Foundation Research On Mechanisms Of Traumatic Brain Injury And Hypothermic Neuroprotection

The research of traumatic brain injury(TBI)has been attracted by clinician and scientific researchers owing to its increased morbidity with the development of economy and transportation,many years wilderness researcher devote themselves to search for the rehabilitative method of TBI,the exploration for vulnerate mechanism,clinical evaluation, auxiliary diagnosis,and treatment of TBI go extends ahead with the mode of foundation binding clinic constantly.They have performed extensive and penetrating investigation from different point of view for the occurrence reasons,biomechanics mechanisms,pathophysiology, pathomorphology,molecular pathology and other aspects of brain trauma. At present,we presume that the secondly brain injured factors be the important reasons for the brain damage.These lectors involve in cerebral ischemia,energy dysmetabolism,overload of ca²⁺,accretion of oxygen-derived free radicals,neurotoxic action of excitatory amino acids, stimulus by inflammatory factor,and so on.Traumatic coma is the difficult spot in the research of TBI,the pathogenesis and treatment always perplex many researchers,how to reveal the mechanism of traumatic coma has already become the new topic that neuroscience worker faced,pay close attention to the clinic issue of traumatic coma is the motive power for foundation research.Long term coma make close correlation with the earlier period in brain induced by brain trauma,initiate with the animal model of acute traumatic coma,we may obtain the pathomechanism correlated with trauma.In test 1 of our research,we established the traumatic coma animal model successfully by midline fluid percussion(MFP),and analysised the expression of calpainâ…¡, Voltage-gated sodium channel-6(Nach6)and the variation of microRNA in midbrain,try to interpret the section of pathogenesis of traumatic coma on the expression of mRNA and regulation of microRNA.Hypothermia treatment always to be the hot spot in the research of treatment about TBI,it has been hundreds of years from the 19 century overseas scholar initiate to extensive elementary investigation and clinical application,the outcome is satisfactory,the fairly penetrating recognition on mechanisms applicability of mild hypothermia neuroprotection has been achieved by experiment and clinical application study.But it is not clearly about the neuroprotective mechanisms,the study on neuroprotective mechanisms of mild hypothermia still to be the extremely attractive topic,many scholars still dedicate to explore this topic.In test 2 of our research,we established the moderate brain injury animal model,detailed analyze and explain the neuroprotective mechanisms of mild hypothermia on myelin sheath constitution,cytoskeleton, inflammatory regulation,immune function,and so on,in order to supplement the neuroprotective mechanisms of mild hypothermia,and provide experimental evidence for further empirical study and clinical application.The research of ultraprofound hypothermia is less than that of mild hypothermia in experimental and clinical study,for the reason of it is difficult to applicated in clinical.But as the whole integrity of the research of hypothermia,ultraprofound hypothermia should not de neglected,otherwise we could not understand the mechanism of hypothermia omnibearing and full-scale clinical application.In test 3 of our research, we research the duration time limit of selective cerebral ultraprofound hypothermia following both carotid arteries clip in monkey based on the research achievement that made by our college cooperation with Shanghai jiaotong university,in order to find the duration time limit of selective cerebral ultraprofound hypothermia following both carotid arteries clip,establish experimental and theory foundation for the further research and clinical application of ultraprofound hypothermia.Test 1.The establishment of animal model of acute traumatic coma in rat and research of the mechanism of itObjective:1.Establish safe animal model of acute traumatic coma in rat.2.Research the expression of calpainâ…¡and Voltage-gated sodiumchannel-6(Nach-6)mRNA in midbrain.3.study the variation of microRNA in mindbrain.Methods:12 Sprague-Dawley(SD)rats were subjected to 2 groups randomly,sham operated group(n=6):rats were anesthetized,the placement of injury tube inside the craniectomy,but not accepted fluid injury. acute traumatic coma group(n=6):rats under anesthesia with amobarbital(50mg/kg),Fixed Head with rat stereotaxic apparatus,a 4.8mm craniectomy centered over the midline parietal bone,exposured cerebral dura mater,the placement of injury tube(2.6mm)inside the craniectomy, dental bone cement fixed the tube,1.7atm fluid pressure impacted cerebral dura mater after conscious(righting reflex recovered).The brain were removed in experimental group 1h after coma,and in control group1h after operation,midbrain tissue were analyzed by RT-PCR to determine the expression of calpainâ…¡and Voltage-gated sodium channel-6(Nach-6)mRNA, and were studied by microRNA chip.Results:1.Established corresponding stable animal model of acute traumatic coma in rat.mortality<20%,coma time>1h.2.Results of RT-PCR: experimental group compared with control group,the relative amount of the expression of calpainâ…¡mRNA increased significantly(p＜0.01),the relative amount of the expression of Nach6 mRNA increased significantly (p＜0.05).3.33microRNA up-regulation above 2 fold,38 microRNA down regulation 50%.Conclusion:1.Calpainâ…¡acted as damage factor to dissolve nerve and myelin sheath.The expression of Nach6 increased in midbrain in traumatic coma rats,sodium ion inflow,resulted in nerve cell and glial cell injuried.They may influence each other,accelerated sodium and calcium ion inflow,effected reticular activating system in midbrain,it may concerned with the pathogenesis of traumatic coma.2.33microRNA up-regulation above 2 fold,38 microRNA down regulation 50%.microRNA may involved in cytoskeleton,cell adhesion,apoptosis,metabolism,ischemia, and involved in the regulation of expression in related gene in the mechanism of traumatic coma.Test 2.study on mechanism of the lateral fluid percussion brain injury and mild hypothermic neuroprotectionPart 1 the establishment of animal model of the lateral fluid percussion brain injury in ratObjective:Established stable animal model of the lateral fluid percussion brain injury.Methods:12 Sprague-Dawley(SD)rats were subjected to 2 groups randomly. sham operated group(n=6):rats were anesthetized,the placement of injury tube inside the craniectomy,but not accepted fluid injury,acute traumatic coma group(n=6):rats under anesthesia with amobarbital (50mg/kg),Fixed Head with rat stereotaxic apparatus,a 4.8mm craniectomy centered over the the left parietal bone,4.0mm lateral to the sagittal suture,the placement of injury tube(2.6mm)inside the craniectomy, dental bone cement fixed the tube,1.7atm fluid pressure impacted cerebral dura mater to establish moderate brain injury,established stable animal model of the lateral fluid percussion brain injury.Results:We use international approved fluid percussion brain injury device to establish animal model of brain injury,pressure is 1.4atm,it appears better stability,better repeatability,low mortality,high degree of confidenceonly one rat was dead(about5.6%).Conclusions:This animal model significant characteristic is:1. accurate quantitation of pressure,better repeatability,2.traumatic condition can be divided according to quantitation of energy,it can replicate mild,moderate,severe brain injury animal model.3.we can observe various kinds of treatment effect on mortality,dysfunction of nervous system after brain injury.Part 2 the expression of calpainâ…¡,MBP,MAP2 mRNA in hippocampusobjective:Study the expression of calpainâ…¡,MBP,MAP2 mRNA in hippocampus.Methods:18 Sprague-Dawley(SD)rats were subjected to 3 groups randomly,sham operated group(n=6):rats were anesthetized,the placement of injury tube inside the craniectomy,but not accepted fluid injury.LFP brain injury group(n=6):rats were assisted ventilation by respirator after injury,maintained body temperature by electrically heated blanket. Mild hypothermia group(n=6):rats'rectal temperature were dropped to 33℃and been maintained in this lever by ice after injury,rectal temperature were monitored by meter,it takes 3 hours for mild hypothermia. hippocampus tissue were analyzed by RT-PCR to determine the expression of calpainâ…¡,MBP,MAP2 mRNA.Results:Brain injury group compared with control group,the relative amount of the expression of calpainâ…¡mRNA increased significantly(p＜0.01);mild hypothermia group compared with brain injury group,the relative amount of the expression of calpainâ…¡mRNA decreased significantly(p＜0.01).brain injury group compared with control group, the relative amount of the expression of MBP mRNA decreased significantly (p＜0.01);mild hypothermia group compared with brain injury group, the relative amount of the expression of MBP mRNA increased significantly (p＜0.01).brain injury group compared with control group,the relative amount of the expression of MAP2 mRNA decreased significantly(p＜0.01); mild hypothermia group compared with brain injury group,the relative amount of the expression of MAP2mRNA increased significantly(p＜0.01).Conclutions:1.Mild hypothermia may provide neuroprotection by inhibit the expression of calpainâ…¡.2.Mild hypothermia may provide neuroprotection by promote the expression of MBP,reduce degradation of MBP and demyelination.3.Mild hypothermia may provide neuroprotection by maintain tabilization of MAP2.4.Mild hypothermia may provide neuroprotection by inhibit the expression of calpainâ…¡and reduce solution and damage of myelin sheath and cytoskeleton in nerve cell and glial cell, reduce degradation of MBP and demyelination,tabilization of MAP2.But there are many factors that maintain normal structure of cytoskeleton, specific mechanisms will be studied in afterwards tests.Part 3 The expression of PPAR-γ,NF-κBmRNA in cortexObjective:Study the expression of PPAR-γ,NF-κBmRNA in cortex.Methods:18 Sprague-Dawley(SD)rats were subjected to 3 groups randomly,sham operated group(n=6):rats were anesthetized,the placement of injury tube inside the craniectomy,but not accepted fluid injury.LFP brain injury group(n=6):rats were assisted ventilation by respirator after injury,maintained body temperature by electrically heated blanket. Mild hypothermia group(n=6):rats' rectal temperature were dropped to 33℃and been maintained in this lever by ice after injury,rectal temperature were monitored by meter,it takes 3 hours for mild hypothermia,cortex tissue were analyzed by RT-PCR to determine the expression of PPAR-γ,NF-κB mRNA in cortex.Results:Brain injury group compared with control group,the relative amount of the expression of PPAR-γmRNA decreased significantly(p＜ 0.01);mild hypothermia group compared with brain injury group,the relative amount of the expression of PPAR-γmRNA increased significantly (p＜0.01).brain injury group compared with control group,the relative amount of the expression of NF-κB mRNA increased significantly(p＜0.01); mild hypothermia group compared with brain injury group,the relative amount of the expression of NF-κB mRNA decreased significantly(p＜0.01).conclusion:Mild hypothermia probably increase the expression of PPAR-γ,it makes the number of PPAR-γcombine with P65/P50 subunit of NF-κB increased,then makes combine activity depressed,inhibit synthesis of NF-κB DNA,at last inhibit the expression of NF-κB,or inhibit NF-κB transcription by reinforce competitive binding cooperation activating factor P300 and CBP.mild hypothermia provide neuroprotection probably through PPAR-γin coordination with NF-κB or act alone make multi-pathway to relieve brain damage.Part 4 The effect of mild hypothermia on immune function in LFP brain injuried ratsobjective:Study the change of CD4⁺,CD8⁺antigen,IL-2,TNF-α,IL-6,IL-10 levels in serum after mild hypothermia treatment for LFP brain injuried rats.Methods:Rats were subjected to 3 groups randomly.Control group(n=6), LFP brain injuried 1-3 group(n=6),mild hypothermia 1-3 group(n=6),time points is:3h,1d,3d.blood was drew from inferior caval vein in different time points after the animal model accomplished,CD4⁺,CD8⁺ levels in serum were determined by using flow cytometry,IL-2,TNF-α,IL-6,IL-10levels in serum were determined though enzyme-linked immunosorbent assay (ELISA).Results:1.T1 group,T2 group,T3group compared with control group, relative amount of CD4⁺T cell decreased significantly(P＜0.05),H1 group compared with T1 group,H2 group compared with T2 group,H3 group compared with T3 group in the same time point,relative amount of CD4⁺T cell increased significantly(P＜0.01);T1 group,T2 group,T3group compared with control group,relative amount of CD8⁺T cell increased significantly (P＜0.05),H1 group compared with T1 group,H2 group compared with T2 group,H3 group compared with T3 group in the same time point,relative amount of CD8⁺T cell decreased significantly(P＜0.01).2.T1 group,T2 group,T3group compared with control group,IL-2 levels in serum decreased significantly(P＜0.05),H1 group compared with T1 group,H2 group compared with T2 group,H3 group compared with T3 group in the same time point,IL-2 levels in serum increased significantly(P＜0.05);T1 group, T2 group,T3group compared with control group,TNF-αlevels in serum increased significantly(P＜0.05),H1 group compared with T1 group,H2 group compared with T2 group,H3 group compared with T3 group in the same time point,TNF-αlevels in serum decreased significantly(P＜0.05); T1 group,T2 group,T3group compared with control group,IL-6 levels in serum increased significantly(P＜0.05),H1 group compared with T1 group, H2 group compared with T2 group,H3 group compared with T3 group in the same time point,IL-6 levels in serum increased significantly(P＜0.05); T1 group,T2 group,T3group compared with control group,IL-10 levels in serum increased significantly(P＜0.05),H1 group compared with T1 group, H2 group compared with T2 group,H3 group compared with T3 group in the same time point,IL-10 levels in serum increased significantly(P＜0.05).Conclusions:1.Mild hypothermia can increase relative amount of CD4⁺T cell and decrease CD8⁺T cell in serum in brain injuried rats.2.IL-2 levels in serum decreased,TNF-α,IL-6,IL-10 levels in serum increased in brain injuried rats.3.Mild hypothermia can increase IL-2,IL-6,IL-10 levels in serum,decrease TNF-αlevels in serum in brain injuried rats.4.Mild hypothermia treatment after brain injury can provide neuroprotection probably by improving body immune state through multi-pathway and inhibit traumatic inflammatory reaction.Test3 Neuroprotection of selective ultro-pround hypothermia in time limit of common temperature brain ischemia in rhesus monkeyObjective:Study the Neuroprotection of selective ultro-pround hypothermia in different time limit of common temperature brain ischemia,approach the limit time of brain ischemia resuscitation.Methods:15 healthy adult rhesus monkey,male,normal nervous function, 4-10 years old,average old 7.90±1.82 years old,4.2-14kg,average weight 8.22±2.15kg,randomly divided into 4 groups:resuscitation of Omin of 2 carotid artery blockage group(n=4),resuscitation of 10min of 2 carotid artery blockage group(n=4),resuscitation of 15min of 2 carotid artery blockage group(n=4),resuscitation of 20min of 2 carotid artery blockage group(n=3),establish animal models of ultro-pround hypothermia resuscitation.Results:All of the monkey in esuscitation of 0min of 2 carotid artery blockage group(n=4)and resuscitation of 10min of 2 carotid artery blockage group(n=4)were survived;2 long-term surviving,1 severe disable, 1 died in 15min of 2 carotid artery blockage group(n=4);it is difficult to resus the monkey in 20min of 2 carotid artery blockage group(n=3), vital sign were sustained 3,7,20 hours then died.Image of obviously ischemia were not found in 0min,10min,15min of 2 carotid artery blockage group in MRI,MRA,DWI,ADC.In fine structure observation,generous nerve cell and organ structure were normal in 0min,10min and survived monkey in 10min group.Minority of nerve cell were died in 15min of 2 carotid artery blockage group.Generous nerve cell were died in 20min of 2 carotid artery blockage group and died monkey in 10 min group.Conclutions:(1)Selective brain ultro-pround hypothermia(14.3-16℃) may achieved by half internal carotid artery cold irritating in mayrhesus monkey.(2)Selective brain ultro-pround hypothermia is safe,reliability and effective,not only act as neuroprotection and prolonged the time limit of ischemia but also avoid organ complication in system,it can be used as a tool of resuscitation.(3)It is safety for resuscitation in 10 min brain ischemia.(4)It is possible for resuscitation in 15 min brain ischemia,but may result in different degree the nerve nerve disfunction and organ complication and high disability and death rate.(5) resuscitation of 20min of 2 carotid artery blockage was invalid due to irreversibility damage.(6)We presume:the gold time of resuscitation for brain ischemia is in 10min,cerebral function will be full recovered without organ complication by ultro-pround hypothermia in 10min;life can be saved in 15min,but may result in different degree nerve disfunction and high disability and death rate.