considered as main producers of adenosine triphosphate (ATP) and reactive oxygen species (ROS). In many cancer cells those organelles become dysfunctional leading to a shift of energy metabolism from oxidative phosphorylation to active glycolysis and an increase of ROS generation. According to Warberg’ theory, cancer damage might occur at the mitochondrial level, affecting tiny structures within each cell implicated in the energy production through ATP. New insight is that mitochondria might be a good therapeutic target for metabolic syndromes, ischemia/reperfusion injury and organs transplantation. Therefore, search for novel molecules able to keep mitochondria functional are of relevant interest. Methodology: Cardiomyocytes (H9C2 cells) were from ATCC (USA) and grown till confluence. The basal cellular respiratory rate, proton and electron leaks as well as ATP production were measured with the High Resolution Oxygraphy (Oroboros, Austria). All compounds: diazoxide (DIAZ), diazoxide –related analogs (1: BPDZ-259, 2: BPDZ-444), and benzopyran derivatives (3: BPDZ-490, 4: BPDZ-711) were tested at final concentration of 10-5 M, except when specified and compared to control samples (cells with or without DMSO). Results and conclusion: The basal respiratory rate of H9C2 cells (5x106/mL) was changed depending on the chemical structure of the tested compounds: e.g. compound 3 strongly enhanced the routine respiration, while 4 displayed a marked lowering effect. In contrast, the addition of similar concentration of benzothiadiazin derivatives (1, 2) had no effect on routine respiration but also on the other respiratory parameters such as oligomycin-induced leak and ATP production. Similar profile was obtained with the reference molecule: diazoxide. Overall, our findings indicate that both diazoxide-like analogues (1 and 2) and diazoxide were without significant effect on basal respiration, ATP production, even on maximal respiration. Interestingly, two derivatives show opposite effects: compound 3 behaves as a uncoupling agent and the other one (4) exhibits a real lowering effect on respiration but that was reversible. The latter effect might be of interest if this kind of molecules could be used for further use as an agent for organ conservation during transplantation. Our results also demonstrate that diazoxide, a well-known Mito-KATP opener, did not exert its effect beside of clinical situation like ischemia/reperfusion injury. [less ▲]Detailed reference viewed: 70 (8 ULg)
La fonction mitochondriale des cellules musculaires squelettiques équines en culture : effet de l'anoxie et des neutrophiles activés.
Doctoral thesis (2012)
Introduction Horses are known to be remarkable athletes. However, to realize such repeated intense exercises, as competition horses do, and could also became a stressing condition leading to muscular ... [more ▼]
Introduction Horses are known to be remarkable athletes. However, to realize such repeated intense exercises, as competition horses do, and could also became a stressing condition leading to muscular dysfunctions but could also decrease their performances. In horses, lesions from ischemia/reperfusion are found in numerous clinical situations but also during intense exercise, the muscular oxygenation decreasing as a function of the exercise intensity. But it is during the reoxygenation, with the subsequent increase of the electron flux within the mitochondrial respiratory electron transport chain at the cessation of exercise, that an important increase of the reactive oxygen and nitrogen species (RNOS) production will be observed. The realization of such intense exercise provokes also an important systemic inflammatory reaction. Recently, some studies realized by our group on competing horses showed that intense exercise can activate the polymorphonuclear neutrophils (PMNs) which degranulate their myeloperoxidase (MPO) and elastase (ELT). The increase of MPO was not only plasmatic but also muscular, where it was associated with a decrease of the mitochondrial complex I activity, showing there a possible link between the MPO activity and the mitochondrial dysfunction in horses performing exercise. The relationship between ischemia/reperfusion, ROS production from mitochondria, muscular damages and inflammatory reaction is largely unexplored in horses. The principal objectives of this work were to study, on a primary culture of equine skeletal muscle cells obtained from muscular microbiopsies, the effect of anoxia/reoxygenation (A/R) and/or of MPO on their ROS production and mitochondrial respiratory function. Results Primary culture of equine skeletal muscle cells from muscular microbiopsies With muscular microbiopsies, we developed an efficient and convenient sampling method, usable in current practice and even on high level competing horses. By using the microbiopsies as explants, we obtained a primary culture of skeletal muscle cells, a suitable experimental model for the in vitro study of equine muscular function. Model of anoxia/reoxygenation applied on cultured equine skeletal muscle cells Thanks to the primary culture, we showed by fluorescence spectroscopy and gas chromatography, an increased ROS production by adherent cells submitted to 2 h of A/R. For the following investigations, 2 other models were designed on detached cells : a first one of cyclic A/R (2 x 30 min) and a second one of a single long period of 1 h, 2 h or 3 h of A/R. The first one showed a decrease of the routine respiration, but also of ROS production and of the mitochondrial complex I specific activity of the cells submitted to cyclic A/R. After a single long period of A/R, on the contrary, the routine respiration, the ROS production and the mitochondrial complex I specific activity of the cells were increased while the ATP production by these cells was decreased. This observation, arguing for an uncoupling of the oxidative phosphorylation, prompted us to investigate the uncoupling protein 3 (UCP3) expression by cultured cells. The indirect immunofluorescence, as well as the electrophoresis coupled with the western blot, showed that they expressed UCP3, in normoxia and after 2 h of A/R, but also that this expression varied as a function of the duration of the A/R, with a maximum observed for 2 h. The oxygraphic phosphorylation control protocol (PCP) applied on detached cells showed an increase of their routine but also of their non phosphorylating (after ATP synthase inhibition) respirations after a single long period of 2 h A/R. Their maximal respiratory capacity, per contra, decreased significantly. When regarding the flux control ratios (FCR, related to maximal respiratory capacity), we confirmed that cells submitted to 2 h of A/R need to function more to keep stable their energetic state (increase of the part of their maximal respiratory capacity used for oxidative phosphorylation because of a lesser efficiency). Inflammatory model Equine neutrophils activation in whole blood All the activation systems used, 12-phorbol 13-myristate acetate (PMA), cytochalasin B with N-formyl-methionyl-leucyl-phenylalanine (CB/fMLP), tumor necrosis factor-alpha (TNF- α), lipopolysaccharides (LPS) and LPS with TNF-α, induced a significant degranulation of total MPO by the PMNs activated in whole blood. Only the combinations CB/fMLP and LPS/TNF-α caused a significant release of active MPO. For the release of ELT by activated PMNs, only the action of the PMA was significant. Use of purified active equine MPO Initially, we showed that MPO incubated with equine skeletal muscle cells was able to bind to the cell membranes, but furthermore, thanks to the differential centrifugation protocol, that we demonstrated that it was able to enter within the cells, because we found it in the cytosolic fraction. Our following experiences showed that it was able, even alone, to increase the ROS produced by the cells, as demonstrated by fluorescence and gas chromatography. Furthermore, MPO worked harder than Horseradish peroxidase (HRP), our "peroxidasic control". The MPO seemed also to alter the mitochondrial respiratory function of cultured cells. Anyway, thanks to the oxygraphic PCP protocol, we showed that MPO alone decreased mostly their routine respiration and their maximal respiratory capacity. Paradoxically, we seen that it increased the part of the maximal respiratory capacity used for routine and non phosphorylating respirations. The second oxygraphic protocol, designed on permeabilized cells, seems to confirm these observations. Compared to control cells, the MPO-treated cells had a increased non phosphorylating respiration and a decreased maximal respiratory capacity, particularly via the mitochondrial complex I. Combined model: anoxia/reoxygenation in inflammatory conditions With fluorescence spectroscopy as well as with gas chromatography, the MPO amplifies the ROS production already increased with the A/R alone. Furthermore, with a 3,3-diaminobenzidine (DAB) coloration, we showed the persistence of the MPO (and of the HRP), within the cells, even after 2 h of A/R. The oxygraphic PCP protocol confirms that MPO seems to exacerbate the damage initiated by A/R. Thus, compared to what we observed with A/R alone, the addition of MPO causes a further decrease of the routine respiration and the maximal respiratory capacity of the cells and increases their non phosphorylating respiration. These observations, along the lines of an important mitochondrial dysfunction, were confirmed when regarding the FCR. Thus, MPO-treated cells, submitted to A/R need to use a greater part of their maximal respiratory capacity for routine respiration, but a greater part of it was also used for non efficient respiration. So, these cells need to work more intensely to keep stable their energetic state, what is reflected in our protocol by an increased part of the maximal respiratory capacity used for oxidative phosphorylation compared to what we observed after A/R alone. Conclusion and perspectives During intense exercise, some physiopathological conditions, inducing important mitochondrial and energetic alterations, seem to gather within the muscle of sport horses. As showed by our work, because of its implication in the mitochondrial regulation at the respiratory level as well as for RNOS production, and of its particular sensibility to nitration, the mitochondrial complex I could play a pivotal role. Although our results clearly indicate that MPO, by exacerbating the deleterious effects of A/R, is detrimental to cellular and mitochondrial functions, further precisions are needed to confirm the specific implication of mitochondrial complex I in the observed phenomena. The results obtained by our group in vivo on competing horses lead the way in this direction but, the adaptation of the oxygraphic protocol on permeabilized cells, combining A/R with the action of MPO, will help to confirm, in vitro, the hypothesis. On another way, the effect of nitration or nitrosation on the mitochondrial complex I will need to be further investigated. Finally, to consider in a largest way the action of the PMNs on the mitochondrial function, it will be interesting to use the whole blood model of PMNs activation in co-culture with equine skeletal myoblasts. By this way, the action of other inflammatory factors or mediators could be studied in conditions nearly to the ones observed in vivo. [less ▲]Detailed reference viewed: 136 (37 ULg)
An in vitro whole blood model to test the effects of different stimuli conditions on the release of myeloperoxidase and elastase by equine neutrophils.
Ceusters, Justine ; Serteyn, Didier ; MINGUET, Grégory et al
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 ▲]Detailed reference viewed: 48 (25 ULg)
EFFECT OF MYELOPEROXIDASE ON MITOCHONDRIAL RESPIRATORY FUNCTION OF PERMEABILIZED PRIMARY EQUINE SKELETAL MYOBLASTS IN CULTURE.
Ceusters, Justine ; Mouithys-Mickalad, Ange ; Franck, Thierry et al
Poster (2012, November)Detailed reference viewed: 39 (10 ULg)
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.
Ceusters, Justine ; Mouithys-Mickalad, Ange ; de la Rebière de Pouyade, Geoffroy et al
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 ▲]Detailed reference viewed: 83 (33 ULg)
Relationship between exercise-induced systemic inflammatory like reaction and racing performance in endurance horses
Serteyn, Didier ; Caudron, Isabelle ; Lejeune, Jean-Philippe et al
in Comparative Exercise Physiology (2012), 8(3/4), 213218
This study showed that systemic inflammatory like reaction is not clearly related to performance but also to horse-related factors such as intinsic capacity or training.Detailed reference viewed: 37 (11 ULg)
Production of free radicals and oxygen consumption by primary equine endothelial cells during anoxia-reoxygenation.
de la Rebière de Pouyade, Geoffroy ; Salciccia, Alexandra ; Ceusters, Justine et al
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 ▲]Detailed reference viewed: 37 (9 ULg)
MODULATING EFFECT OF THE RESPIRATORY RATE OF CARDIOMYOCYTES (H9C2 CELLS) AND MITOCHONDRIAL SWELLING BY DIAZOXIDE ANALOGUES: STRUCTURE–FUNCTION RELATIONSHIPS
Ceusters, Justine ; Mouithys-Mickalad, Ange ; De Tullio, Pascal et al
Poster (2011)Detailed reference viewed: 26 (6 ULg)
EFFECT OF DIFFERENT ANOXIA/REOXYGENATION MODELS ON MITOCHONDRIAL COMPLEX I ACTIVITY OF CULTURED EQUINE SKELETAL MUSCLE CELLS: ESR AND OXYGRAPHIC STUDIES.
Ceusters, Justine ; Mouithys-Mickalad, Ange ; Niesten, Ariane et al
Poster (2010)Detailed reference viewed: 25 (9 ULg)
Specific immuno-extraction followed by enzymatic detection (SIEFED) of myeloperoxidase and mitochondrial complex I in muscular microbiopsies: preliminary results in endurance horses
Franck, Thierry ; Votion, Dominique ; Ceusters, Justine et al
in Equine Veterinary Journal. Supplement (2010), 42(Suppl. 38), 296-302Detailed reference viewed: 36 (15 ULg)