| ObjectiveBrain injury could be seen frequently in forensic science and clinic practice,It is difficult but useful to determine time course of brain injury.calpain1 and calpain2 are two important members of calcium activated neutral protease family,which plays a major role in tissue morphogenesis,wound healing and inflammation.To illuminate the relationship between the expression of calpainl and calpain2 and the time course of traumatic brain injury,the expression of calpain was studied by immunohistochemistry and Western blot techniques on model of focal brain injury in rats.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).Focal brain injury was performed according to the method established by Wu Xu.Following injury,rats were housed individually and allowed free access to food and water.Finally,rats were decapitated under diethyl ether anesthesia at 15min,12,24 hours,3,5,7,10and 15 days after brain injury(5 rats each group).5 rats were assigned to sham and control group respectively.2.Immunohistochemical staining of calpain1 and calpain2Brain tissues were fixed with 4%paraformaldehyde for 12 hours,and then embedded in paraffin.5μm-thick coronal sections were obtained with a microtome. After deparaffinizing and hydrating sections were incubated with methanol-H2O2 to block the endogenous peroxidases and normal rabbit serum to block nonspecific-binding sites.Sections were then incubated overnight in a humidified chamber at 4℃with polyclonal antibody against calpain1(1:400 diluted,Abcam), calpain2(1:400 diluted,Sigma)followed by incubated with biotinylated goat anti- rabbit IgG for 20 minutes,and then SP agent 20 minutes.DAB was used as chromogen for visualization of the reaction.Finally,the sections were counterstained with hematoxylin,dehydrated and mounted.The results were analyzed by the software of Motic Images Advanced 3.2 to get the area and average optical density(AOD) of positive staining.Additionally,HE staining was performed routinely.3.Analysis of calpainl and calpain2 by Western blotProtein extracted from brain tissue was quantified by Bradford method,and then run in SDS-PAGE,transferred to PVDF membrane.Membrane was blocked with 5% milk in TBS,then incubated with calpain antibody(1:1000 diluted) overnight at 4℃, and HRP labeled goat anti-rabbit IgG(1:2500 diluted) for 2 hours at room temperature. At last,protein bands were visualized by DAB kit.GAPDH was used as an internal control.Results1.Result of ImmunohistochemistryThe mmunostained cells were observedboth in control and sham group adjacent to injury.The calpain1 positive staining was remarkably increased and reached the maximal level in 12h after focal brain injury,decreased after 24h,markedly increased again in day 5,and then gradually decreased from day 7 to 15;the calpain2 positive staining was significantly increased in 12h and decreased in 24h,markedly increased again in day 5,and then gradually decreased from day 7 to 15.2.Result of Western blotCalpain1 and calpain2 both expressed in control and sham group by Western blot analysis.Calpain1 was detected staining bands both at 80kDa and 58kDa,calpain2 was detected staining bands at 80kDa.By analyzing the average gray value of immunoreactive bands of calpain,it shows the same changes as immunohistochemical staining.Conclusions1.Both calpain1 and calpain2 could be detected in normal rat brain;The expressions of calpainl and calpain2 protein had a possible relationship with the extended periods of time after brain injury.2.The expressions of calpainl and calpain2 might be used to determine time course of traumatic brain injury for forensic medicine.3.The expressions of calpain1 and calpain2 might have a possible relationship with secondary brain injury. |
