The nitroxide 4-methoxy TEMPO inhibits neutrophil-stimulated kinase activation in H9c2 cardiomyocytes.

After acute myocardial infarction (AMI), neutrophils are recruited to the affected myocardium. Hypochlorous acid (HOCl) produced by neutrophil myeloperoxidase (MPO) damages cardiomyocytes and potentially expands the primary infarct. Rat cardiomyocyte-like cells were incubated with isolated human neutrophils treated with chemical activators in the absence or presence of nitroxide 4-methoxy-Tempo (MetT; 25 µM) for 4, 6 or 24 h; studies with reagent HOCl served as positive control. Treating cardiomyocytes with activated neutrophils or reagent HOCl resulted in a marked increase in protein tyrosine chlorination and a decline in protein tyrosine phosphatase (PTP) activity. On balance our data also supported an increase in phosphorylation of MAPK p38 and ERK1/2 suggestive of an intracellular hyperphosphorylation status and this was accompanied by decreases in cell viability, as judged by assessing caspases-3/7 activity. For cells exposed to activated neutrophils receptor-mediated uptake of transferrin decreased although total matrix metalloproteinase (MMP) activity was unaffected. Addition of MetT ameliorated protein tyrosine chlorination, decreased MAPK activity and restored receptor-mediated transferrin uptake and PTP activity in cardiomyocytes. Overall, adverse effects of neutrophil-derived HOCl on cultured cardiomyocytes were ameliorated by MetT suggesting that nitroxides may be beneficial to inflammatory pathologies, where neutrophil recruitment/activation is a prominent and early feature.

Randomized clinical trial of the effect of intraoperative humidified carbon dioxide insufflation in open laparotomy for colorectal resection.

BACKGROUND: Animal studies have shown that peritoneal injury can be minimized by insufflating the abdominal cavity with warm humidified carbon dioxide gas. METHODS: A single-blind RCT was performed at a tertiary colorectal unit. Inclusion criteria were patient aged 18 years and over undergoing open elective surgery. The intervention group received warmed (37°C), humidified (98 per cent relative humidity) carbon dioxide (WHCO2  group). Multiple markers of peritoneal inflammation and oxidative damage were used to compare groups, including cytokines and chemokines, apoptosis, the 3-chlorotyrosine/native tyrosine ratio, and light microscopy on peritoneal biopsies at the start (T0 ) and end (Tend ) of the operation. Postoperative clinical outcomes were compared between the groups. RESULTS: Of 40 patients enrolled, 20 in the WHCO2 group and 19 in the control group were available for analysis. A significant log(Tend /T0 ) difference between control and WHCO2 groups was documented for interleukin (IL) 2 (5·3 versus 2·8 respectively; P = 0·028) and IL-4 (3·5 versus 2·0; P = 0·041), whereas apoptosis assays documented no significant change in caspase activity, and similar apoptosis rates were documented along the peritoneal edge in both groups. The 3-chlorotyrosine/tyrosine ratio had increased at Tend by 1·1-fold in the WHCO2 group and by 3·1-fold in the control group. Under light microscopy, peritoneum was visible in 11 of 19 samples from the control group and in 19 of 20 samples from the WHCO2 group (P = 0·006). The only difference in clinical outcomes between intervention and control groups was the number of days to passage of flatus (2·5 versus 5·0 days respectively; P = 0·008). CONCLUSION: The use of warmed, humidified carbon dioxide appears to reduce some markers related to peritoneal oxidative damage during laparotomy. No difference was observed in clinical outcomes, but the study was underpowered for analysis of surgical results. Registration number: NCT02975947 ( www.ClinicalTrials.gov/).

ANTECEDENTES: Los estudios en animales han demostrado que la lesión peritoneal se puede minimizar insuflando gas de dióxido de carbono caliente y humidificado (warm, humidified carbon dioxide gas,WHCO2(g) ) en la cavidad abdominal. El objetivo de este ensayo fue investigar los marcadores de inflamación peritoneal y de daño oxidativo en pacientes sometidos a cirugía colorrectal y abdominal tratados dióxido de carbono calentado humidificado en comparación con controles. El objetivo secundario fue evaluar los resultados clínicos perioperatorios. MÉTODOS: Se llevó a cabo un ensayo aleatorizado, controlado y simple ciego en una unidad colorrectal terciaria. Se incluyeron pacientes de > 18 años de edad sometidos operaciones electivas por vía abierta. El grupo de intervención recibió CO2(g) calentado (37°C) y humidificado (98% humedad relativa). Para la comparación de los grupos, se determinaron múltiples marcadores de inflamación peritoneal y daño oxidativo, incluyendo citocinas y quimiocinas, apoptosis (actividad Caspasas -3 y -7 y DeadEndTM TUNEl sistema fluorométrico), la tasa 3-clorotirosina/tirosina nativa (HPLC-MS) y microscopía electrónica de biopsias peritoneales al inicio (T0 ) y al término (Tfinal ) de la operación. Los resultados clínicos postoperatorios se compararon entre los grupos. RESULTADOS: De los 40 pacientes incluidos en el estudio, se dispuso de datos para el análisis en 20 pacientes asignados al grupo de CO2 y en 19 asignados al grupo control. Se observó una diferencia significativa Log(Tend/T0) entre los grupos respecto a IL-2 (grupo control: 5,34, grupo CO2: 2,78, P = 0,028) y IL-4 (grupo control: 3,53, grupo CO2: 2,00, P = 0,04), en tanto que los análisis relativos a la apoptosis no pusieron de manifiesto cambios significativos en la actividad de la caspasa, y se observaron tasas de apoptosis similares a lo largo del borde peritoneal en ambos grupos. La tasa 3-clorotirosina/tirosina nativa aumentó en 1,05 veces en el grupo del CO2 y en 3,1 veces en el grupo control. Por microscopía óptica el peritoneo era visible en el 57,9% de los sujetos del grupo control y en el 95% de los que recibieron tratamiento con WHCO2(g) (P = 0,006). La única diferencia en los resultados clínicos entre los grupos de intervención y control fue el número de días para el paso de gases (2,5 en el grupo de CO2 versus 5,0 días en el grupo control, P = 0,008). CONCLUSIÓN: El uso de WHCO2(g) parece disminuir algunos de los marcadores relacionados con el daño peritoneal por estrés oxidativo durante la laparotomía. Aunque no se observaron diferencias en los resultados clínicos, el estudio no tenía la suficiente potencia para analizar los resultados quirúrgicos.