| ObjectivePancreas transplantation is frequently complicated by the development of acute pancreatitis, largely as a result of ischemia - reperfu-sion injury secondary to cold preservation. During the reperfusion period , endothelial dysfunction, activation of endogenous enzymes, leucocyte recruitment and activation all lead to the generation of oxygen -derived free radical. Nitric oxide (NO) is a free radical with strong reactivity, and it also has the function of second messenger and neuro-transmitter. Nitric oxide may be oxided to nitrate and nitrite; otherwise it may lose an electron to form the nitrosonium cation ( NO * ) , which can combine with the superoxide radical to form peroxynitrite ( ONOO- ' ), a highly active free radical, and it has fierce cytotoxici-ty. However, nitric oxide can vasodilate significantly, and can remit the vasospasm of graft, decrease the occurrence of vascular crisis, which can protect pancreas graft from thrombosis or pancreatitis due to transplantation. Therefore, the nitric oxide has ambivalent functions in the ischemia - reperfusion of pancreas transplantation, and attracts lots of interests. Nitric oxide is synthesized from L - argmine by the enzyme nitric oxide synthase ( NOS). NOS has two principal forms: constitutive NOS (cNOS) and inducible NOS (iNOS) , and the iNOS has more important pathological role. In this study, we hypothesis thatonly iNOS is responsible for pancreatitis due to transplantation, and selective iNOS inhibitor can alleviate ischemia - reperfusion injury of pancreas.Material and MethodMaterialMale Wistar rats weighing 250 ?20g were randomly assigned to three experiment groups. In group 1 ( sham operation) , the animals underwent laparotomy without pancreas ischemia - reperfusion. In animals of group 2 ( ischemia - reperfusion) , the splenic artery was occluded reversibly by microsurgical clip for 30 minutes. (In rat, the blood of pancreas body and tail is mainly supplied by splenic artery. ) The animals were killed after 0,2, 4, 6, 12, 24 hours of reperfusion. In animals of group 3 ( aminoguanidine + ischemia - reperfusion) , aminoguanidine( AG) was added to the infusion to intravascular as the onset of reperfusion to provide a dose of 40, 60, 80 mg/kg weight. The animals were killed after 4 and 6 hours of reperfusion. The pancreases was harvested and divided into two segments, one was fixed in 10% PBS formalin, the other was preserved in deep low temperature of - 70 . The blood was withdraw without coagulant and then cen-trifugated at a rate of 2000 turns/minute for ten minutes, and the serum was preserved in low temperature of - 20 . Method1. Nitrate reductase detected serum nitric oxide level.2. Iodine - starch chromatometry detected serum amylase activity.3. NOS Test Kit detected pancreas cNOS and iNOS activity.4. HE Staining: One pancreas segment was fixed in 10% PBSformalin, dehydrate through a grade ethanol series, cleared in xylene and embedded in paraffin blocks. The blocks were cut into 4 um sections and stained with haematosylin and eosin. Sections were evaluated by light microscopic examination.5. Immunohistochemistry Staining: The primary antibody, anti -iNOS polyclonal, were produced in rabbit. The StreptAvidin - Biotin Complex immunoperoxidase staining system was used, and the presence of positive staining was reddish - brown.6. Statistical analysis: Continuous data were expressed as mean ?standard deviation, and data were analysed using analysis of variance with contrasts to define groups difference.Result1. Serum nitric oxide level: NO level increased significantly following 30 minutes pancreas ischemia and 4 hours reperfusion, and then dropped to normal level with the extending of reperfusion. With the administration of aminoguanidine, a selective iNOS inhibitor, NO level was much lower than the control group.2. Serum amylase activity: Amylase activity increased after reperfusion , especially fowling 4, 6, 12, 24 hours reperfusion. With the administration of aminoguanidine, amyla...
|
PDF Full Text Request