| BACKGROUND & AIMS Severe acute pancreatitis (SAP) is acute hemorrhagic and necrotizing pancreatitis (AHNP) on pathology. SAP comes on rapidly and be in a extremely dangerous state. Its pathological characteristic is extensive necrosis and hemorrhage in pancreas. The clinical prognosis of SAP is so bad that it often combined with the damage or disfunction of the organs beside pancreas, or present such syndrome as pyocyst or false cyst in local pancreas. As before, SAP animal models were induced by such techniques as pancreatic ligature, intraductal injection of sodium taurocholate, undercapsule injection of sodium taurocholate, and so on. However, all these methods are wounding, difficult and high mortality of experimental animals. Lampel used caerulein to induce no wound animal model of acute pancreatitis for the first time. Since then, the caerulein-induced-pancreatitis model was widely applied for experimental studying of pancreatitis. Whenas, the pathological characteristic of this kind of model is mild pancreatitis but not severe acute pancreatitis. In our experiment, a kind of no wound model of severe acute pancreatitis on mice was induced by intraperitoneal injections of caerulein plus lipopolysaccharide. MATERIAL & METHODS1. Animals and groupings Female NIH mice, weighing 25.0?.0g, were used in this study and divided into four groups, i.e. Cn +LPS group, Cn group, LPS group and NS group, each consisting eight mice.2. Induction of animal models Animals were fasting but drinking water for eighteen hours before experiment. Cn +LPS group animals were received intraperitoneal injections of caerulein at a dose of SO ug/kg ?h for 6 hours and an intraperitoneal injection of 10 mg/kg LPS was applied after the last caerulein injection. Cn group animals were receivedintraperitoneal injections of caerulein at a dose of 50 jig/kg ?h for 6 hours. LPS group animals were received intraperitoneal injections of normal saline at a dose of 50 ml/kg - h for 6 hours and an intraperitoneal injection of 10 mg/kg LPS was applied after the last NS injection. NS group animals were received intraperitoneal injections of NS at a dose of 50 ml/kg ?h for 6 hours.3. Secological determination Three hours after the injections, blood was taken for serum amylase determination by iodine-amylum colorimetry and serum nitric oxide (NO) concentration was measured by nitrate reductase method.4. Materials Mice were put to death after blood was taken. The pancreas was carefully dissected and weighed. Different pieces of pancreas were excised to be fixed in 10% formalin solution, 2.5% glutaradehyde solution at 4X?, or be stored in icebox at ?0"C. The organs out of pancreas including stomach, the end of ileum, liver, kidney, spleen, heart and lung were dissected and fixed in 10% formalin solution.5. Examination of SOD and MDA To prepare 10% plasma of pancreatic tissue, then measure the protein quantity by biuret method. Superoxide dismutase (SOD) was examined by xanthine oxidase method and malondialdenhyde (MDA) by thiobarbituric acid (TBA) method.6. Preparation for LM Samples were fixed in 10% formalin solution. Paraffin-embedded section were cut and stained with hematoxylin and eosin.7. Preparation for TEM The pancreas tissue were cut in 1mm3 and fixed in 2.5% glutaradehyde solution for 4-6 h, postfixed in !%OsO4 for 1 h , at 4"C. After block-staining in 2% uranyl acetate, dehydration in a graded ethanol series and acetone, the tissue were embeded in Epon 812. Ultrathin sections were prepared on Leica LKB2088 ultramicrotome, double stained with uranyl acetate and leat citrate and examined in Philips TECNAI 10 by 80 Kv.8. Histopathological grading According to Schmidt's standard, using the double-blind mothed.9. Statistical analysis Comparison of the difference between the mean values and the grading data of different groups were made by /-test and Ridit analysis respectively.RESULTS1. General observation In Cn+LPS group, peritoneal fluid wa... |
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