| BackgroundTraumatic brain injury(TBI)has been an important issue that threatens human health.Brain trauma not only leads to primary damage to the cerebral cortex,but also cause death of secondary hippocampal neurons,resulting in epileptic seizures,memory and learning disabilities after traumatic brain injury so far,the treatment of craniocerebral trauma is still lack of exact and effective drugs.Therefore,to further explore the pathogenesis of TBI and pathophysiological processes,to seek more effective and effective treatment has become the focus of attention of scholars at home and abroad.One of the important pathophysiological mechanisms is that after the primary craniocerebral trauma,a large number of glutamate-based excitatory neurotransmitters is quickly released,and lead to an excitotoxic function to the not directly hit nerve cells.However,the use of exogenous drugs in the treatment of craniocerebral trauma has little effect,in this case,how to start and develop endogenous brain protection mechanism has become a research hotspot.A large number of basic studies have shown that synaptic membrane release of glutamate at the same time,can actively release a protective peptide neurotransmitter N-acetyl aspartylglutamate(NAAG),which provides a negative feedback to inhibit glutamate on the presynaptic membrane freed.Unfortunately,the NAAG in synaptic clefts is rapidly hydrolyzed by NAAG peptidases on the surface of glial cells after release into N-acetylaspartate(NAA)and glutamate,losing its endogenous Protective effects.Studies have shown that NAAG is mainly inactivated by GCPⅡ.Therefore,inhibition of GCPⅡ activity is expected to become a new strategy for endogenous neuroprotection.On the basis of this theory,we successfully established the GCPⅡ gene knockout mouse model,and proved that it could reduce the degeneration of hippocampal neurons and glial cells after traumatic brain injury without affecting the normal development of mice.In this study,the GCPⅡ gene was used to detect the neurotransmitter in the hippocampus of the mice with GCPⅡ gene deletion by using the high-field magnetic resonance model.The neurotransmitter content was analyzed and the GCPⅡ gene was removed.Mechanism of neuroprotective effects and its mechanism on brain edema after traumatic brain injury in mice.This study is divided into three parts.Part Ⅰ: Establishment and classification of mouse controlled cortical strike model;Part Ⅱ: Differences in glutamate and NAA content in hippocampus of GCPⅡ gene knockout mice and wild type mice after moderate craniocerebral trauma;Part Ⅲ: Effects of GCPⅡ gene knockout on secondary cerebral edema after traumatic brain injury in mice.Part Ⅰ The establishment and classification of the mouse controlled cortical impact modelObjective To establish a TBI model in mice by using a precision cortical impactor.The brain injury was graded according to the results of the test to establish a stable and repeatable model of traumatic brain injury in mice,and provides a reliable basis for post-related research.Methods Forty-eight male C57/BL mice(provided by the Shanghai Southern Model Biology Research Center,8-12 weeks of age,weighing 20-25 g)were randomly divided into 4 groups according to the random number method,3 TBI groups and 1 sham TBI group.A controlled cortical impact model was made using the Pin Point ? PCI3000,a precision cortical impactor(Hatteras Instruments Inc.USA).Among them,the depth of the TBI groups was set to 0.5 mm,1.0 mm and 1.5 mm respectively;the strike speed was set to 3.0 m / s;the duration of the attack was 180 ms,strike the cortex vertically and suture the skin without the bone flap given back.Sham TBI mice were only given the same location as the traumatic group mice,remove the bone flap without attacking,then suture the skin.The basic vital signs,coma time and nerve reflex were observed after injury.24 h after injury,the Evans Blue method was used to estimate the degree of blood-brain barrier injury in mice,and the different grading models of mice were primarily established.Results The degree of craniocerebral injury in mice was positively correlated with the depth of strike.With the increase of the depth of attack,the cortical lesion increased,the recovery time of mice gradually extended(p <0.01),pain reflex and reflex reflex inhibition time gradually extended(p <0.01).Evans Blue test showed that there is no significant blood-brain barrier damage in the sham TBI group.With the deepening of the injury,the degree of damage to the blood-brain barrier was significantly increased.Simulated mild craniocerebral injury at a depth of 0.5 mm when the speed and duration of the attack were the same;the moderate depth of craniocerebral trauma was simulated at a depth of 1.0 mm;the severe craniocerebral trauma was simulated at a depth of 1.5 mm.Conclusion A stable and reliable controlled cortex impact model can be established by using the precise brain injury device,and graded according to the depth of impact.This provides a reliable model basis for the further study of the pathophysiology of traumatic brain injury.Part Ⅱ Differences in glutamate and NAA levels in hippocampus of GCPⅡ gene knockout mice and wild type mice after moderate craniocerebral traumaObjective To investigate the effect of GCPⅡ gene knockout on glutamate and NAA level in mice after traumatic brain injury on the model of moderate brain injury.Methods 12 male GCPⅡ gene knockout mice and 12 male wild-type mice were randomly divided into wild type sham trauma group(WT + Sham),wild type trauma group(WT + TBI),gene knockout sham TBI group(KO + Sham),gene knockout trauma group(KO + TBI).Build moderate craniocerebral trauma models by the application of a precision cortical impactor.24 hours after injury,the ipsilateral hippocampus region(including CA2 / 3 region)in each mouse group was detected by using a 7 Tesla magnetic resonance scanning instrument(Biospec USR70 / 20,Bruker,Germany)and the Mest Re Nova software(Mestrelab Research,Spain)were used to analyze the content of neurotransmitters in the hippocampus of mice,and then analyze the results statistically.Results The relative contents of glutamate in the ipsilateral hippocampus were significantly increased(p <0.05),and NAA were significantly decreased;while there was no significant differences in the contralateral hippocampus(p> 0.05)of the wild type mice compared with the wild type sham TBI group(vs WT+ Sham).GCPⅡ gene knockout mice showed similar changes after craniocerebral trauma(p <0.05,vs KO + Sham),but the degree of elevation of glutamate and reduction of NAA was significantly lower than that of wild type(p <0.05,vs WT + TBI).Conclusions GCPⅡ gene knockout can significantly reduce the elevation of the concentration of glutamate,and release the decrease of concentration of NAA in the ipsilateral hippocampus after traumatic brain injury in mice.Part Ⅲ Effects of GCPⅡ gene knockout on brain edema after traumatic brain injury in miceObjective To investigate the effect of GCPⅡ gene knockout on brain edema after traumatic brain injury in mice based on the model of moderate brain injury.Methods Twelve male GCPⅡ gene knockout mice and 12 male wild-type mice were randomly divided into wild type sham trauma group(WT + Sham),wild type trauma group(WT + TBI),gene knockout sham TBI group(KO + Sham),gene knockout trauma group(KO + TBI).6 mice per group.Build moderate craniocerebral trauma models by the application of a precision cortical impactor.Twenty-four hours after brain injury,each mouse was subjected to T2 imaging using a 7 Tesla magnetic resonance scanner(Biospec USR70 / 20,Bruker,Germany).The brain tissue edema was observed in each group and after that the mice were sacrificed and the brain tissue was taken out.The ratio of dry and wet weight was measured and analyzed statistically.Results Edema of the injured and the surrounding cortical area were significantly higher in the mice after traumatic brain injury compared with the sham TBI groups.The cortical edema in the GCPⅡ gene group was significantly less than that in the wild type trauma group.The results of wet and dry weight analysis showed that the water content of GCPⅡ brain tissue was significantly lower than that of wild type traumatic brain injury(p <0.05,vs WT + TBI).Conclusion GCPⅡ gene can significantly reduce the degree of edema in the brain injury area and the surrounding cortex of mice. |
PDF Full Text Request