The GABA And Glutamate System Changes And Its Influences On Sympathetic Hyperactivity In Nuclei Paraventricularis After Traumatic Diffused Axonal Injury

Traumatic Brain Injury(TBI)is one of the most common injury during wartime or peacetime,and more than 40% of which has no apparent skull fracture or intracranial focal injury.Diffuse Axonal Injury(DAI)is one of the most common types,and one of its most familiar complication is Paroxysmal sympathetic hyperactivity(PSH).With high mortality,high morbidity,long LOS(length of stay)and high cost,the PSH remains a serious clinical issue with unclear pathogenesis.Studies have confirmed that the Paraventricular Nucleus(PVN)in hypothalamus is pivotal in modulating sympathetic activity,and the integration of its inhibitory and excitatory signal decides the excitability of symathetic pathway.As the most common and most important transmitters in PVN,Glutamate and Gamma Amino Acid Butyric Acid(GABA)may have significant effect on regulating sympathetic signal.The present study is to explore sympathetic change pattern in post-DAI PSH rats,to investigate glutamatergic and GABAergic system variations in PVN of DAI rats and to sequentially find out the relationship of the above both.Part I Sympathetic change pattern in DAI rats.Objective:1.To establish the rat DAI model.2.To explore the sympathetic changing pattern by observing the basic vital signs.Methods:Employ 80 SD(Sprague Dawley)rats and randomly devided them into DAI group with 60 rats and control group with 20.Rats in DAI group were narcotized and blowed by the DAI model established machine twice,the control group were narcotized without blow.The survived DAI rats were then randomly devided into 3Hr(DAI), 24Hr(DAI),72Hr(DAI)group,and the rats in control group were devided with same pattern,i.e.into 3Hr(Ctrl),24Hr(Ctrl),72Hr(Ctrl)group,rats in each group were undertook MAP(mean arterial pressure)and HR(Heart Rate)measuement.Results:1.37 out of 57 rats remained alive in DAI group and the near-midline structure such as hypothalamus,hippocampe and medulla were ?-APP(+).2.Compare to Ctrl rats,the HR of DAI rats had increasing trend within 3h post-trauma(p=0.077),and had significant increase up to 24h(p=0.024),then stabled in high level till at least 72 h post-injury(p<0.001).the HR of DAI rats at 24 h post-injury had no difference with 3h post-injury(p=0.116),and no HR difference was seen between 24 h post-injury and 72 post-injury(p=0.668)in DAI rats.3.The MAP of DAI rats was higher than Ctrl rats 3h(p=0.033),24h(p<0.001)and 72h(p<0.001)post-injury.The MAP of DAI rats was higher at 24 h post-injury than 3h post-injury(p=0.011),and the MAP of DAI rats 72 h post-injury had no difference with 24 h post-injury(p=0.118).Conclusion:The sympathetic activity increased 3h post-injuty and kept increasing within 24 h post injury,then stabled in high level till at least 72 h post-injury.Part II Quantitative variations of glutamatergic and GABAergic receptors in PVN of DAI rats.Objective: To investigate glutamatergic and GABAergic receptors quantitative variations in PVN.Methods:We employed the SD rats in part I,and the PVN of rats were taken out to determine the mRNA and protein level of GABAR?6,mGluR5 and NMDAR1 receptors using RT-qPCR and Western-blot.Results:1.At 3hr post-DAI,compared with control group,the mRNA of GABAR?6 was down-regulated(P=0.004),whereas the mRNA of NMDAR1 and mGluR5 remained stable(P>0.05).At 24 hr post-DAI,compared with control group,the mRNA of GABAR?6 was down-regulated at 3hr post-DAI(P=0.001),whereas the mRNA of NMDAR1 and mGluR5 remained stable(P>0.05).At 72 hr post-DAI,compared with control group,the mRNA of GABAR?6 was down-regulated at 3hr post-DAI(P=0.023),whereas the m RNA of NMDAR1 and mGluR5 remained stable(P>0.05).2.At 3hr post-DAI,compared with control group,the protein level of GABAR?6 was down-regulated but without significance(P=0.083),and the protein level of NMDAR1 was down-regulated(P=0.019)whereas the protein level of mGluR5 remained stable(P>0.05).At 24 hr post-DAI,compared with control group,the protein level of GABAR?6 was down-regulated(P<0.001),the protein level of NMDAR1 returned to normal range(P>0.05)and the protein level of mGluR5 remained stable(P>0.05).At 24 hr post-DAI,compared with control group,the protein level of GABAR?6 was down-regulated(P=0.014)whereas the protein level of NMDAR1 and mGluR5 remained stable(P>0.05).Conclusion:1.The mRNA of GABAA receptors in PVN presents quantitative decrease in acute DAI rats,whereas the mRNA of NMDAR1 and mGluR5 remained stable.2.The protein level of GABAA receptors in PVN presents quantitative decrease in acute DAI rats,whereas protein level of NMDAR1 and mGluR5 were maintained in normal range.Part III Correlation study of functional change of glutamatergic and GABAergic system in PVN of DAI rats and sympathetic hyperactivity.Objective:To explicite the functional change of glutamatergic and GABAergic system in PVN before and after DAI injury and its roles in sympathetic hyperactivity.Methods:Employ 45 SD(Sprague Dawley)rats and randomly devided them into DAI group with 33 rats and control group with 12.Rats in DAI group were narcotized and blowed by the DAI model established machine twice,the control group were narcotized without blow.The survived DAI rats were then randomly devided into bicuculline(DAI),MPEP-MK801(DAI),MK801-MPEP(DAI)group,and the rats in control group were devided with same pattern,i.e.into bicuculline(Ctrl), MPEP-MK801(Ctrl),MK801-MPEP(Ctrl)group,The rats were undertook microinjection into PVN with 50 pm bicuculline,MEPE or MK801 with different ways according to the group rats in,and the changes of MAP,HR and HRV(SDNN,SDNN index)were observed.Results:1.18 rats in DAI group had higher MAP(P<0.001)and HR(P<0.001)and lower SDNN(P=0.009)and SDNN(P=0.038)index than the 12 rats in control group.2.6 rats in bicuculline(DAI)group and 4 in bicuculline(Ctrl)group received bicuculline microinjection and all of them exhibited higher MAP and HR and lower HRV.However,the HR(P=0.008)and SDNN(P=0.013)changes in bicuculline(DAI)group were more significant than bicuculline(Ctrl)group rats,and the MAP(P=0.077)and SDNN index(P=0.102)changes were more than bicuculline(Ctrl)group but without statistic significance.3.6 rats in MPEP-MK801(DAI)group and 4 in MPEP-MK801(Ctrl)group received sequential MPEP-MK801 microinjection.Rats in MPEP-MK801(Ctrl)group didn't exhibited significant change in every item.Rats in MPEP-MK801(DAI)group exhibited lower MAP(P<0.001)and HR(P<0.001)and higher SDNN(P=0.001)and SDNN index(P=0.026)after first injection of MPEP with statistic significance,and exhibited lower MAP and HR and higher SDNN and SDNN index after second injection of MK801 without statistic significance.4.6 rats in MK801-MPEP(DAI)group and 4 in MK801-MPEP(Ctrl)group received sequential MK801-MPEP microinjection.Rats in MK801-MPEP(Ctrl)group didn't exhibited significant change in every item.Rats in MK801-MPEP(DAI)group exhibited lower MAP(P<0.001)and HR(P<0.001)and higher SDNN(P=0.002)and SDNN index(P=0.002)after first injection of MK801 with statistic significance.After second injection of MPEP,the rats exhibited lower MAP(P=0.026)and higher SDNN index(P=0.009)with statistic significance and lower HR and higher SDNN without statistic significance.Conclusion:1.The present study suggested that activity decrease of GABAA receptor in PVN of acute DAI rats may be involved in the pathological mechanism of sympathetic hyperactivity.2.The present study suggested that activity increase of NMDAR and mGluR5 receptors in PVN of acute DAI rats may be involved in the pathological mechanism of sympathetic hyperactivity.