| ObjectiveClinical studyTo study the nature of the Glycerol of the cerebral extracellular in patients with mild traumatic brain injury (mTBI) and severe traumatic brain injury (sTBI) .To Study the Effect of Hypothermia on Cell Membrane Damage In Patients with Severe Traumatic Brain Injury.Experimental studyTo study the relationship between Glu and Gly and the change of phosphatidyl inositol system associating the change of Gly, to explore whether hypothermia therapy is due to these changes.In vitro studyThe aim of this study was to investigate whether the increase of Glu leads to the increase of Gly, the excess express of Bax, and the cell membrane injury.MethodsIn the first part, 6 mild traumatic brain injury (GCS13-14) patients and 31 severe traumatic brain injury patients were included in the study, all 31 patientswith severe traumatic brain injury (GCS<8) were randomly divided into the hypothermic group and the control group. In mild traumatic brain injury patients , microdialysis catheters were inserted into the right frontal cortex. In the severe traumatic brain injury patients , Microdialysis catheters were inserted into the cerebral cortex of perilesion and relative normal brain tissue.The probes were perfused at 0.3μl/min. The efflux dialysate was collected in 1 hour fractions that were frozen at -70℃. Every microdialysis probe was tested in vitro to measure the total recovery rates.Membrane length is 10mm and membrane diameter is 0.6mm.All sample was analyzed using the CMA Microdialysis analyzer. Intracerebral pressure of all patients, cerebral blood flow and brain temperature in the cases with sTBI patients was monitored.In the second part, 266 Sprague-Dawley rats weighing 300-350g were randomly divided into four group: control group, hypothermia group, normothermia group and hypothermia control group.hypothermia group and normothermia group were subjected FPI . The rectal temperature was measured and maintained for 6 hours at a constant level of around 32 ℃ in hypothermia group and hypothermia control group ,Sham control was subjected to all the same procedures except for the actual insult .Glu and Gly were examined using microdialysis at 0.5,6,12,24,48 and 72 hour after brain injury ,all rats were killed by decapitation under intraperitoneal anesthesia .Cortical tissue speciments at the site of the perilesion were excised ,aliquated,frozen,and kept at -80 ℃ .Express of protein and mRNA of mGluRl,5 and PLC was examined using Western blot and RT-PCR analysis.In the third part, Glutamate-induced hippocampal neurons apoptosis modelwas established.Cytotoxicity( Level of Gly and LDH) and apoptotic molecular regulator Bax were measured.Effect of hypothermia (29°C,31°C,33°C and 35°C) on apoptosis and cell membrane injury were evaluated.As control,cells without induction of apoptosis were incubated under the same conditiond as the apoptosis group.ResultsClinical studyl.The level of Glycerol In patients with mTBI(GCS13-14) was 43.95 ± 10.89umol/L and remained constant;2:In the NT group, Relationship between outcome and Gly,CBF,and CCP: In comparison with the severe or moderate disability group , pGly of the persistent vegetative state or death group was significantly increased,but CBF and CCP were significantly decreased;In comparison with the good recovery, Gly of the severe or moderate disability group was significantly increased, but CBF and CCP were significantly decreased;3:Effect of ICP,CBF and CCP on pGly: pGly of ICP>15 mmHg group,CCP<70mmHg group and CBF<50AU group was respectively higher than ICP<15 mmHg gruop> CCP>70mm group and CBF 5O-15OAU group;4: In the NT group,In patients with diffuse axial injury ,the mean pGly was 203.83 + 24.81umol/L,which was significantly higher than the levels for patients with epidural hematoma(n=2, 53.27+11.87,) , subdural hematoma(n=3, 87.93+4.3l),or contusion (n=8, 161.54 + 57.93);5: In the NT group, The level of Glycerol remained elevated throughout the monitoring period,in the third day postinjury,the highest level of Glycerol was seen;6: In NT group,nGly was is higher than the bassline values of Glycerol;7: In NT group, the pGlycerol in perilension were higher than in relative normal brain;8:In comparison with the NT group , the pGlycerol of HT group at 12hafter hypothermia therapy was significantly decreased and remained constant until rewarming period;9 : During the hypothermia therapy, nGly remained constant;10: After the hypothermia therapy 12 hours,in comparison with the nGly ,pGly did not differ.Experimental study1.Change of Glu and Gly:at 0.5h after brain injury, Glu and Gly of HT group and NT group were higher than that of control group and hypothermia control group;at 6h after brain injury, Glu and Gly of HT group were higher than that of control group and Glu and Gly of NT were higher than that of hypothermia group;at 12h after brain injury, Glu and Gly of NT were higher than that of hypothermia group, Glu and Gly of control group and hypothermia group were higher than that of hypothermia control group;at 24,48and 72h after brain injury, Glu and Gly of NT were higher than that of the three additional group.2.Change of mGluRl:at 0.5h after brain injury, mGluRl of NT group and control group were higher than that of HT group and hypothermia control group;at 6h after brain injury, mGluRl of NT group were higher than that of the additional three group;at 12h after brain injury, mGluRl of NT were higher than that of hypothermia group and control group, mGluRl of control group and hypothermia group were higher than that of hypothermia control group;at 24,48 and 72h after brain injury, mGluRl of NT group were higher than that of the three additional group. 3.Change of mGluR5:at 0.5,24,48 and 72h after brain injury, there was no difference between the HT group ,the NT group ,the control group and the hypothermia control group;at 6h after brain injury, mGluR5 of the NT group and the contral group was higher than that of the HT group and the hypothermia controlgroup;at 12h after brain injury, mGluR5 of NT were higher than that of the three additional group;4.Change of PLC:at 0.5,12,24 and 48h after brain injury, there was no difference between the HT group ,the NT group ,the control group and the hypothermia control group;at 6 and 48h after brain injury, PLC of the NT group was higher than that of the the three additional group, hi vitro studyAt each temperature examined from 31°C to 35°C , significant decreases in cytotoxicity and the express of Bax were observed, respectively. In 29 "C group , significant increases in cytotoxicity and the express of Bax were observed. Conclusion: Clinical study1: the cerebral extracellular Gly of patients with mild traumatic brain injury(GCS13-14)during wakefulness were close to the baseline value of the Gly;2: Gly is a important marker for membrane phosphlipid degradation and ischaemia,which reflects the severity of primary or secondary insult;3 : hypothermia protects brain in patients with sTBI by improving function and metabolism of brain cell membrane in perilension.Experimental study1.After traumatic brain injury, the change of Glu and Gly was similar, neural tissue cell membrane damage may be caused by Glu.2.Cell membrane damage caused by mGluRl,5 and PLC was associated with change of the phosphoinositide-mediated messengers.3. Hypothermia therapy attenuates the phosphoinositide-mediated messengers and neurotoxicity.In vitro studyHypothermia (31-35°C) inhibited express of Bax and cytotoxicity, although hypothermia below 29°C may induce apoptosis and cells membrane injury in intact cells. |
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