Ischaemic postconditioning reduces serum and tubular TNF-α expression in ischaemic-reperfused kidney in healthy rats.

OBJECTIVE: We studied the protective effects of postconditioning (PS) in healthy and hypercholesterolemic rats after renal ischaemia-reperfusion (IR) injury. We aimed to examine cytokine expression and apoptosis in tissue damage after revascularisation (TNF-α levels in serum and tissue). METHODS: Male Wistar rats (n = 32) were divided into four groups. The animals of normal feed groups (NF) were fed with normal rat chow and the cholesterol feed groups (CF) were fed with 1.5% cholesterol containing diet for 8 weeks. Anaesthetized rats underwent a 45-min cross-clamping in both kidney pedicles. Ischaemia was followed by 120-min reperfusion with or without PS protocol (group PS vs. IR). Postconditioning was induced by four intermittent periods of ischaemia-reperfusion of 15-s duration each. Serum cholesterol, triglyceride, urea and creatinine levels were determined. Proinflammation was characterized by the measurement of serum TNF-α. Tissue injury in kidney was determined by formaline-fixed, paraffin-embedded tissue sections. Tissue TNF-α levels were determined by immunohistochemistry. RESULTS: Significant elevation was observed in serum TNF-α level after IR injury in normal feed groups, which was reduced by PS. In CF group neither the elevation nor the postconditioning induced reduction were as significant as in the NF groups. In normal feed group PS caused a significant reduction in tissue TNF-α level which was significantly higher in CF. CONCLUSIONS: Ischaemic postconditioning proved to be an effective defense against IR in NF groups, but it was ineffective in CF groups in kidney tissue.

Effect of a glutathione S-transferase inhibitor on oxidative stress and ischemia-reperfusion-induced apoptotic signalling of cultured cardiomyocytes.

Oxidative stress and ischemia-reperfusion (I/R) injury are crucial in the pathogenesis of cardiovascular diseases. The antioxidant glutathione S-transferase (GST) is responsible for the high-capacity metabolic inactivation of electrophilic compounds and toxic substrates. The main objective of the present study was to examine the effect of GST inhibition (with the administration of ethacrynic acid [EA]) on the viability and apoptosis of cardiomyocytes when these cells are exposed to various stress components of I/R and mitogen-activated protein kinase (c-Jun N-terminal kinase, p38 and extracellular signal-regulated kinase [ERK]) inhibitors. The primary culture of neonatal rat cardiomyocytes was divided into six experimental groups: control group of cells (group 1), cells exposed to H(2)O(2) (group 2), I/R (group 3), I/R and EA (group 4), H(2)O(2) coupled with EA (group 5), and EA alone (group 6). The viability of cardiomyocytes was determined using a colorimetric MTT assay. The apoptosis ratio was evaluated via fluorescein isothiocyanate-labelled annexin V and propidium iodide staining. c-Jun N-terminal kinase, p38, Akt/protein kinase B and ERK/p42-p44 transcription factors were monitored with flow cytometry. c-Jun N-terminal kinase activation increased due to GST inhibition during I/R. EA administration led to a significant increase in p38 activation following both H(2)O(2) treatment and I/R. ERK phosphorylation increased when GST was exposed to I/R. A pronounced decrease in Akt phosphorylation was observed when cells were cotreated with EA and H(2)O(2). GST plays an important role as a regulator of mitogen-activated protein kinase pathways in I/R injury.

Examination of protective effect of ischemic postconditioning after small bowel autotransplantation.

Ischemia/reperfusion (I/R) injury is a serious condition that results from some surgical procedures, including intestinal transplantation. Ischemic postconditioning is defined as brief periods of reperfusion alternating with reocclusion applied during the early minutes after reperfusion. The objective of this study was to investigate the effect of ischemic postconditioning before small bowel autotransplantation. Total orthotopic intestinal autotransplantation was performed in 30 white domestic pigs. Grafts were stored in cold University of Wisconsin solution for 1, 3, or 6 hours. Duration of reperfusion was 3 hours in all grafts. Before reperfusion, the intestine was postconditioned via 3 cycles of ischemia for 30 seconds and reperfusion for 30 seconds (ischemic postconditioning protocol). Tissue from the small intestine was obtained after laparotomy (control group) and at the end of reperfusion periods. To monitor oxidative stress, tissue concentrations of malondialdehyde and reduced glutathione, and activity of superoxide dismutase were determined at spectrophotometry. Tissue damage on sections stained with hematoxylin- eosin was evaluated using a quantitative method (Scion Image software; Scion Corp, Frederick, Maryland). Our results demonstrated that ischemic postconditioning significantly decreased the reperfusion-ended lipid peroxidation value (mean +/- SEM, 142.0 +/- 7.1 micromol/g vs 125.0 +/- 2.1 micromol/g; P < .05). Moreover, the capacity and activity of endogenous antioxidant protective systems (glutathione 789+/-8.0 micromol/g vs 934 +/- 5.7 micromol/g, and superoxide dismutase 110 +/- 9 IU/g vs 126 +/- 4 IU/g; P < .05) remained higher in the ischemic postconditioning groups compared with tissues without ischemic postconditioning. At quantitative analysis, tissue injury was increased by the duration of cold preservation. The greatest injury was observed in the mucosal and submucosal layers and in the depth of crypts after 6 hours of preservation. Ischemic postconditioning significantly decreased intestinal wall injury in each group (P < .05). It was concluded that ischemic postconditioning before reperfusion mitigated oxidative stress and histologic damage during small bowel autotransplantation.

Ischaemic postconditioning reduces peroxide formation, cytokine expression and leukocyte activation in reperfusion injury after abdominal aortic surgery in rat model.

OBJECTIVE: We studied the protective effects of ischaemic postconditioning (PS) on ischemia-reperfusion injury of the lower extremities in a rat model of abdominal aortic intervention. We aimed to examine the evoked oxidative stress, cytokine expression and leukocyte activation after revascularisation surgery. METHODS: Anesthetized animals (48 Whistar rats) underwent a 60 min infrarenal aorta cross-clamping. After the ischaemic period, an intermittent 4 times 15 s reperfusion--15 seconds ischaemic episodes--were applied (ischaemic postconditioning: group PS). Then we started a 120 min reperfusion in the aorta. In untreated group animals underwent a long ischaemia (60 min) and the following reperfusion (group IR). Peripherial blood samples were collected before operation, and in early (5, 10, 15, 30, 60 and 120 min) reperfusion periods. Serum peroxide level, TNF-alpha concentration, myeloperoxidase (MPO) activity and PMA-induced leukocyte ROS production were measured. RESULTS: In PS group, plasma peroxide level elevation was significantly lower in very early reperfusion (5-30 min) comparing to non-conditioned IR group (10.04+/-1.9 microM/l vs. 16.91+/-3.67 microM/l, p<0.05). PS also reduced serum TNF-alpha concentration (167.41+/-31.26 microg/ml vs. 116.55+/-12.04 microg/ml, p<0.05), MPO activity (1.759+/-0.239 microM/ml vs. 1.22+/-0.126 microM/ml, p<0.05) and leukocyte activation detected by PMA-induced leukocyte ROS production (5.7+/-0.96 AU/10(3) cells vs. 4.63+/-0.69 AU/10(3) cells). CONCLUSIONS: Ischaemic postconditioning could reduce ROI production after IR in early reperfusion period, thus limiting ROI mediated tissue lesion, cytokine-leukocyte activation and inflammatory responses. PS seems to be an effective tool in vascular surgery to reduce reperfusion injuries after revascularization interventions.