| Objective: Diffuse axonal injury (DAI) is characteristic by White matternerve bundle multiple small damage, the major clinical manifestations of DAIis deep coma. The research of neuronal axonal injury has100years, thinkingthe pathogenesis of DAI is closely related of shearing which produced bytrauma, but the exact mechanism of the damage is still not fully solved.It isoften accompanied many kinds of complications, such as neurogenicpulmonary edema, abnormal muscle tone and epilepsy. In the forensicinspection as lacking the objective evidence, often cause judicial disputesbecause the injured claims getting not support. Therefore, the researching ofDAI has a historic,forensic medicine and social significance.Microglia as the main immune medium of the central nervous system(CNS), which plays an important role in regeneration, development,inflammatory disease of the central nervous system and the brain trauma. Theactivation of microglia is considered to be one core inflammation during thebrain damage. Wasserman JK studies suggesting that microglia cells, whichthrough complex "activation" process and "formation" change, accumulationthe edge of ischemic injury area. We can doubt whether continuous activatedmicroglia influence on neuron membrane potential which leading the repairfunction defect of neurons? The research of this aspect has not been seen. Cellbioelectricity is the foundation of the life activities, which plays an importantrole in the neurons regenerate and repair. Basing on reasons above, we studiedthe influence of microglia on neurons resting potential and action potentialduring the diffuse axonal injury. Our target is to reveal the damage role ofmicroglia.Methods: Healthy adult, weighing300±20g, male Sprague-Dawley (SD)rats were used in the present study. SD rats (n=70)were randomly divided intothree groups: Sham group,injury group and minocycline treatment group; Each group include six phase:6,12,24,48,72and120hours. Diffuse braininjury was induced in adult rats, using the experimental head trauma modeldescribed by Marmarou A.Sham group was given the operation and the treatment before batting.Injury group, the animals were anesthetized by Chloral Hydrate, A10-mm-diameter stainless steel disc with a thickness of3mm was then fixedwith gluewater on the exposed skull, in order to allow a homogeneousdistribution of the impact force. Thereafter, on the next day, the rats wereetherization, placed on a foam bed in a prone position. An acceleration impactwas applied by dropping a weight of450g from a height of2m onto themetallic disc on the skull through a vertical tube. No depression fractures ofthe skull, causing focal injuries, were seen. The minocyline treatment group,After the head trauma, minocycline (45mg/kg) was given to treatment groupby intraperitoneal injection, then once a day until all the rats been killed. Asthe same time, injury group were injected the same doses of saline (0.9%).The rats were then returned to their cages with food and water adlibitum.The animal was decapitated and the brain removed and placed in ice-coldACSF, Pause for3-5minutes while the tissue cools down. Cut slices (400μmthick) with vibrating slicer. Incubate the slices at30-35°C for at least60minutes before beginning recordings. Whole-cell patch clamp recordings wereperformed, to observe the frontal cortex of neurons resting potential and thechanges of action potential.Data were collected and analyzed with Clamp fit10.0(Axon Instruments)Origin7.5and Microsoft Excel.Data were expressed as the mean±S.E.Analyzed with ANOVA and LSD using SPSS13.0statistical program. A levelof p<0.05was considered as statistical significance.Results:1DAI model in SD rats was successfully reproductionApnea and coma occurred immediately after impact, the average time ofapnea is15.17±3.73s, the average time of coma is6.35±1.11min. Accordingto the standard of ethological score in rats, all injured rats got a6.09±1.21 scores of the total15scores.HE staining in brain tissue morphological observation:Control group andSham group rats organization structure is clear, there was no alterationbetween control group and sham group. After24h of brain stem injury, there issignificant congestion and edema in the brain stem (Fig1).Bielschowsky staining in brain tissue: The organization structure of brainstem is clear between Control and Sham group. The axon is long and straightwith smooth surface. At24h of injury, obviously waving and swelling of axonwere observed in brain stem (Fig2).2changes of the membrane potential at each time point of DAI12h and24h group, the body of cortex neuron in the state ofdepolarization, The resting membrane potential reduced to-63.03±0.81mV,the spike of action potential up to14.43±0.65/1000ms, compared with thecontrol has a statistical significance(p<0.05); Application of minocycline,which is the microglia inhibitor, each index improved obviously (Fig5,Fig6).48h group, the resting membrane potential of cortex was-72.16±0.51mV,the peak of amplitude was106.5±1.92mV, compared with the control grouphas not statistically significant. However, the spike of action potential was11.8±0.67/1000ms, The cortex neurons still performance exciting, comparedwith the control has a statistical significance(p<0.05), applying microgliainhibitor minocycline, neuronal excitability has improved(Fig7).72h group,the cortex neurons in obvious hyper-polarization stating, theresting membrane potential up to-76.3±0.8mV, the peak of amplitude was112.67±2.54mV, compared with the control has a statistical significance(p<0.05);Application of minocycline, which is the microglia inhibitors, eachindex improved obviously (Fig8).Conclusions: Diffuse axonal injury model in SD rats was successfullyestablished described by Marmarou A et al, who establish the experimentalhead trauma model. Resting potential and action potential were observed inthe absence and presence of minocycline. we assume that the continuousactivated microglia play excitability toxicity and damage effect in DAI. |
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