The Relationship Between Expression Of HIF-1α And Estimation Of Cerebral Contusion Interval In Rats

Objective:To examine the sequential expression trend of HIF-la protein and HIF-lamRNA in the brain after cerebral contusion of rats by immunohistochemical SABC, Western Bloting and real-time fluorescent quantitative RT-PCR,evaluate the application for estimating the early time of cerebral contusion in forensic medicine aspect.Methods:The 64 SD rats were divided into control group and experimental group randomly,(n=8). Based on Feeney's method, the rat model of cerebral contusion was established. Animals were sacrifice at lh,6h,12h,48h,72h,7d,14d after cerebral contusion.Rats in control group were cut scalp and opend window in right parietal bone without cerebral contusion,were sacrifice after 1h.The brain tissue was cut in coronal section and fixed in 4% paraform for immunohistochemical.The brain tissue were cut respectively in each experimental group and then the samples were stored in-80°liquid nitrogen for the isolation of protein and total RNA. the expression of HIF-1a protein was detected by immunohistochemical SABC and Western Bloting.Quantitative Determination of immunohistochemical staining sections of positive cellsand band integral optical density value were detected by image analysis technique.Total RNA was isolated from each sample and reverse transcript to cDNA,then their reaction outcome were quantitated by real-time fluorescent quantitative PCR.The results were assessed by statistic analysis.Results:(1) The result of HE:The morphology of neuronal cell is normol, cell nuclear is round and nuclear staining is light with clear nucleolus in control group.experimental group can be seen scattered intraparenchymal hemorrhage, subarachnoid space and lateral ventricle hemorrhage.Deeply stained nuclei and condensation of neuron cells, edema appears around the gap to varying degrees are visible in different parts of neurons of the selected aspect, and gradually developed into nucleus broken and dissolved, neuronal necrosis disappeared, the surrounding glial cell proliferation;(2) The result of immunohistochemical SABC:Only few HIF-la-positive cells at control group,The positive product can be seen in cytoplasm and nucleus The expression of HIF-1αprotein could be detected at 1h after injury,The quantity of HIF-la-positive cells increased remarkably after 12h after injury(P<0.05);peaked at 48h, Little expression was found at 7d after injury.The 14d group was similar as the control group.(3) The result of Western Bloting:Bands can see at 1h after Cerebral contusion, widened significantly at12h, widest bands at 48h,then began to decrease,the band is almost invisible at 14d.Each experimental group compara to control group except 14d groups, the difference had statistical significance(P<0.05).Significant statistic difference was found in multiple comparison (P<0.05)except groups between 12h and 48h(P>0.05).(4) The result of real-time fluorescent quantitative PCR:The expression of HIF-1αmRNA was obviously increased at 1h(P<0.01)after cerebral contusion and arrived the peak value.then fell but another peak appear at 48h.the control group and the 14d group showed negative staining.Conclusion:The time-dependent expression of HIF-1αprotein and HIF-1αmRNA in the brain tissue after cerebral contusion is different,the peak of.HIF-1αmRNA more early than HIF-1αprotein,they may be used for the estimation of posttraumatic intervals after cerebral contusion in forensic practice.