| objective:To detect SOD activity and MDA level of the brain tissue in severe acute pancreatitis(SAP) rats and explore the role of oxygen free radicals(OFR) in pancreatic encephalopathy(PE):To investigate the expression of tumor necrosis factor-α(TNF-α) and interleukin-1β(IL-1β) in rats brain tissue and explore the relationship to the central dopaminergic pathway of PE pathology, as well as further study the pathogenesis of PE for the foundation and provide experimental evidence for the search for new therapeutic targets. Methods:72 male Sprague-Dawley(SD) rats, weighing 230-330g, were randomized into control group(n=36) and PE group(n=36), each group subdivided into 3h,6h, and 12h group(n=6). A rat PE model was induced by retrogradely injected 5% sodium taurocholate into the bile-pancreatic duct. For control group, retrograde infusion of normal saline into the bile-pancreatic duct were performed as described above. The macro changes of all rats’pancreas and brain were observed. The pancreas and brain were removed and fixed in 4% paraformaldehyde. Histopathological changes were observed by both hematoxylin-eosin(HE) staining under light microscopy. The positive expression of TNF-α, IL-1β, tyrosine hydroxylase(TH) and dopamine receptor-2(D2) in frontal cortex tissues were detected by immunohistochemical methods and analyzed by image pro plus 6.0 software. The remaining 36 rats after the success in modeling the corresponding time points(3h,6h,12h) were killed, and were removed the brains immediately. Frontal cortex homogenates were prepared in ice bath and were centrifuged at 10,000r/min,10 miniuts, then superoxide dismutase activity and malondialdbelryde level were determined with chemical-colorimetric method. Partial occipital lobes were scaled respectively in wet weight after the rats were decapitated in control group and PE group, placed in 60℃oven bake for 24h, to remove moisture, scaled dry weight respectively and to calculate the radio of brain wet/dry at last. Results:1. The macrochanges of rats’ pancreas and brian were as follow. Pancreas hyperemia, edema, necrosis of pancreas, and bloody ascites were observed in all PE groups rats, While no obvious changes were observed in control groups rats. Gyri became flatten, sulci were shallowed, brain ventricles were narrowing, cerebral superficial blood vessels dilatation and congestion were observed in all rats of PE groups:While no obvious changes were observed in control groups rats.2. The histopathologic alerations pancreatic tissues of the rats in PE were as follow. Erythrocytes and inflammatory cells were observed in the interstitium of pancreas in all PE groups rats, and the degree was aggravated with the time prolonging. Inflammatory cells were observed in the interstitium of pancreas in 3h group rats, and the degree of necrosis was pligher; more erythrocytes and inflammatory cells were observed in the interstitium of pancreas and serve swelling and necrosis of pancreatic acinar cell were noticed, the range of pancreatic lobule necrosis enlarged, and even spread to the whole pancreatic lobule, but its structures were clear in 12h group rats. While no obvious changes were observed in control groups rats.3. The histopathologic alerations of brain tissues in PE group rats were as follow. Neuron swelling, ballooning degeneration, and leukocyte accumulation andattached to the wall of microvessels were observed in the brain tissues in all PE groups rats, and the degree was aggravated with the time prolonging. While no obvious changes were observed in control groups rats.4. Test superoxide dismutase activity and malondialdbelryde level of homogenate of brain tissues:Compared with control groups, the SOD activity was significantly lower in PE groups (P<0.01), but the MDA level was significantly higher(P<0.01), and with the time prolonging the SOD activity gradually decreased to reach the lowest at 12h, but the MDA level gradually increased to reach the highest at 12h.5. Determination the water content of brain: Compared with control groups, the water content of rats brain were higher in PE groups(P<0.05). With the progress of disease, the water content of brain increased gradually, and the highest at 12h.6. The expression of TNF-a, IL-1β, TH, D2 in frontal cortex:TNF-a, IL-1βexpressed in the cytoplasm of inflammatory cells, neurons, glial cells, etc.TH mainly expressed in the cytoplasm of dopaminergic neurons. D2 widely expressed in the nuclei and/or cytoplasm of neurons. Under high power lens(×400), TNF-a, IL-1β, TH, D2 were more specific expression in PE groups rats brain tissue. Positive signal of TNF-a, IL-1β, TH, and D2 in PE 3h group enhanced more than the control group. The average optical density values respectively were 6.12±0.58,5.38±0.66,10.32±0.74,8.23±0.28. With the progress of disease, positive cells of TNF-a, IL-1β, TH, D2 and intensity increased to reache the peak at 12h. The average optical density values respectively were 10.43±1.49, 12.68±1.40,14.95±0.61,10.38±0.94. Compared with control groups, there were significant differences(P<0.01). Conclusion:1. With the progress of disease, the level of MDA gradually increased, while SOD activity decreased in brain tissue in PE groups rats. Above all tips with the progress of disease, oxygen free radicals increased and permeated the blood-brain barrier into the brain, so possibly result in neurons damage.2. TNF-α, IL-1βof brain tissues didn’t express or rarely expressed in control groups rats, but expressed obviously in PE groups rats, suggesting that inflammatory cytokines excessively released and directly or indirectly involved in brain damage in PE.3. TH, D2 of brain tissues expressed low in control groups, but expressed obviously in PE groups rats. With the progress of disease expression of positive cells gradually increased and enhanced, suggesting that synthesis of DA increased and receptor was regulation up in PE. |
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