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.

Influence of pituitary adenylate cyclase-activating polypeptide on the activation of mitogen activated protein kinases following small bowel cold preservation.

Cold preservation prior to small bowel transplantation can moderate tissue oxidative injury. This stress triggers several intracellular pathways via mitogen activated protein (MAP) kinases. MAP kinases include the extracellular signal related kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAP kinase. Pituitary adenylate cyclase-activating polypeptide (PACAP) plays a central role in intestinal physiology. We sought to investigate the effect of PACAP on the activation of MAP kinases during cold preservation of the small bowel. Total orthotopic intestinal autotransplantation was performed on 40 Wistar rats. Perfused grafts were stored in University of Wisconsin (UW) solution for 1 (GI), 2 (GII), 3 (GIII), or 6 hours (GIV) without or with 30 PACAP, namely 1 (GV), 2 (GVI), 3 (GVII), or 6 hours (GVIII). After 3 hours of reperfusion in all groups, the activation of MAP kinases were measured using immunocytochemistry of small bowel tissue. Among the UW preserved grafts (GI-GIV), phosphorylated ERK1/2 level were decreased, while phosphorylated JNK1/2 and p38 MAP kinase activation were elevated compared with control levels. In GV-GVIII PACAP we observed enhanced phospho-ERK1/2 appearance with decreased JNK and p38 MAP kinase activity at the end of the reperfusion periods. We concluded that cold preservation decreased phosphorylated ERK1/2 levels and increased JNK1/2 and p38 MAP kinase activities, which meant that cold storage triggered apoptotic cell death. In contrast, PACAP treatment induced signalling pathways protective against oxidative injury by MAP kinases in bowel tissue.

Changes and effect of PACAP-38 on intestinal ischemia-reperfusion and autotransplantation.

Tissue injury caused by cold preservation and reperfusion during small bowel transplantation remains an unsolved problem. Increasing evidence suggests that pituitary adenylate cyclase-activating polypeptide (PACAP) has protective effects in several ischemia-reperfusion (I/R) models. This study investigated the effect of PACAP-38 on oxidative stress in autotransplanted intestine. We established sham-operated, I/R, and autotransplanted groups in Wistar rats (n = 55). We applied ischemia for 1 (GI), 2 (GII), or 3 hours (GIII). In autotransplanted groups, we performed total orthotopic intestinal autotransplantation. Grafts were preserved in University of Wisconsin (UW) solution for 1 (GIV), 2 (GV), 3 (GVI), or 6 (GVII) hours and in PACAP-38-containing UW for 1 (GVIII), 2 (GIX), 3 (GX), or 6 (GXI) hours. Reperfusion lasted 3 hours in each group. Endogenous PACAP-38 values were measured by radioimmunoassay. Oxidative stress parameters malondialdehyde (MDA), reduced glutathione (GSH), and superoxide dismutase (SOD) were measured in tissue homogenates. Concentration of endogenous PACAP-38 significantly decreased in GI to GIII compared with the sham-operated animals following I/R periods (P < .05). Cold preservation in UW and reperfusion of the intestine increased the level of tissue MDA in GIV to GVII, which correlated with the duration of cold storage. The content of GSH decreased in GIV to GVII to levels that were significantly different between GIV and GVIII and between GVII and GXI. SOD activity decreased dramatically in GIV to GVII with significantly higher activity in GIX to GXI. Our findings confirmed that I/R decreased endogenous PACAP-38 concentration. Administration of PACAP-38 to UW solution mitigated the oxidative injury during intestinal 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.