| Objective: Diffuse axonal injury (DAI) is seen as wide-spread damage characterized by morphological changes to axons throughout the brain and brain stem in the white matter of brain. Severe DAI is characterized by immediate onset of coma at the time of injury, followed by persistent coma and vegetative state or severe persistent disability. DAI can occur itself or concomitantly with other brain injury. It is extremely difficult to detect noninvasively and is poorly defined as clinical syndrome. Recent studies have shown that the functional disturbance induced by DAI was evoked by the shear or tensile forces generated by the primary injury, but also caused by the"secondary attack",or neuron secondary injury,happened in the following time after the injury.We have found that quantity of microglia was activated after DAI. Inflammatory and oxidative damage induced by microglia plays an important role in the secondary injury after DAI. But the mechanism of nerve damage is not only the direct action of cytokines,but also the toxicity induced by the active microglia. Recent studies indicate that microglia is the important place for metabolism of excitatory amino acids while taking the support and nutritional role in central nervous system. The activation of microglia can release a lot of EAA which cause nerve injury in many nervous system diseases, but the role of active microglia induced EAA- excitotoxicity in the secondary injury has not been clearly.We induced DAI in Sprague-Dawley (SD) rats using an injury model adapted from Marmarou et al. in 1994. We want to observe the activation of microglia and the inhibition of microglia given minocycline after DAI by immunohistochemical method, then observed the content of excitatory amino acids by HPLC. So, we can explore the role of EAA in microglia-mediate second brain injury in DAI model.Methods: SD rats(weighing 260±28g) were randomly divided into three groups: Sham group ,injury group and minocycline treatment group; Each group include nine phase: 0h, 1h, 3h, 6h, 12h, 24h, 48h, 72h and 7d.Sham group was given the operation and the treatment before batting. Injury group and minocyline treatment group were used an injury model adapted from Marmarou A to induce DAI. After the head trauma ,minocycline (45mg/kg )was given to treatment group by intraperitoneal injection, then once a day until all the rats been killed. As the same time the injury group was injected same doses of saline (0.9%). The brain was removed, sections 5μm thick were cut and stained with HE staining to verify the brain tissue changes. Immunohistochemical technique was used to examine the expression of CD11b to determine the expression of microglia in cortex and brain stem. HPLC was used to detect the content of Glu and Asp in cortex and brain stem.The data were presented as Mean±SD , analyzed with ANOVA and LSD using SPSS13.0 statistical program. A level of p<0.05 was considered as statistical significance.Results:1 Estabished animal model of DAI:There was no alteration in brain tissue in sham group. The loose tissue was most severe by HE staining observation. In injury group congestion and edema were observed in cortex and brain stem at 12h after injury. The findings were severe in the 24h and 48h injured rats. Compared to the injury group, the congestion and edema were slighter in treatment group from 12h to 72h.2 Immunochemistry in brain tissue: The activation of microglia at different time points: In sham group CD11b, the surface marker of microglia, expressed little. CD11b expressed higher after injury compared with that of control group (p<0.05).The expression of CD11b in cortex got the peak at 12h.,while in the brain stem at 24h, and decreased at 72h, but was still higher than that of sham group (p<0.05). The trend of expression in treatment group was similar to injury group, but the quantity and appearance was different. There was not significantly difference in treatment group compared to injury group until 6h post-injury. (p>0.05)At 12h-72h, the expression in treatment group were significantly reduced than injury group. (p<0.05)3 The consequence of detecting the level of Glu and Asp with HPLC: The level of Glu and Asp of injury group after injury were higher compared with that of sham group,(P<0.05)and reached peak at 12h post-injury in cortex, while 24h in brain stem; then reduced after that. Though the trend of these two groups was similar, but it was much different between them. Compared to injury group, there was no statistically significant difference before 6h post-injury. (p>0.05) But it was significant reduced at 12h-72h. (p<0.05)Conclusions: DAI model in Sprague-Dawley rats was successfully established with high reproducibility, which could be used for the further study of DAI. We had observed the therapeutic effect of minocycline which can restrain activation of microglia and the relationship between the activation of microglia and the expression of Glu and Asp. Conclusively, excitotoxicity induced by microglia plays an important role in the secondary injury after DAI.
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