Study On Changes Of MMP-3 Expression After Brain Contusion In Rats

IntroductionBrain 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. Matrix metalloproteinase-3 (MMP-3) is a member of matrix metalloproteinase family, which plays a major role during tissue morphogenesis, wound healing and inflammation, and regulates the course of brain contusion. To explore the applicability of MMP-3 to determine the age of brain contusion, the expression of MMP-3 was studied by immunohistochemistry and Western blot techniques on model of brain contusion in rats.Materials and Methods1. Establishment of animal modelFifty adult male Sprague-Dawley rats (200-250 g 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 6, 12, 24 hours, 3, 5, 7, 10 and 14 days after the onset of contusion (5 rats each group). For control purposes 5 rats were given only skull operation, the rest 5 rats were killed directly without any treatment.2. Immunohistochemical detection of MMP-3Brains were fixed with 4% polyoxymethylene for 12 hours, 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 overnight in a humidified chamber at 4℃with polyclonal antibody against MMP-3(1:400 diluted, Santa Cruz), followed by incubated with biotinylated rabbit anti-goat IgG for 20 minutes, and then SP agent 20 minutes. DAB was used as chromogen for visualization of the reaction. Finally, the sections were stained with hematoxylin, dehydrated and mounted. Additionally, H.E. staining was performed routinely.3. Analysis of MMP-3 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 MMP-3 antibody (1:1000 diluted) overnight at 4℃, and HRP labeled rabbit anti-goat IgG (1:2500 diluted) for 2 hours at room temperature. At last, protein bands were visualized by ECL kit. GAPDH was used as a loading control.Results1. Result of ImmunohistochemistryWe observed no staining in control and sham operation group. The positive staining of MMP-3 appeared 6 hours after contusion, and extent of it becomes stronger 24 hours after contusion. The extent of staining reached the maximum at 5 days post-contusion, and then it decreased, positive staining could still be observed 14 days after contusion. 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. By statistical analyzing, there were significant differences between groups.2. Result of Western blotNo MMP-3 expression was detected in control and sham group by Western blot analysis. Expression of MMP-3 was at a low level during the contusion age 6h to 12h, and maximized in group 5d. In group 7d, the expression decreased, but it still could be observed in group 14d. By analyzing the average gray value of immunoreactive bands of MMP-3, it shows that there were significant differences between groups.Conclusions1. MMP-3 could not be detected in normal rat brain; 2. The expression of MMP-3 appears 6 hours after brain contusion;3. There is a relationship between the expression of MMP-3 and the time course after brain contusion. MMP-3 would be used as an indicator for estimating the age of brain contusion in forensic pathology.