The Mechanism Of BBG Inhibiting Inflammatory Response In Rats After Traumatic Brain Injury Research

Objective To investigate the relationship between ionic channel P2X7and theinflammatory after TBI. This study applied P2X7inhibitor BBG (brilliant blue G)following TBI to inhibit the activation of microglia, and then detected the expression ofTNF-α, IL-1β and PKCγ in hippocampus subregion in rats. In order to find newtherapeutic targets from inhibiting neuroinflammatory after TBI, and also provide anexperimental basis for clinical treatment of TBI.Method A total of120male Sprague-Dawley (SD) rats were randomly divided to3groups:Sham group (n=40), TBI group (n=40), BBG group (n=40). Furthermore, each group wasdivided into6h,12h,24h and48h. TBI model were reproduced by Marmarou’s fallingmethod. Following indexes were observed:H﹠E staining was used to observemorphological changes in hippocampus subregion.Wet/dry method was used to measurebrain water content. The cellular localization of PKCγ was observed byimmunofluorescence microscope.The expression of PKCγ, IL-1β and P2X7were evaluatedby Western blotting. Another32rats were selected to detect their ability of learning andmemory at7-11day following TBI by Morris water maze test.The Western Blot resultswere quantified by using the Gel-Doc analysis software. SPSS16.0was used to analyzeexperimental data. Q-test was selected to determine the significance between two groups. P＜0.05was considered as statistical significance.Result1Pathological changes observed by H﹠E: Neurons in the hippocampus of Shamgroup had a larger number, intact structure, arrangedt regularly, no significantpathological changes. In the TBI group, brain tissue was loose and edema. The gapsurrounding cells and capillaries widened significantly after trauma. Neurons in thehippocampus structure in TBI group, the number reduced significantly, nuclearcondensated, nuclear Jen part disappeared. In BBG group, the tissue is slightly edema,the gap surrounding cells and capillaries only slight widened. In hippocampus, neuronalstructures are relatively intact, the number of neurons was significantly increasedcompared with TBI group.2Results of spatial learning and memory ability: TBI groupperformed navigation test7-10days after the trauma, rats escape time significantlyincrease in the average latency compared with the Sham group (P<0.05). Compared withTBI group, the average escape latency of BBG group was significantly decreased in9,10days following TBI (P<0.05). In the search space experiment in11days post-TBI, thetimes of rats across the platform, the platform quadrant length and time ratio of the TBIgroup rats were significantly reduced (P<0.05). Compared with the TBI group, the timesof rats across the platform and the two ratios were significantly increased in BBG group (P<0.05).3Brain tissue water content detected: Sham group have no difference in tissuewater content at each time point. Brain tissue water content of TBI group increases withtime, was significantly higher than Sham group in12-72h. BBG group were significantlylower than the TBI group rats in12-72h (P<0.05).4Expression of P2X7by western blot:P2X7has a strong expression in Sham group, the expression had no difference at all timepoints. Compared with the Sham group, P2X7protein expression was significantly lower12h after TBI (P<0.05), and reached its lowest point at24h, although P2X7has beenraised later, the expression of it still was lower than the Sham group72h post trauma(P<0.05). The expression levels of P2X7in BBG group is slightly higher than the TBIgroup in24-48h (P<0.05).5Expression of IL-1β and TNF-α by western blot: Only asmall amount of IL-1β and TNF-α expression in Sham group, and the expression levelhad no difference at all time points. Compared with the Sham group, the expression ofIL-1β and TNF-α was increased from6h after injury (P<0.05), peaked at12h (P<0.05),continuous high expression at24h (P<0.05), at48h after TBI IL-1β and TNF-α proteinexpression decreased, but still significantly higher than the Sham group (P<0.05). Theexpression levels of IL-1β and TNF-α in BBG group is significantly lower than the TBIgroup in12-48h (P<0.05).6Result of PKCγ and NeuN by immunofluorescence. Thegreen fluorescent of PKCγ accumulated in cytoplasm labeled by FICT, the redfluorescent of NeuN was labeled by TRITC in neuronal nucleus. At24h after TBI,PKCγ(green) and NeuN (red) were collocated in hippocampus.7Expression of PKCγ bywestern blot: In Sham group, we could not observed the change of PKCγ in all points.The value of PKCγ gradually increased from12h and peaked at24h after traumacompared with the Sham group(P<0.05). Although PKCγdecreased later, it was stillhigher than the Sham group at72h post trauma (P<0.05). The changes of PKCγexpression in BBG group was slightly lower than the TBI group in24-48h (P<0.05).Conclusion Following TBI in rats, the P2X7inhibitor BBG could affect the expressionof inflammatory cytokines, thereby regulating neurons inflammatory. It played aneuroprotective effect via anti-excitotoxicity, and improving brain learning and memoryof TBI rats.