Changes Of Expression Of Homer Protein Expression After Focal Brain Contusion In Rats

IntroductionWith economy and transportation developing rapidly, the incidence of traumatic brain contusion is increasing year by year, which have severely threatening the human life. So the studies on time-course, mechanism and extent of brain contusion are of importance. Homer is the protein predominantly localized in the post-synapse in mammalian neurons and act as adaptor proteins for many postsynaptic density proteins. Initially, it was found in the nervous system, acting in signal transductions, synaptic form, receptor transportation and intra-cellular receptor location. Excitatory synaptic actions including epilepsy, high frequency stimulation, injury and development can induce the increase of expression of Homer. As yet, there are many studies about the mechanism of brain injuries, one of which is to induce intra-cellular Ca2+ releasing through mGluRs, in which mechanism Homer plays an important role. So it is necessary that a further research about Homer in brain focal contusion in rats be done.Materials and Methods1. Establishment of animal modelFifty adult male Wistar rats (220-250g 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 0.5h,3h,6h,12h, 1d,3d,5d and 7d after the onset of contusion. 2. Immunohistochemical detection of HomerBrain 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 goat serum to block nonspecific-binding sites. Sections were then incubated in a humidified chamber at 4℃overnight with monoclonal antibody against Homer(1:200 diluted, BEJING BIOSYNTHESIS BIOTECHN-OLOGY), followed by incubation with biotinylated goat anti-mouse IgG for 30 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, H.E. staining was performed routinely.3. Analysis of Homer 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 Homer antibody(1:800 diluted) overnight at 4%, and HRP labeled goat anti-mouse IgG(1:5000 diluted) for 2h at room temperature. At last, protein bands were visualized by DAB.Results1. Result of ImmunohistochemistryThe ratio of the number of Homer to total number of the cells nearby the wound were evaluated and calculated, The weak staining in normal group and sham operated group was observed. The positive staining of Homer increased 0.5h after contusion, kept 6h and reached peak at 12h, and then started to decrease, and went back to the basal level at 7d.2. Result of Western blotBy statistical analyzing, there were significant differences between each experiment groups and normal groups. Weak Homer expression was detected in normal group and sham operated group. Expression of Homer began to increase at 0.5h after contusion, and reached the maximum at 12h, and then started to decrease, and remained in the basal level at 7d.Conclusions1. Homer could be detected weakly in normal rat brain.2. The expression of Homer increased from 0.5h in perimeter domain around the wound, and reached peak at 12h, and then decreased, and remained in the basal level at 5d,7d.3. There is a relationship between the expression of Homer and the time course after brain contusion can be seen.