Surgical Closure of Equine Abdomen, Prevention, and Management of Incisional Complications.

Although rarely fatal, complications of ventral midline laparotomy incision in equine patients increase hospitalization cost and duration and may jeopardize return to athletic function. Therefore, many techniques have been developed to reduce their occurrence and expedite their resolution when they occur. Our technique of celiotomy incision closure includes the use of tension sutures (vertical U mattress) of polyglactin 910 on the linea alba, which is then apposed by polyglactin 910 interrupted sutures or a simple continuous pattern suture with a stop midway before routine closure of the superficial layers. The celiotomy incision is protected by an elastic bandage during the immediate postoperative period. This technique has been associated with favorable results: 5.3% confirmed incisional infections after a single celiotomy and 26.7% after repeat celiotomy. The overall incisional complication (serous/sanguineous discharge, hematoma, infection, hernia formation, and complete wound breakdown) occurrence was 9.5% and 33.3% after single and repeat laparotomy, respectively. In cases considered more susceptible to infection (early relaparotomy or laparotomy incisions longer than 30 cm), negative pressure therapy was found easy to apply on closed incisions. No detrimental effects were observed. However, the potential prophylactic benefit of this therapy needs to be confirmed in a larger group. In infected laparotomy wounds requiring drainage, the use of negative pressure therapy seemed to have a positive effect on the formation of granulation tissue. However, there was no control group to allow statistical confirmation. Finally, one case of complete breakdown of the laparotomy incision was managed by stainless steel retention sutures, the application of negative pressure therapy, and a hernia belt. At re-evaluation 15 months post-surgery, several small hernias were detected, but the horse had returned to his previous level of sports performance and had not shown any episode of colic.

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.

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.

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.

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.

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.

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.

Onset and duration of cis-atracurium neuromuscular block during fentanyl and lidocaine infusions in isoflurane-anaesthetised dogs.

BACKGROUND: This retrospective study assessed the onset and duration of the neuromuscular block (NMB) induced by cis-atracurium 0.15 mg/kg intravenously with and without fentanyl or lidocaine infusions in 45 isoflurane-anaesthetised dogs. METHODS: Dogs with neuromuscular function assessed by a calibrated train-of-four (TOF) monitor with stimulation (every 13 s) of the peroneal nerve were included. The onset and duration of the NMB were defined as the time from cis-atracurium administration until TOF=0 and the time during TOF=0 display, respectively. RESULTS: The NMB onset was shorter during fentanyl (mean±sd) (1.9±0.7 minutes; P=0.0042) and lidocaine (2.0±0.7 minutes; P=0.0154) compared with control (2.9±0.8 minutes). The NMB duration was shorter in the fentanyl (27.5±7.3 minutes; P=0.0491), but not in the lidocaine group (32.3±6.9 minutes; P=0.0790), compared with control (33.7±9.1 minutes). The NMB onset was poorly but significantly correlated with the dose of fentanyl and lidocaine administered before cis-atracurium (r=-0.3396; P=0.0225). The fentanyl and lidocaine groups received more crystalloid and colloid boluses than the control. CONCLUSIONS: Fentanyl and lidocaine shortened the NMB onset and the former decreased the NMB duration. Further prospective studies are required to clarify whether this was associated with an indirect decrease in blood pressure or a direct interaction between cis-atracurium and fentanyl and lidocaine.

Gastrointestinal effects of general anaesthesia in horses undergoing non abdominal surgery: focus on the clinical parameters and ultrasonographic images.

The ultrasonographic images of the gastrointestinal tract in horses can be influenced by fasting and sedation but the proper effect of general anaesthesia (GA) on them has not been determined yet. This study aimed to evaluate the effects of GA on ultrasonographic images of the gastrointestinal tract in horses and to compare these effects with a clinical evaluation. Twenty horses undergoing non-abdominal surgeries were evaluated by ultrasonography before and 4 times within 24 h after GA. Each ultrasonographic exam focused on the stomach, the duodenum and on 5 locations on the jejunum. The four-quadrant auscultation and the postoperative faecal output were also recorded. Pre and post anaesthetic values were compared using linear mixed effects models. None of the horses presented colic signs or reduced faecal output. During the first 2 post anaesthetic evaluations, the gut sounds were significantly decreased and, when taking all jejunal locations together, the jejunal diameter and visualisation frequency significantly increased. No intestinal loop appeared thickened and most of their diameters remained within the normal range. Our results suggest that the effects of GA on the ultrasonographic images of the small intestine are mild and of short duration and can therefore be differentiated from a pathological process.

Comparison of two portable clinical analyzers to one stationary analyzer for the determination of blood gas partial pressures and blood electrolyte concentrations in horses.

Portable blood gas analyzers are used to facilitate diagnosis and treatment of disorders related to disturbances of acid-base and electrolyte balance in the ambulatory care of equine patients. The aim of this study was to determine whether 2 portable analyzers produce results in agreement with a stationary analyzer. Blood samples from 23 horses hospitalized for various medical reasons were included in this prospective study. Blood gas analysis and electrolyte concentrations measured by the portable analyzers VetStat and epoc were compared to those produced by the cobas b 123 analyzer via concordance analysis, Passing-Bablok regression and Bland-Altman analysis. Limits of agreement indicated relevant bias between the VetStat and cobas b 123 for partial pressure of oxygen (pO2; 27.5-33.8 mmHg), sodium ([Na+]; 4.3-21.6 mmol/L) and chloride concentration ([Cl-]; 0.3-7.9 mmol/L) and between the epoc and cobas b 123 for pH (0.070-0.022), partial pressure of carbon dioxide (pCO2; 3.6-7.3 mmHg), pO2 (36.2-32.7 mmHg) and [Na+] (0.38.1 mmol/L). The VetStat analyzer yielded results that were in agreement with the cobas b 123 analyzer for determination of pH, pCO2, bicarbonate ([HCO3-]) and potassium concentration [K+], while the epoc analyzer achieved acceptable agreement for [HCO3-] and [K+]. The VetStat analyzer may be useful in performing blood gas analysis in equine samples but analysis of [Na+], [Cl-] and pO2 should be interpreted with caution. The epoc delivered reliable results for [HCO3-] and [K+], while results for pH, pCO2, pO2 and [Na+] should be interpreted with caution.

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 laryngeal nerve shows lesions of demyelination. The benefit of applying stem cells to demyelinated nerves has been demonstrated in various animal models. The aim of the study was to test the feasibility and safety of a peri-neuronal injection of autologous muscle-derived mesenchymal stem cells to the left recurrent laryngeal nerve in healthy horses by using an electrical nerve stimulator. Muscle-derived stems cell are obtained from five healthy Standardbred horses by sampling 20 mg of muscle tissue with a semi-automatic 14 G biopsy needle from the triceps muscle. Movements of the larynx are monitored via upper-airway video endoscopy. The left recurrent laryngeal nerve is approached with an insulated nerve block needle. Nerve stimulation is applied, starting at 2 mA, and the successful abduction of the left arytenoid is monitored. The stimulation intensity is reduced progressively. When a loss of the motor response is observed at 0.5 mA, 107 autologous muscle-derived stem cells are injected. Two examiners, who are blinded to the time point, score the laryngeal function of the horses prior to the treatment and at day 1, day 7, and day 28 after the injection of the cells. In a sixth horse, 1 mL of 2% lidocaine is injected to further confirm the correct positioning of the needle. This leads to a temporary paralysis of the left arytenoid cartilage. This study proves that the recurrent laryngeal nerve can be approached with the help of an electrical nerve stimulator and that the electrical stimulation of the nerve is well tolerated by the horses. No modification of the laryngeal function was observed in any of the horses after the injection of the stem cells. Further studies should be conducted to describe the effects of a peri-neuronal injection of autologous muscle-derived mesenchymal stem cells to horses suffering from RLN.

Use of Nasotracheal Intubation during General Anesthesia in Two Ponies with Tracheal Collapse.

Ponies with tracheal collapse may have an increased anesthetic risk due to airway obstruction during induction and recovery. To our knowledge, there are no anesthetic descriptions of these patients, despite a reported 5.6% incidence and 77% mortality rate. Two Shetland ponies with tracheal collapse, a 12-year-old male (pony 1) and a 27-year-old female (pony 2), were referred for right eye enucleation due to a perforating corneal ulcer and severe recurrent uveitis, respectively. Pony 1 was stressed, had lung stridor and hyperthermia, and developed inspiratory dyspnea with handling. Radiography confirmed collapse of the entire trachea as well as inflammation of the lower airways. Corticosteroids and bronchodilators were administered by nebulization for 1 week before surgery. Pony 2 had a grade III/VI mitral murmur and a clinical history of esophageal obstructions and tracheal collapse requiring tracheostomy. Both ponies were premedicated with acepromazine and xylazine; anesthesia was induced with midazolam and ketamine. Nasotracheal intubation was performed in left lateral recumbency with extension of the neck and head and was guided by capnography. The nasotracheal tube consisted of two endotracheal tubes attached end-to-end to create a tube of adequate length and diameter. Pony 2 was orotracheally intubated during surgery and later reintubated with a nasotracheal tube. Anesthesia was maintained with isoflurane using volume-controlled ventilation. Analgesia was provided by a retrobulbar blockade with mepivacaine and lidocaine. Cardiovascular support consisted of lactated Ringer's solution and dobutamine. After surgery, the ponies were administered xylazine and supplemented with oxygen through the nasotracheal tube. Recovery was assisted by manual support of the head and tail. Successful extubation was achieved following butorphanol administration after approximately 1 h in standing position. Both ponies were discharged from the clinic a few days after surgery.

Sevoflurane modulates the release of reactive oxygen species, myeloperoxidase, and elastase in human whole blood: Effects of different stimuli on neutrophil response to volatile anesthetic in vitro.

Volatile anesthetics have been shown to modulate polymorphonuclear neutrophil (PMN) functions. The aim of this study was to examine the impact of clinically relevant concentrations of sevoflurane (SEVO), a volatile anesthetic, on the release of reactive oxygen species (ROS), myeloperoxidase (MPO), and elastase (EL) from human activated PMNs. For this purpose, samples of whole blood were collected from healthy volunteers and exposed in vitro to 2.3% or 4.6% SEVO in air. To assess for a stimulus-dependent effect of the volatile anesthetic, PMNs were activated using different validated protocols. Artificial stimulation of neutrophils involved either a combination of cytochalasin B (CB) and N-formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol 12-myristate 13-acetate (PMA). In addition, a combination of lipopolysaccharide (LPS) and tumor necrosis factor alpha (TNF-α) was also tested as a natural activation mean of PMNs. The production of ROS by PMNs was assessed by L-012 chemiluminescence. Total MPO and EL released in supernatant were measured by enzyme-linked immunosorbent assay (ELISA). Furthermore, degranulation of the active fraction of MPO was also measured by specific immunological extraction followed by enzymatic detection (SIEFED). Overall, SEVO enhanced the release of ROS, MPO, and EL following artificial stimulation of PMNs but the volatile anesthetic inhibited the degranulation of active MPO and EL after neutrophil exposure to LPS and TNF-α. This study highlighted that the effect of SEVO on activated PMNs is dependent on the conditions of cell stimulation. These properties should be taken into consideration in future studies investigating immunomodulatory effects of volatile anesthetics.

On the causes and consequences of the uncoupler-like effects of quercetin and dehydrosilybin in H9c2 cells.

Quercetin and dehydrosilybin are polyphenols which are known to behave like uncouplers of respiration in isolated mitochondria. Here we investigated whether the effect is conserved in whole cells. Following short term incubation, neither compound uncouples mitochondrial respiration in whole H9c2 cells below 50µM. However, following hypoxia, or long term incubation, leak (state IV with oligomycin) oxygen consumption is increased by quercetin. Both compounds partially protected complex I respiration, but not complex II in H9c2 cells following hypoxia. In a permeabilised H9c2 cell model, the increase in leak respiration caused by quercetin is lowered by increased [ADP] and is increased by adenine nucleotide transporter inhibitor, atractyloside, but not bongkrekic acid. Both quercetin and dehydrosilybin dissipate mitochondrial membrane potential in whole cells. In the case of quercetin, the effect is potentiated post hypoxia. Genetically encoded Ca++ sensors, targeted to the mitochondria, enabled the use of fluorescence microscopy to show that quercetin decreased mitochondrial [Ca++] while dehydrosilybin did not. Likewise, quercetin decreases accumulation of [Ca++] in mitochondria following hypoxia. Fluorescent probes were used to show that both compounds decrease plasma membrane potential and increase cytosolic [Ca++]. We conclude that the uncoupler-like effects of these polyphenols are attenuated in whole cells compared to isolated mitochondria, but downstream effects are nevertheless apparent. Results suggest that the effect of quercetin observed in whole and permeabilised cells may originate in the mitochondria, while the mechanism of action of cardioprotection by dehydrosilybin may be less dependent on mitochondrial uncoupling than originally thought. Rather, protective effects may originate due to interactions at the plasma membrane.

Comparison of metabolic profiles and bioactivities of the leaves of three edible Congolese Hibiscus species.

Methanolic and dichloromethane extracts from the leaves of Congolese Hibiscus species were characterised by chromatographic and spectroscopic techniques and their in vitro biochemical activities against ROS production were evaluated in cellular models and on an enzyme, myeloperoxidase (MPO), involved in inflammation. Hibiscus acetosella has a chemical fingerprint different from Hibiscus cannabinus and Hibiscus sabdariffa both having similar fingerprints. Major compounds were polyphenols, represented mainly by caffeoyl-hydroxycitric acid for H. acetosella and neochlorogenic acid for the two other species. All extracts displayed high cellular antioxidant activity with IC50 values ranging from 0.5 to 3 µg mL-1 using lucigenin on neutrophils. Dichloromethane extracts showed more efficient effects on extracellular ROS production and MPO activity. Antioxidant and anti-inflammatory activities of caffeoyl-hydroxycitric acid were significantly higher than those of neochlorogenic acid. The bioactivities of Hibiscus species were positively correlated with their phytochemical content and could justify their use as local nutraceutical resources and medicines.

Modified Thomas splint-cast combination for the management of limb fractures in small equids.

OBJECTIVE: To describe the management and outcome of limb fractures in small domestic equids treated with a modified Thomas splint-cast combination (MTSCC). STUDY DESIGN: Retrospective case series. ANIMALS: Client owned horses and donkeys. METHODS: Medical records, including radiographs, were reviewed for details of animals diagnosed with a limb fracture and treated by external coaptation using a MTSCC (2001-2012). Follow-up >6 months after discharge was obtained via telephone consultation with owners or veterinarians. RESULTS: Nine horses and 4 donkeys were identified with fractures of the tibial diaphysis (n = 4), ulna (n = 3), distal metatarsus (n = 2), proximal metacarpus (n = 1), radial diaphysis (n = 1), calcaneus (n = 1), and distal femoral physis (n = 1). Follow-up was available for 12 equids, of which 8 (67%) recovered from the fracture and became pasture sound. Six equids developed obvious external deformation of the affected limb. CONCLUSION: Selected small equids with long bone fractures, and without athletic expectations, can be managed with external coaptation using an MTSCC. The owner should be informed that the treatment is considered a salvage procedure.