The Role Of Microglia To NADPH Oxidase Activation In RVLM And The Effect To Sympathetic Activity After DAI

Severe traumatic brain injury can cause high mortality and morbidity.Further more,some patients with severe head injury have excessive paroxysmal sympathetic hyperactivity(PSH)symptoms,the main symptoms of which are fever,hypertension,tachycardia,shortness of breath,sweating and dystonia.It ultimately leads to extracranial organ function failure and brings great challenge to the treatment of patients with severe brain injury.But its pathogenesis and physiopathology still have not been known to us.The study have found that rostral ventrolateral medulla(RVLM)is the important center of sympathetic excitement.Oxidative stress in the RVLM is the important pathophysiological basis of hypertension and heart failure.NADPH oxidase is the key enzyme of oxidative stress,and NOX2(gp91phox)is the main submit of oxidative stress.The microglia activation can activate NADPH oxidase,and generate oxidative stress,which eventually lead to the body's sympathetic hyperactivity.But so far,the research reports about microglia activation in RVLM after TBI has not been seen.The relationship between Microglia activation and the body's sympathetic hyperactivity after TBI remains unclear.To understand the mechanism of microglia activation on the sympathetic hyperactivity in RVLM after TBI,this paper mianly discuss microglia activation in RVLM after diffuse axonal injury and the role of oxidative stress on the sympathetic hyperactivity.Part 1 Microglia activation in RVLM after DAI in ratsObjective:1?To explore microglia activation in RVLM after DAI;2?To clarify wheather minocycline can inhibit microglial cells activationon in RVLM after DAI.Method:54 healthy adult male SD rats weighted 250g-300 g were randomly divided into normal control group(n=6),DAI group(n=36),DAI + minocycline group(n=12).Rats of DAI group and DAI 48 h + minocycline group receive diffuse axonal injury.Then survival rats of DAI group were randomly divided into three subgroups(24 h,48 h and 72 h subgroup respectively),each subgroup include 6 survival rats.At 4 h before injury and 4 h,8 h,12 h after injury,microglia inhibitor minocycline(10 mg/kg)was intravenously injected into rat body of DAI+ minocycline 48 h group.At 24 h,48 h and 72 h after injury,rats were fixed and perfused.Immunofluorescence was used to mark the microglia marker Iba-1 in the RVLM,through which we can know whether there is microglia activation and the role of minocycline on microglia activation in the RVLM.Result:1?At 24 h,48 h,72 h after DAI,we found that microglia activation in the RVLM showed a trend of rise.2?We can found that microglia activation in RVLM was significantly attenuated by minocycline,and there was statistically significant difference.Conclusion:1?Microglia were significantly activated in the RVLM after DAI;2?Microglia cells activation were significantly inhibited by minocycline.Part 2 The role of microglia on NADPH oxidase activation in the RVLM after DAIObjective:1?To explore whether there is increased for NOX2(gp91phox)expression in RVLM after DAI in rats;2?To explore whether minocycline can inhibit the expression of NOX2 in the RVLM after DAI;3?To definitude the role of microglia on NADPH oxidase activation in the RVLM after DAI.Method:78 healthy adult male SD rats weighted 250-300 g were randomly divided into normal control group(n=6),DAI group(n=36),DAI + minocycline group(n=36).Rats of DAI group and DAI + minocycline group received diffuse axonal injury.Then survival rats of DAI group and DAI + minocycline group were randomly divided into three subgroups(24 h,48 h and 72 h subgroup respectively),each subgroup include 6 SD rats.At 4 h before injury and 4 h,8 h,12 h after injury,microglia inhibitor minocycline(10 mg/kg)was intravenously injected into rats of DAI+ minocycline group.We took out the whole brain of rats quickly at three points(24 h,48 h,72 h)after injury,then put them into liquid nitrogen for 2 hours,then in-80 refrigerator follow.Western?-blot technology was used to detect the change of gp91 phox expression in the RVLM between different groups.Result:1?Compared with the normal control group,expression of NOX2(gp91phox)were obviously increased in the RVLM after DAI,and there was statistically significant difference(P<0.05);2?Expression of NOX2(gp91phox)of DAI + minocycline group were significantly lower than that of DAI group with statistical significance difference(P<0.05).Conclusion:1? Expression of NOX2(gp91phox)were significantly increased in RVLM after DAI;2? The expression of NOX2(gp91phox)was obviously inhibited by minocycline in RVLM after DAI;3? With the metabolism of minocycline in rats,inhibitory effect on NOX2(gp91phox)is also gradually weakened;4? Microglia plays an important role on NADPH oxidase activation in the RVLM after DAI.Part 3 The role of microglia on the body's sympathetic excitatory after DAIObjective:1?To explore the sympathetic excitatory(blood pressure,heart rate)changes after DAI;2?To make sure whether minocycline can reduce body's sympathetic excitement;3?To explore the effects of microglia activation on body's sympathetic activity.Method:104 healthy adult male SD rats weighted 250-300 g were randomly divided into normal control group(n=8),DAI group(n=48),DAI + minocycline group(n=48).Rats of DAI group and DAI + minocycline group received diffuse axonal injury.Then survival rats of DAI group and DAI + minocycline group were randomly divided into three subgroups(24 h,48 h and 72 h subgroup respectively),each subgroup include 6 SD rats.At 4 h before injury and 4 h,8 h,12 h after injury,microglia inhibitor minocycline(10 mg/kg)was intravenously injected into rats of DAI+ minocycline group.At 24 h,48 h and 72 h after injury,blood pressure and heart rate were monitored continuously through femoral artery catheter for 2 h,and the average value of blood pressure and heart rate were analysised by using the MPA system.Result:1?Compared with the normal group,sympathetic excitatory(blood pressure,heart rate)is higher after DAI with statistically significant difference;2?Sympathetic excitatory can be inhibited by minocycline at 24 h after DAI most obviously.Conclusion:1?Sympathetic excitability increase in a period of time after diffuse axonal injury;2?Minocycline can inhibit the sympathetic excitatory after DAI,with the metabolism of minocycline in rats,inhibitory effect on body's sympathetic excitatory is also gradually weakened.The result suggests that activated microglial cells play an important role on the body's sympathetic hyperactivity.