The Study On Changes Of Expression Of PSD-95 After Brain Contusion In Rats

IntroductionAs national economy developing rapidly,construction industry and transportation industy spring up,traumatic brain contusion is the main cause of death in young crowd. The mechanism of brain contusion and its prognosis are always the emphasis on clinical and the forensic research.There are a lot of theory and hypothesis including Calcium Overload,icrocirculation,on which the therapy depended less can pass to theâ…¢-stage clinical test to guid the clinical work,Brain contusion is the most important injury in forensic science,and estimating the age of brain contusion is very useful for detecting criminal cases,though it is a difficult problem,Therefore.it is necessary to study the molecular mechanism of brain contusion for furtherly guiding the clinical work and forensic expertise,by appling the immunohistochemisty and the western-blot to exam the expression of PSD-95 after brain contusion.Materials and Methods1.Establishment of animal modelFifty adult male Sprague-Dawley rats(200-250g body weight) were obtained from Department of Laboratory Animal Science,China Medical University.The rats were initially anesthetized with diethyl ether,and then anesthesia was maintained with 2%pentobarbital sodium(30mg/kg).Brain contusion was performed according to the method established by Wu Xu.Following surgery,rats were housed individually and allowed free access to food and water.Finally,rats were killed under diethyl ether anesthesia at 3h,6h,12h,1d,3d,5d,7d and 10d after the onset of contusion.2.Immunohistochemical detection of PSD-95 Brain samples were fixed with 4%polyoxymethylene for 12h,and then embedded in paraffin.Five-μm-thick coronal sections were obtained with a microtome.Paraffin was removed and sections were incubated with methanol-H₂O₂ to block the endogenous peroxidases and normal rabbit serum to block nonspecific-binding sites.Sections were then incubated in a humidified chamber at 4℃overnight with polyclonal antibody against PSD-95(1:400 diluted,BEIJING BOISYNTHESIS BIOTECHN-OLOGY), followed by incubated with biotinylated rabbit anti-goat IgG for 20 min,and then with SP agent 20 min.DAB was used as chromogen for visualization of the reaction.Finally, the sections were stained with hematoxylin,dehydrated and mounted.Additionally,HE staining was performed routinely.3.Analysis of PSD-95 by western blotProtein extracted from brain tissue was quantified by Bradford method,and then run in SDS-PAGE,transferred to PVDF membrane,which was blocked with 5%milk in TBS,then incubated with PSD-95 antibody(1:200 diluted) overnight at 4℃,and HRP labeled rabbit anti-goat IgG(1:2500 diluted) for 2h at room temperature.At last, protein bands were visualized by ECL kit.Results1.Result of immunohistochemistryThe ratio of the number of PSD-95 to total number of the cells in the wound were evaluated and calculated,The weak staining in normal group was observed.The positive staining of PSD-95 increased 3h after contusion,kept 6h and reached highly level 12h.The positive staining reached the maximum at 5d post-contusion,and then decreased.2.Result of western blotBy statistical analyzing,there were significant differences between each experiment groups and normal groups.Weak PSD-95 expression was detected in normal group by Western blot analysis.Expression of PSD-95 was at a high level during the contusion age 3h to 12h,then decreased in 1-3d,the expression increased again in 5d and decreased in 7d.Conclusions1.PSD-95 could be detected weakly in normal rat brain.2.The expression of PSD-95 increased in 3-12h after brain contusion,then decreased in 1-3d and increased again in 5d,7d appeared weak expression,two peaks can be seen in 12h and 5d.3.There is a relationship between the expression of PSD-95 and the time course after brain contusion can be seen.