| Objective: Diffuse axonal injury (DAI) is seen as wide-spread damage in the white matter of brain characterized by morphological changes to axons throughout the brain and brain stem. 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 may largely account for the clinical manifestations of brain trauma. However, DAI is extremely difficult to detect noninvasively and is poorly defined as clinical syndrome. Maxwell summarized that in human DAI the majority of axons do not undergo immediate disruption at the time of injury (primary axotomy), but rather are exposed to non-disruptive axonal injubrain abscessleading, after several hours, to disruption (secondary axotomy).Microglia plays an important role in nervous system development, repair and regeneration of nerve tissue, neuronimmunity and synaptic transmission etc. Microglia responded most early to ischemic brain injury. The activated microglia could create considerable free radicles which could cause lipid peroxidation and injury neuron . Now the reaction of oxidative damage mediated by microglia is not clear in DAI rats . We induced DAI in Sprague-Dawley (SD) rats using an injury model adapted from Marmarou et al. in 1994. We want to the reaction of oxidative damage mediated by microglia is not clear in DAI rats . So, we can explain the relationship between microglia and the second brain injury in DAI animal model better.Methods: Induction of head trauma: SD rats, each weighing 239g to 281g, were randomly divided into 11 groups: control group, sham group, 30min, 1h, 3h, 6h, 12h, 24h, 48h, 72h and 7d group. Amidline scalp incision was performed followed by periosteal elevation to expose the central area of the skull vault between the coronal and lambdoid sutures. A stainless-steel disc 1 cm in diameter was firmly fixed to this central portion of the skull vault. When the trauma device was ready, the rat was placed in the prone position on a foam bed with the disc centered immediately under the lower end of the Plexiglas tube of the trauma device. The weight (450g) was allowed to drop freely from the designated height (2m) through the Plexiglas tube onto the disc; the foam bed together with the rat was moved away from underneath the tube immediately after impact to ensure a single hit. The rat was then transferred back to the operating table and observed for a couple of minutes. The skull vault was inspected for the presence of any fracture. The scalp was sutured. Rats that died on impact and those with skull fractures were excluded from the study.Animals in the control groups were surgically prepared for impact in the same way as above, but were not subjected to the head trauma . The brain was removed, sections 5μm thick were cut and stained with HE, Bielschowsky, Cresyl violet and Weil's myelin staining to verify the axonal changes.Immunocytochemical technique was used to examine the expression of nNOS,iNOS and COX-2.The data were presented as Mean±SD and analyzed with ANOVA and LSD using SPSS13.0 statistical program. A level of P<0.05 was considered as statistical significance.Results: 1 Change of pathological section:HE staining in brain stem: There was no alteration in brain tissue in control group and sham group. In injured group slight edema were observed in brain stem at 30min after injury. The findings were severe in the 24h and 48h injured rats.Bielschowsky staining in brain stem: There was no alteration in brain tissue in control group and sham group. In strike group, waving and enlargement of axons and reaction balls were observed in brain stem in the 12h injured rats. The findings were severe in the 48h and 72h injured rats.2 Immunochemistry in brain stem:The changes of the expression ofβ-APP at different time points in brain stem: In normal and sham control group there was little expression ofβ-APP. The number and diameter of positive axons increased and achieved peak at 48h and 72h postinjury,there were a lot of microglias around the ruptured axons and the positive neurons . The activation of microglia at different time points in brain stem: In control group and sham group CD11b, the surface marker of microglia, expressed little. Activated microglias changed their morphology to reactive or amoeboid forms in the injured rats.The changes of the expression of nNOS at different time points in brain stem : In control group and sham group nNOS expresses little in brain tissue. The expression of nNOS increased evidently at 30min ,compared with that of control group (P<0.05). The nNOS expression increased further at 6h, and get to the top at 12h and 24h . After 48h the protein expression began to decrease compared with 12h group, and decreased further at 72h and 7d, but was still higher than that of control group (P<0.05).The changes of the expression of iNOS at different time points in brain stem : In control group and sham group iNOS expresses little in brain tissue. The expression of iNOS increased evidently at 30min ,compared with that of control group (P<0.05). The iNOS expression get to the top at 72h . the protein expression began to decrease at 7d , but was still higher than that of control group (P<0.05).The changes of the expression of COX-2 at different time points in brain stem: In control group and sham group COX-2 expresses little in brain tissue. The expression of COX-2 increased evidently at 30min, compared with that of control group (P<0.05). The COX-2 expression increased further at 6h, and get to the top at 24h. After 48h the protein expression began to decrease compared with 24h group, and decreased further at 72h and 7d, but was still higher than that of control group (P<0.05).Conclusions: DAI was induced in SD rats using an injury model adapted from Marmarou et al. in 1994. The expression of iNOS and COX-2 by microglia increased evidently in the injured region of brain stem after DAI. The relationship between the reaction of oxidative stress mediated by microglia and the second brain injury in DAI animal model was approved. |
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