The Correlation Between The Expression Of GFAP?S100B And The Injury Time After TBI In Rats

Background and purposeTraumatic brain injury(traumatic brain injury TBI)is a complex and severe central nerve injury caused by direct or indirect action on the head.With the development of society and the increasing of social activities,the cases of traumatic brain injury occur frequently.It mainly happened in traffic accident,construction site high fall,youth violence,middle and old people fall and so on.The pathogenesis of TBI is complex,and it is the result of the common action of primary injury and secondary injury.At present,there are some progresses in the clinical study of TBI damage mechanism and treatment prognosis,and it can also be diagnosed according to CT and MRI.In the field of forensic medicine,the key point and difficulty of TBI research is to determine the damage time.Traditionally,the damage time determination based primarily on the pathological and behavioral changes after TBI,but this method is too subjective,not objective,time to the damage of judgment is not accurate.With the continuous improvement of molecular biology technology,the determination of damage time through the detection of biomarkers becomes the focus of forensic medical research.Astrocytes in the central nerve damage plays a vital role,GFAP and S100 B were specific markers in astrocytes,and also involved in immune response and repair after brain injury.In this experiment,the model of craniocerebral injury was established,the immunohistochemical staining method and the fluorescent quantitative PCR technique were used to establish the model of brain injury.The expression of GFAP S100 B in different time after injury and the relationship between the expression of S100 B and the time of injury were detected to provide a theoretical basis for the estimation of time of craniocerebral injury in forensic examination practice.MethodsWe purchased 110 healthy SD(Sprague Dawley,SD)rats(250-300g),male and female,from experimental animal center of Zhengzhou University Medical School [license number: SCXK(yu)2015-0004 NO.41003100004232.Cage in the forensic laboratory of Basic Medical College of Zhengzhou University animal room,fed for two weeks.According to the requirements of the experiment,all rats were randomly divided into the control group(20)and the experimental group(90).The animals in the experimental group were divided into 3h,6h,12 h,1d,3d and 7d according to different damage time.During the experiment,skull fracture or death was not included in the experimental group.The rat model of rat closed brain injury was prepared by using Feeney rat brain injury.The rats in the control group were the same as the experimental group except that they did not take the crack operation.The rats in the control group and the experimental group were divided into staining group,fluorescence quantitative PCR group and Western Blot group respectively to determine the positive cell expression,mRNA expression and protein expression of GFAP and S100 B respectively.(1)The process of animal treatment of the immunohistochemical staining group was as follows: The rat brain was fixed with polyformaldehyde.(via cardiac perfusion operation),paraffin embedding section and dyeing.HE staining observed the morphological changes in the cerebral cortex of rats,and observed the differences in morphological changes between the experimental group and the control group,and the expression rate of GFAP and S100 B positive cells was detected by immunohistochemical staining.(2)q-PCR group:The injured area and the surrounding 5 mm brain tissue were collected and stored in liquid nitrogen.The total RNAs were extracted,then reversed into cDNAs.Finally,the expressions of GFAP mRNA and S100 B mRNA were measured.(3)Western blot group: Extract the brain tissue samples,extract protein concentration,SDS-PAGE electrophoresis,transmembrane,one anti incubation,two anti incubation,exposure,and color display.Result1.Behavioral observation: compared with the control group,the rats in the experimental group were partially reflected and disappeared,with the presence of cortical syndrome,shallow breathing and even pause.2.Results of brain tissue samples and water content: rats in the experimental group,lesion and peripheral contusion,hemorrhage and edema.The water content of brain tissue changes with the time of injury.The brain damage of rats was gradually increased and the water content of the brain increased gradually,reaching a peak at 1d,and then began to decline.Compared with the control group,the water content of brain tissue in each group was statistically significant.(p<0.05)3.Results of HE : 3h began to show unclear cell structure;Localized bleeding,swelling,volume enlargement;The space around the cell increased;Glial cells proliferate;Damage to 3d began to recover gradually,and the damage of 7d was found to be basically normal,but it was still different from the control group.4.Results of immunohistochemical staining: after TBI,the positive expression rate of GFAP S100 B increased at 3h,and 1d reached its peak.After 3d,it began to decrease,and 7d was still higher than the control,which was statistically significant.(p<0.05)5.Results of q-PCR:After TBI,the expression of GFAP mRNA and S100 B mRNA began to increase at 3h,and 3h-1d continued to increase,1d to the highest peak,3d began to decline,and 7d was still higher than the control group.The difference was statistically significant.(p<0.05)6.Results of Western blot: After TBI,the expression of GFAP and S100 B protein began to increase at 3h,1d to the highest peak,and then decreased,and 7d was still higher than the control group.The difference was statistically significant.(p<0.05)Conclusion1.The improved Feeney free-fall hit device has the advantages of simple fabrication,convenient operation,low mortality of experimental animals,and can be repeated stable operation,which is of practical value for exploring the experimental study of brain injury in rats.2.The expression changes of GFAP and S100 B after craniocerebral injury in rats can provide a basis for accurate diagnosis of craniocerebral injury in clinic.3.after traumatic brain injury in rats,GFAP,called S100 B both with the distributed regularity of injury time,and have similar expression trend,the two proposed for forensic inspection practice provides the reference for the inference of craniocerebral injury.