Associativity Study Between NF-κB Expression And Dating Of Wound After Cerebral Contusion Of Rat

Objective:To examine the expression trend ofNF-KB in the brain after cerebral contusion of rat by immunohistochemical SABC,western blot and real-time fluorescent quantitative RT-PCR and their relation with the wound ages. In order to explore the application for estimating the early time of brain contusion in forensic medicine aspect.Methods:The 64 Wistar rats were divided into control group (contain 8 rats) and experimental group (contain 56 rats) randomly. Experimental group were divided into7 sub-group by dating of wound.The cerebral contusion was performed on the side of middle of skull of experimental animal by using a gravitational force of a free falling weight. Tissue samples were fixed;paraffin embedded,sectioned and then stained by H.E.(hematoxylin and eosin) performed to identify the pathologic change of NF-κB at different internals within 14 days after brain injury. The brain tissue of 100mg were cut respectively at 1h,4h,12h,48h,72h,7d,14d (8rats in each subgroup) and then the samples were stored in liquid nitrogen. Rats in control group were dealt as same as rats in experimental group except for gravitational force. To examine the expression trend of NF-κB in the brain after cerebral contusion of rat by immunohistochemical SABC,western blot and real-time fluorescent quantitative RT-PCR, analyzed by use of the computer image analysis technology, Contrast with uninjured group.The results were assessed by statistic analysis.Results:(1) The purity,concentration and integrity all can suffice the next experiments by UV-Visible Spectrophotometer. (2) The amplification effect of NF-κB to housekeeping gene (rpL32) were concordant by standard curve, it indicated that the housekeeping gene and primers were suitable to the experiment.(3) immunohistochemical SABC, The positive cell of NF-κB was detected at 1h after injury, increased at 4h, peaked at 48h, decreased at 72h,and returned to the level of controls at 14d. Little positive cell was found at 7d. (4)In western blot, the protein of NF-κB expression increased at lh,reached peak at12-48h,dreased at 72h, but still higher than that of the controls. and returned to the level of controls at 14d. (5) In real-time fluorescent quantitative RT-PCR, NF-κBmRNA expression increased remarkably;but decreased at4h,fell to low the lowest at 48h, increased and peaked at 72h,weakened at 7dConclusion:The time-dependent expression of NF-κB in the brain tissue after cerebral contusion of rat could be applied in dating of wound for brain contusion. Immunohistochemical is simple and convenient. western blot and Real-time fluorescent quantitative RT-PCR is accurate and sensitive and refer to forensic study.