Optimization of the Amplification of Equine Muscle-Derived Mesenchymal Stromal Cells in a Hollow-Fiber Bioreactor.

The main causes of mortality in horses are the gastrointestinal pathologies associated with septic shock. Stem cells have shown, through systemic injection, a capacity to decrease inflammation and to regenerate injured tissue faster. Nevertheless, to achieve this rapid and total regeneration, systemic injections of 1 to 2 million cells per kilogram of body weight must be considered. Here, we demonstrate for the first time the feasibility and expansion capacity of equine muscle-derived mesenchymal stromal cells (mdMSCs) in a functionally closed, automated, perfusion-based, hollow-fiber bioreactor (HFBR) called the Quantum™ Cell Expansion System (Terumo Blood and Cell Technologies). This feature greatly increases the number of generated cells with a surface area of 1.7 m2. The expansion of mdMSCs is very efficient in this bioreactor. The maximum expansion generated twenty times more cells than the initial seeding in nine days. The best returns were observed with an optimal seeding between 10 and 25 million mdMSCs, using the Bull's eye loading method and with a run duration between 7 and 10 days. Moreover, all the generated cells kept their stem properties: the ability to adhere to plastic and to differentiate into chondroblasts, osteoblasts and adipocytes. They also showed the expression of CD-44 and CD-90 markers, with a positive rate above 93%, while CD-45 and MHCII were non-expressed, with a positive rate below 0.5%. By capitalizing on the scalability, automation and 3D culture capabilities of the Quantum™, it is possible to generate large quantities of high-quality equine mdMSCs for gastrointestinal disorders and other clinical applications.

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.

Free Radical Inhibition Using a Water-Soluble Curcumin Complex, NDS27: Mechanism Study Using EPR, Chemiluminescence, and Docking.

There is a growing interest in the use of natural compounds to tackle inflammatory diseases and cancers. However, most of them face the bioavailability and solubility challenges to reaching cellular compartments and exert their potential biological effects. Polyphenols belong to that class of molecules, and numerous efforts have been made to improve and overcome these problems. Curcumin is widely studied for its antioxidant and anti-inflammatory properties as well as its use as an anticancer agent. However, its poor solubility and bioavailability are often a source of concern with disappointing or unexpected results in cellular models or in vivo, which limits the clinical use of curcumin as such. Beside nanoparticles and liposomes, cyclodextrins are one of the best candidates to improve the solubility of these molecules. We have used lysine and cyclodextrin to form a water-soluble curcumin complex, named NDS27, in which potential anti-inflammatory effects were demonstrated in cellular and in vivo models. Herein, we investigated for the first time its direct free radicals scavenging activity on DPPH/ABTS assays as well as on hydroxyl, superoxide anion, and peroxyl radical species. The ability of NDS27 to quench singlet oxygen, produced by rose bengal photosensitization, was studied, as was the inhibiting effect on the enzyme-catalyzed oxidation of the co-substrate, luminol analog (L012), using horseradish peroxidase (HRP)/hydrogen peroxide (H2O2) system. Finally, docking was performed to study the behavior of NDS27 in the active site of the peroxidase enzyme.

Subconjunctival autologous muscle-derived mesenchymal stem cell therapy: A novel, minimally invasive approach for treating equine immune-mediated keratitis.

OBJECTIVE: To establish the safety of subconjunctival injections of autologous muscle-derived mesenchymal stem cells (mdMSCs) in healthy horses and to evaluate their effect in four horses (six eyes) with severe chronic equine immune-mediated keratitis (IMMK) that was unresponsive to medical treatments. METHODS: MdMSCs were cultured from minimally invasive muscle biopsies. In the safety group, four healthy horses received two subconjunctival injections of 2.5 and 5 million cells, respectively, at 1-month interval, to the same eye. Ocular side effects were monitored for 1 month following each injection. In the treatment group, six eyes received four to seven subconjunctival mdMSCs injections (2.5 or 5 million cells per injection) every 4 weeks, approximatively. Medical treatment was discontinued 1 week before and throughout the entire treatment period. A scoring system was used to assess the evolution of the ocular lesions. RESULTS: In the safety group, all horses exhibited mild to moderate chemosis and conjunctival hyperemia at the injection site, lasting 24-48 h. In the treatment group, all eyes initially responded positively to therapy, with a reduction in lesion scores observed after the first injection. Four eyes achieved control of the lesions with repeated injections during the 9.2 months of follow-up. CONCLUSION: The first subconjunctival injection of mdMSCs resulted in improvement of the ocular lesions. Repeated injections were found to be safe, minimally invasive and showed promise in managing refractory cases of equine IMMK. Further studies are warranted to demonstrate the long-term benefits of these injections and to optimize the therapeutic protocol.

Targeting Myeloperoxidase Activity and Neutrophil ROS Production to Modulate Redox Process: Effect of Ellagic Acid and Analogues.

Malaria is an infectious disease caused by a Plasmodium genus parasite that remains the most widespread parasitosis. The spread of Plasmodium clones that are increasingly resistant to antimalarial molecules is a serious public health problem for underdeveloped countries. Therefore, the search for new therapeutic approaches is necessary. For example, one strategy could consist of studying the redox process involved in the development of the parasite. Regarding potential drug candidates, ellagic acid is widely studied due to its antioxidant and parasite-inhibiting properties. However, its low oral bioavailability remains a concern and has led to pharmacomodulation and the synthesis of new polyphenolic compounds to improve antimalarial activity. This work aimed at investigating the modulatory effect of ellagic acid and its analogues on the redox activity of neutrophils and myeloperoxidase involved in malaria. Overall, the compounds show an inhibitory effect on free radicals as well as on the enzyme horseradish peroxidase- and myeloperoxidase (HRP/MPO)-catalyzed oxidation of substrates (L-012 and Amplex Red). Similar results are obtained with reactive oxygen species (ROS) produced by phorbol 12-mystate acetate (PMA)-activated neutrophils. The efficiency of ellagic acid analogues will be discussed in terms of structure-activity relationships.

Evaluation of the appearance of osteochondrosis lesions by two radiographic examinations in sport horses aged from 12 to 36 months.

Osteochondrosis is a developmental orthopedic disease characterized by a defect of enchondral ossification. This pathological condition develops and evolves during growth and is influenced by various factors, in particular genetic and environmental. However, little research has been conducted on the dynamic of this condition in horses after the age of 12 months. The retrospective study presented here investigates changes in osteochondrosis lesions through two standardized radiographic examinations carried out on young Walloon sport horses after one year of age (mean age at first and second examination was 407 (±41) and 680 (±117) days respectively). Each examination, analyzed independently by three veterinarians, included latero-medial views of the fetlocks, hocks, stifles, plantarolateral-dorsomedial hocks view and additional radiograph if the operator deemed it necessary. Each joint site was graded as healthy, osteochondrosis (OC) or osteochondrosis dissecans (OCD) affected. A group of 58 horses was studied, among them 20 presented one or more osteochondrosis lesions for a total of 36 lesions present during at least one examination. In this population, 4 animals (6.9%) presented osteochondrosis during only one examination (2 at the first examination and 2 at the second one). Moreover, it was possible to demonstrate the appearance, disappearance and more generally the evolution of 9/36 lesions (25%) within the different joints. The results of the study suggest that, although substantial main limitations, osteochondrosis lesions can evolve after the age of 12 months in sport horses. Knowing this is useful in helping to decide the appropriate radiographic diagnosis timing and management.

Analytical Performance Evaluation of the New GEM® Premier™ 5000 in Comparison to the Epoc® Blood Gas Analyzer in Horses.

Different blood gas analyzers are used in equine practice. Every machine needs to be validated, as they have not been designed for use in horses. The aim of this study was to compare the newly marketed GEM5000 machine to the formerly validated epoc machine for blood gas analysis in horses. In this prospective, non-blinded, comparative laboratory analyzer study, 43 equine blood samples were analyzed on both analyzers and values were compared between the two machines via Lin's concordance analysis, Passing-Bablok regression analysis and Bland-Altman plots. Duplicate measurements were conducted on the GEM5000 machine to evaluate precision. The GEM5000 failed to achieve the required precision for tHb, Hct and iCa2+, but achieved acceptable precision for all other parameters. Concordance correlation analysis revealed poor correlation for Na+, Cl-, iCa2+, K+, Hct and tHb, while there was an at least moderate agreement for all other parameters. Passing-Bablok regression revealed significant constant bias for pCO2, pO2, Cl-, and iCa2+ and significant proportional bias for pCO2, iCa2+ and SO2. Bland-Altman analysis revealed significant systematic bias for Na+, Cl-, iCa2+, K+, Hct, tHb and SO2. This study shows that while precision of the GEM5000 is good, values should not be used interchangeably with data obtained from other blood gas analyzers.

Equine Muscle Derived Mesenchymal Stem Cells Loaded with Water-Soluble Curcumin: Modulation of Neutrophil Activation and Enhanced Protection against Intracellular Oxidative Attack.

We investigated the antioxidant potential of equine mesenchymal stem cells derived from muscle microbiopsies (mdMSCs), loaded by a water-soluble curcumin lysinate incorporated into hydroxypropyl-ß-cyclodextrin (NDS27). The cell loading was rapid and dependent on NDS27 dosage (14, 7, 3.5 and 1 µM). The immunomodulatory capacity of loaded mdMSCs was evaluated by ROS production, on active and total myeloperoxidase (MPO) degranulation and neutrophil extracellular trap (NET) formation after neutrophil stimulation. The intracellular protection of loaded cells was tested by an oxidative stress induced by cumene hydroperoxide. Results showed that 10 min of mdMSC loading with NDS27 did not affect their viability while reducing their metabolism. NDS27 loaded cells in presence of 14, 7 µM NDS27 inhibited more intensively the ROS production, the activity of the MPO released and bound to the NET after neutrophil stimulation. Furthermore, loaded cells powerfully inhibited intracellular ROS production induced by cumene as compared to control cells or cyclodextrin-loaded cells. Our results showed that the loading of mdMSCs with NDS27 significantly improved their antioxidant potential against the oxidative burst of neutrophil and protected them against intracellular ROS production. The improved antioxidant protective capacity of loaded mdMSCs could be applied to target inflammatory foci involving neutrophils.

Characterization of the Proteins Secreted by Equine Muscle-Derived Mesenchymal Stem Cells Exposed to Cartilage Explants in Osteoarthritis Model.

BACKGROUND: Osteoarthritis (OA) is a highly prevalent joint degenerative disease for which therapeutic treatments are limited or invasive. Cell therapy based on mesenchymal stem/stromal cells (MSCs) is therefore seen as a promising approach for this disease, in both human and horses. As the regenerative potential of MSCs is mainly conferred by paracrine function, the goal of this study was to characterize the secreted proteins of muscle-derived MSCs (mdMSCs) in an in vitro model of OA to evaluate the putative clinical interest of mdMSCs as cell therapy for joint diseases like osteoarthritis. METHODS: An equine osteoarthritis model composed of cartilage explants exposed to pro-inflammatory cytokines was first developed. Then, the effects of mdMSC co-culture on cartilage explant were studied by measuring the glycosaminoglycan release and the NO2- production. To identify the underlying molecular actors, stable isotope-labeling by amino acids in cell culture based secreted protein analyses were conducted, in the presence of serum. The relative abundance of highly sequenced proteins was finally confirmed by western blot. RESULTS: Co-culture with muscle-derived MSCs decreases the cytokine-induced glycosaminoglycan release by cartilage explants, suggesting a protecting effect of mdMSCs. Among the 52 equine proteins sequenced in the co-culture conditioned medium, the abundance of decorin and matrix metalloproteinase 3 was significantly modified, as confirmed by western blot analyses. CONCLUSIONS: These results suggest that muscle-derived MSCs could reduce the catabolic effect of TNFα and IL-1ß on cartilage explant by decreasing the secretion and activity of matrix metalloproteinase 3 and increasing the decorin secretion. mdMSCs capacity to reduce the catabolic consequences of cartilage exposure to pro-inflammatory cytokines. These effects can be explained by mdMSC-secreted bioactive such as TIMP-1 and decorin, known as an inhibitor of MMP3 and an anti-inflammatory protein, respectively.

Propofol metabolites and derivatives inhibit the oxidant activities of neutrophils and myeloperoxidase.

In previous studies, propofol has shown immunomodulatory abilities on various in vitro models. As this anesthetic molecule is extensively used in intensive care units, its anti-inflammatory properties present a great interest for the treatment of inflammatory disorders like the systemic inflammatory response syndrome. In addition to its inhibition abilities on important neutrophils mechanisms (chemotaxis, reactive oxygen species (ROS) production, Neutrophil Extracellular Traps (NETs) formation, …), our group has shown that propofol is also a reversible inhibitor of the oxidant myeloperoxidase (MPO) activity. Propofol being subject to rapid metabolism, its derivatives could contribute to its anti-inflammatory action. First, propofol-ß-glucuronide (PPFG), 2,6-diisopropyl-1,4-p-benzoquinone (PPFQ) and 3,5,3',5'-tetraisopropyl-(4,4')-diphenoquinone (PPFDQ) were compared on their superoxide (O2.-) scavenging properties and more importantly on their inhibitory action on the O2.- release by activated neutrophils using EPR spectroscopy and chemiluminescence assays. PPFQ and PPFDQ are potent superoxide scavengers and also inhibit the release of ROS by neutrophils. An Enzyme-Linked Immunosorbent Assay (ELISA) has also highlighted the ability of both molecules to significantly decrease the MPO degranulation process of neutrophils. Fluorescence enzymatic assays helped to investigate the action of the propofol derivatives on the peroxidase and chlorination activities of MPO. In addition, using SIEFED (Specific Immunological Extraction Followed by Enzyme Detection) assays and docking, we demonstrated the concentration-dependent inhibitory action of PPFQ and its ability to bind to the enzyme active site while PPFG presented a much weaker inhibitory action. Overall, the oxidation derivatives and metabolites PPFQ and PPFDQ can, at physiological concentrations, perpetuate the immunomodulatory action of propofol by acting on the oxidant response of PMN and MPO.

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.

Comparison of single-breath continuous positive airway pressure manoeuvre with inhaled salbutamol to improve oxygenation in horses anaesthetized for laparotomy.

OBJECTIVE: To compare the efficacy of single-breath continuous positive airway pressure manoeuvre (CPAP-M) with inhaled salbutamol, and a combination of both. STUDY DESIGN: Randomized, clinical study. ANIMALS: A total of 62 client-owned horses (American Society of Anesthesiologists status III-V) anaesthetized for laparotomy. METHODS: Horses were premedicated with intravenous (IV) xylazine (0.4-0.6 mg kg-1), anaesthesia was induced with midazolam (0.06 mg kg-1 IV) and ketamine (2.2 mg kg-1 IV) and maintained with isoflurane in oxygen using volume-controlled ventilation without positive end-expiratory pressure. If PaO2 was < 100 mmHg (13.3 kPa), either a CPAP-M (50 cmH2O for 45 seconds) or salbutamol (0.002 mg kg-1) was administered. The intervention was considered successful if PaO2 reached 100 mmHg (13.3 kPa). If PaO2 remained < 100 mmHg (13.3 kPa), treatments were switched. PaO2/FiO2 ratio and estimated shunt fraction (F-shunt) were derived from data obtained from arterial blood gas measurements. Dynamic compliance (Cdyn) was calculated from variables recorded at the moment of arterial blood analysis. Fisher's exact tests compared success rates between treatments, and linear models were performed to test whether the treatment modified the values of the measurements; p < 0.05. RESULTS: Salbutamol was the first intervention in 28 horses and was effective in 22 horses. CPAP-M was the first intervention in 34 horses and was effective in 26 horses. CPAP-M after salbutamol was performed in six horses, with four responders, and salbutamol after CPAP-M was administered to eight horses, with one responder. Salbutamol, but not CPAP-M, significantly decreased F-shunt. Both salbutamol and CPAP-M significantly increased Cdyn. CONCLUSIONS AND CLINICAL RELEVANCE: Salbutamol and CPAP-M were comparably effective in improving oxygenation and Cdyn in anaesthetized horses with PaO2 < 100 mmHg (13.3 kPa). Whether combining both treatments might be beneficial needs to be confirmed on a larger number of horses.

Muscle Derived Mesenchymal Stem Cells Inhibit the Activity of the Free and the Neutrophil Extracellular Trap (NET)-Bond Myeloperoxidase.

Mesenchymal stem cells (MSCs) are known to migrate to tissue injury sites to participate in immune modulation, tissue remodelling and wound healing, reducing tissue damage. Upon neutrophil activation, there is a release of myeloperoxidase (MPO), an oxidant enzyme. But little is known about the direct role of MSCs on MPO activity. The aim of this study was to investigate the effect of equine mesenchymal stem cells derived from muscle microinvasive biopsy (mdMSC) on the oxidant response of neutrophils and particularly on the activity of the myeloperoxidase released by stimulated equine neutrophils. After specific treatment (trypsin and washings in phosphate buffer saline), the mdMSCs were exposed to isolated neutrophils. The effect of the suspended mdMSCs was studied on the ROS production and the release of total and active MPO by stimulated neutrophils and specifically on the activity of MPO in a neutrophil-free model. Additionally, we developed a model combining adherent mdMSCs with neutrophils to study total and active MPO from the neutrophil extracellular trap (NET). Our results show that mdMSCs inhibited the ROS production, the activity of MPO released by stimulated neutrophils and the activity of MPO bound to the NET. Moreover, the co-incubation of mdMSCs directly with MPO results in a strong inhibition of the peroxidase activity of MPO, probably by affecting the active site of the enzyme. We confirm the strong potential of mdMSCs to lower the oxidant response of neutrophils. The novelty of our study is an evident inhibition of the activity of MPO by MSCs. The results indicated a new potential therapeutic approach of mdMSCs in the inhibition of MPO, which is considered as a pro-oxidant actor in numerous chronic and acute inflammatory pathologies.

Effect of Fentanyl Infusion on Heart Rate Variability and Anaesthetic Requirements in Isoflurane-Anaesthetized Horses.

Controversy continues to surround the use of opioids in equine anaesthesia, with variable effects reported. This blinded clinical study aimed to investigate the influence of a low-dose fentanyl continuous rate infusion (CRI) on isoflurane requirements, parasympathetic tone activity (PTA), and anaesthetic parameters in horses during general anaesthesia. All of the twenty-two horses included in the research underwent a standard anaesthetic protocol. Eleven horses in the fentanyl group (Group F) received a loading dose of fentanyl at 6 µg/kg, followed by a CRI of 0.1 µg/kg/min during anaesthesia. A further 11 horses in the control group (Group C) received equivalent volumes of normal saline. Anaesthetic parameters and PTA index were recorded during anaesthesia. The achieved mean fentanyl plasma concentration was 6.2 ± 0.83 ng/mL. No statistically significant differences between groups were found in isoflurane requirements, MAP values, and mean dobutamine requirements. However, horses in Group F required a significantly lower dose of additional ketamine to maintain a sufficient depth of anaesthesia. Significantly higher PTA values were found in the fentanyl group. Further research is warranted to determine the limitations of PTA monitoring, and the influence of various anaesthetics on its values.

Effects of Juglone on Neutrophil Degranulation and Myeloperoxidase Activity Related to Equine Laminitis.

Experimental laminitis, characterized by a failure of the dermal-epidermal interface of the foot, can be induced in horses by the oral administration of a black walnut extract (BWE). In the early phase of this severe and painful disease, an activation of neutrophil occurs, with the release of myeloperoxidase (MPO), a pro-oxidant enzyme of neutrophils, in plasma, skin, and laminar tissue. Juglone, a naphthoquinone derivative endowed with redox properties, is found in walnuts and has been incriminated in this neutrophil activation. We report for the first time the inhibitory activity of juglone on the degranulation of neutrophils induced by cytochalasin B and formyl-methionyl-leucyl-phenylalanine as monitored by the MPO release (>90% inhibition for 25 and 50 µM). Moreover, it also acts on the peroxidase activity of MPO by interacting with the intermediate "π cation radical," as evidenced by the classical and specific immunological extraction followed by enzymatic detection (SIEFED) assays. These results are confirmed by a docking study showing the perfect positioning of juglone in the MPO enzyme active site and its interaction with one of the amino acids (Arg-239) of MPO apoprotein. By chemiluminescence and electron paramagnetic resonance techniques, we demonstrated that juglone inhibited reactive oxygen species (ROS) and superoxide anion free radical produced from phorbol myristate acetate (PMA)-activated polymorphonuclear neutrophils (PMNs). These results indicate that juglone is not the trigger for equine laminitis, at least if we focus on the modulation of neutrophil activation.

Unlocking the Real Potential of Black Soldier Fly (Hermetia illucens) Larvae Protein Derivatives in Pet Diets.

Black soldier fly larvae (BSFL)-derived proteins are gaining popularity as sustainable pet food ingredients. According to the literature, these ingredients have strong antioxidant and antimicrobial activities. Due to the ability of BSFL protein derivatives to donate hydrogen atoms and/or electrons to counterpoise unstable molecules, they could possibly help in the prevention of osteoarthritis. During this study, the antiarthritic potential of BSFL protein derivatives was evaluated using the following assays: (1) proteinase inhibition, (2) erythrocyte membrane stability, (3) reactive oxygen species (ROS) production by activated macrophages, (4) ROS production by monocytes, and (5) cellular toxicity. Additionally, the glucosamine content of these ingredients was also evaluated. Chicken meal is commonly used in pet food formulations and was used as an industrial benchmark. The results obtained during this study demonstrated the strong antiarthritic potential of BSFL protein derivatives. We found that BSFL protein derivatives are not only useful in preventing the development of arthritis but could also help to cure it due to the presence of glucosamine. We also found that chicken meal could contribute to the development of arthritis by increasing ROS production by monocytes.

Priming of mesenchymal stem cells with a hydrosoluble form of curcumin allows keeping their mesenchymal properties for cell-based therapy development.

Mesenchymal stem cells are increasingly studied for their use as drug-carrier in addition to their intrinsic potential for regenerative medicine. They could be used to transport molecules with a poor bioavailability such as curcumin in order to improve their clinical usage. This natural polyphenol, well-known for its antioxidant and anti-inflammatory properties, has a poor solubility that limits its clinical potential. For this purpose, the use of NDS27, a curcumin salt complexed with hydroxypropyl-beta-cyclodextrin (HPßCD), displaying an increased solubility in aqueous solution, is preferred. This study aims to evaluate the uptake of NDS27 into skeletal muscle-derived mesenchymal stem cells (mdMSCs) and the effects of such uptake onto their mesenchymal properties. It appeared that the uptake of NDS27 into mdMSCs is concentration-dependent and not time-dependent. The use of a concentration of 7 µmol/L which does not affect the viability and proliferation also allows preservation of their adhesion, invasion and T cell immunomodulatory abilities.

Clinical safety of computed tomography-guided injection of autologous muscle-derived mesenchymal stem cells in the intervertebral disc in dogs.

Background: Pre-clinical randomized controlled animal trials have been conducted to evaluate the effect of mesenchymal stem cell (MSCs) transplantation on intervertebral disc (IVD) degeneration. MSCs can be obtained from different tissues, but systematic studies concerning the effects of muscle-derived MSCs injections on canine naturally degenerated IVD are still lacking. The aim of this study is the assessment of the clinical safety of this technique and its effects on the imaging features of the lumbosacral IVD. Methods: Eight adult healthy Beagle dogs were used in this study. In the preliminary phase, viability of muscle-derived MSCs in presence of contrast medium was assessed. In the clinical assessment phase, MSCs were injected in the lumbosacral IVD by computed-tomography (CT) guidance, after the injection of contrast medium to assess the correct intradiscal needle position. Regular clinical examinations were performed and pre- and post-injections (CT) and magnetic resonance imaging (MRI) features of the IVD were assessed. Results: The percentage of viability of MSCs in the presence of contrast medium ranged from 90 to 98%. 3x106 MSCs were obtained from six dogs and injected in the IVD. No major or minor complications were reported during the procedure and no abnormalities were noticed during the clinical examinations. No statistically significant variations were noticed between the pre- and post-injections imaging features. Conclusion: This technique is clinically safe and it is not associated with any progression of the IVD degeneration, detected by CT and MRI imaging.

Modulation of mitochondrial respiration rate and calcium-induced swelling by new cromakalim analogues.

A cellular model of cardiomyocytes (H9c2 cell line) and mitochondria isolated from mouse liver were used to understand the drug action of BPDZ490 and BPDZ711, two benzopyran analogues of the reference potassium channel opener cromakalim, on mitochondrial respiratory parameters and swelling, by comparing their effects with those of the parent compound cromakalim. For these three compounds, the oxygen consumption rate (OCR) was determined by high-resolution respirometry (HRR) and their impact on adenosine triphosphate (ATP) production and calcium-induced mitochondrial swelling was investigated. Cromakalim did not modify neither the OCR of H9c2 cells and the ATP production nor the Ca-induced swelling. By contrast, the cromakalim analogue BPDZ490 (1) induced a strong increase of OCR, while the other benzopyran analogue BPDZ711 (2) caused a marked slowdown. For both compounds, 1 displayed a biphasic behavior while 2 still showed an inhibitory effect. Both compounds 1 and 2 were also found to decrease the ATP synthesis, with pronounced effect for 2, while cromakalim remained without effect. Overall, these results indicate that cromakalim, as parent molecule, does not induce per se any direct effect on mitochondrial respiratory function neither on whole cells nor on isolated mitochondria whereas both benzopyran analogues 1 and 2 display totally opposite behavior profiles, suggesting that compound 1, by increasing the maximal respiration capacity, might behave as a mild uncoupling agent and compound 2 is taken as an inhibitor of the mitochondrial electron-transfer chain.

Complications associated with closure of the linea alba using a combination of interrupted vertical mattress and simple interrupted sutures in equine laparotomies.

OBJECTIVES: (1) Evaluate the occurrence and variables associated with incisional morbidities (IMs) after ventral median laparotomy when using interrupted vertical mattress sutures (IVMS) and (2) determine the occurrence of abdominal bandage-associated complications in horses. METHODS: Occurrence of IM and bandage-associated complications were determined after single laparotomies (SL group; n=546 horses) and repeat laparotomies (RL group: multiple laparotomies within four weeks; n=30 horses) in horses that survived ≥7 days postoperatively. Univariate analysis and multivariate logistic regression were performed to evaluate variables associated with IM. RESULTS: The IM rate was 9.52 per cent in the SL group and 33.33 per cent in the RL group. The actual infection rate was 5.31 per cent in the SL group and 26.67 per cent in the RL group. Overall, long-term clinically relevant wound complications was 1.68 per cent. After multivariate analysis, increased anaesthesia duration was associated with IM and performing an enterotomy and postoperative intravenous lidocaine administration were associated with incisional infection in the SL group; no parameter remained significant in the RL group. Bandage-related complications were recorded in 2.95 per cent of the cases. CONCLUSIONS: These results suggest that the use of IVMS for closure of the linea alba is another viable option for closure and that an abdominal bandage does not appear to cause significant complications.