Establishment Of Grading Model Of Acute Repeated Brain Injury And Study On The Expression Of NSE And GFAP Protein In Brain

Objective:To establish a graded animal model of acute repeated traumatic craniocerebral injury in rats.The graded model was used to study the expression of NSE and GFAP protein in the brain of rats after acute repetitive TBI.Methods:63 male SD rats were randomly divided into control group,single impact group and repeated impact group.According to the impact pressure,the latter two impact groups were divided into three subgroups: 0.2 MPa,0.3 MPa and 0.4 MPa,so there were 9 rats in each subgroup and the control group(n=9).Rats were anesthetized with ether.A pneumatic BIM-IV bio-impacter was used to adjust the impact air pressure and use the air pressure to drive the impact head to hit the top of the rat to cause injury.After anesthesia,the rats were hit once in single impact group,and hit twice in1 min at the same pressure in repeated impact group.Physiological signs of rats in each group were recorded and scored after injury.The coma time of rats was observed and modified neurological severity score(m NSS)was performed at 1 hour,3 hour and6 hour after injury.Beside,brain MRI was detected at 6 hours after injury and Abbreviated Injury Scale(AIS)was calculated.Pathological changes of brain tissue were examined by HE staining.The protein expression levels of NSE and GFAP in brain of rats in each group were detected by Western blot.Results:(1)The coma time of repeated 0.3 MPa subgroup was(35.11 ±18.57)min,which was higher than that of single 0.3 MPa subgroup [(12.78 ±6.02)min],P < 0.05.(2)The m NSS score of repeated 0.2 MPa subgroup [3(2.5,3)],0.3 MPa subgroup [9(8,10)] and 0.4 MPa subgroup [10(10,10)] was higher than that of single 0.2 MPa subgroup [2(1.5,2)],0.3 MPa subgroup [4(3.5,4)] and 0.4 MPa subgroup [9(9,10)]respectively,P < 0.05.(3)MRI showed that the injury was limited to subdural space,subarachnoid space and bilateral ventricles in single 0.3 MPa subgroup.Bilateral motor cortex,cingulate gyrus,corpus callosum and other parenchyma injuries were found in single 0.4 MPa subgroup.Focal lesions were found in bilateral motor cortex,cingulate gyrus,corpus callosum and external capsule in repeated 0.3 MPa subgroup.And in repeated 0.4 MPa subgroup,there were massive parenchyma injuries in motor cortex,cingulate gyrus and corpus callosum.(4)The AIS score of repeated 0.3 MPa subgroup[3(3,3.75)] was higher than that of single 0.3 MPa subgroup [2(2,2)].And the AIS score of repeated 0.4 MPa subgroup[4.5(4,5)] was higher than that of single0.4 MPa subgroup [3(3,3)],P<0.05.(5)Single 0.3 MPa subgroup of parietal arachnoid erythrocyte aggregates,between which a small number of glial cell infiltrates were seen.The superficial cortical layer was edematous and granular.The nerve cell gap was widened,and a small portion of neuronal cells were indistinct,fragmented or even disappeared,while most of them had normal structure.Single 0.4MPa subgroup saw subarachnoid hemorrhage in the parietal lobe,between which a large number of glial cell infiltrates were seen.The superficial cortical layer was edematous and granular.The nerve cell gap was widened,and some neuronal cells had indistinct structure,fragmented nuclei or even disappeared.Repeated 0.3 MPa subgroup of parietal subarachnoid hemorrhage,between which a large number of glial cell infiltrates were seen.The superficial cortical layer was edematous and granular.The nerve cell gap was widened,and some neuronal cells had indistinct structure,fragmented nuclei or even disappeared.Repeated 0.4 MPa subgroup was seen in the parietal arachnoid and lateral ventricles with massive aggregation of erythrocytes,between which a large number of neuroglial cell infiltrates were seen.The superficial layer of the cortex was edematous and granular.The neuronal gaps were widened,and most of the neuronal cells had unclear structure,cell fragmentation or even disappearance,while a small portion had normal structure.(6)NSE expression: The expression of NSE protein in the 0.4 MPa subgroup was significantly higher than that in the blank control group(P < 0.05).The expression of NSE protein in the subgroup of 0.3 MPa repeated impact and 0.4 MPa subgroup of repeated impact was higher than that of the blank control group,and the difference was statistically significant(P<0.05).The expression of NSE in the 0.3 MPa subgroup with a single impact was lower than that in the 0.3 MPa subgroup with repeated impacts,and the difference was statistically significant(P<0.05).(7)GFAP expression: The expression of GFAP protein in the 0.4 MPa subgroup with a single impact was higher than that in the blank control group,and the difference was statistically significant(P<0.05).The expression of GFAP protein in the subgroup of 0.3 MPa repeated impact and 0.4 MPa subgroup of repeated impact was higher than that of the blank control group,and the difference was statistically significant(P<0.05).The expression of GFAP in the 0.3MPa subgroup with a single impact was lower than that in the 0.3 MPa subgroup with repeated impacts,and the difference was statistically significant(P<0.05).The expression of GFAP in the 0.4 MPa subgroup with a single impact was lower than that in the 0.4 MPa subgroup with repeated impacts,and the difference was statistically significant(P<0.05).Conclusions:(1)The rat models of closed mild repeated brain injury,closed severe repeated brain injury and open very severe repeated brain injury were successfully established.This model can provide a replicable animal model for the study of biomechanics and pathological mechanism of secondary collision TBI in traffic accidents.(2)Compared with single brain injury,the neurological function of repeated brain injury was more severely impaired,and the pathologically damaged area of brain tissue was larger and deeper.The damage consequence of repeated brain injury is related to the amount of repeated injury.However,the damage accumulation effect may be nonlinear.(3)NSE and GFAP may be used as early damage detection indicators for r TBI severity in animal experiments.