Study Of S100B And GFAP Brain In Primary Brain-stem Injury Diagnosis In Rats

Primary brain-stem injury usually refers to the brain-stem was the chief part of the head injury resulted from violence, and immediately occurred in a longer duration of loss of consciousness and even death. Pathological changes of brain-stem injury such as transaction, contusion, hemorrhage, diffuse axonal injury(DAI) and edema, etc, should occur after brain-stem injury. Besides that, the incompatibility between morphology changes and malfunction can be observed in most cases. For example, hemorrhage, DAI and edema could result in death with unobvious changes. It is difficult to diagnose in clinic medicine and forensic pathology according to general imaging examination and pathological investigation when the microleision exists in the brain-stem, because the high mortality, short survival time after injury and the symptom is not typical in the primary brain-stem injury when compared with other type of brain injury. At present, to establishment of earlier period index system has not been found, thus, it is very difficult to diagnose the cause of death after craniocerebral injury immediately. Therefore, we need an effective, reliable detective method for the diagnosis of primary brain-stem injury to identify the cause of death, which become an urgent problem in forensic pathology.Many studies have shown, S100B is very sensitive and specific to brain damage. S100B is a calcium-binding protein, in mammalian central nervous system, mainly by the synthesis and secretion of glial cells, particularly astrocytes and oligodendrocytes. S100B is highly specific for brain. During brain damage, microglial cells secreted IL-1, which strongly stimulated stellate cell activation and proliferation, and resulting in a large number of S100B. In 1965, McGeer proved in vivo and in vitro studies, IL-1 could increase the levels of S100B protein, to more than 3 times higher. In addition, IL-6, TNF-αhave promoting an effect to expressing of S100B protein. At the same time, S100B protein oneself also promotes the satellite cell hypertrophy and multiplication produces the more S100B proteins, thus forms the formation of positive feedback regulation. Moreover, S100B in demyelization disease, motor neuron disease (multiple sclerosis,acute infectious polyradiculoneuritis) and Alzheimer's disease also present the high expression. However, S100B in primary brain-stem injury expression has not been reported.Glial fibrillory acidic protein (GFAP) is the principal astrocyte cytoskeleton protein is also the specificity symbol protein. In the normal condition, has the support and metabolism to the nerve cell. After brain-stem injury, the astrocyte has the multiplication response, was considered as sensitive indicator of brain-stem injury. At present it had the thorough research in the brain-stem injury, is an applies widely and reliable tumor marker in forensic practice. That very important to elucidate the effects and the expression levels of S100B and GFAP in the primary brain-stem injury, and investigate the application value in the forensic inference of early brain-stem injury time and neuron reparative processes.In the present study, we intend to establish a model of traumatic brain-stem injury in accordance with our preliminary study, and to study the pathological changes of brain-stem of rats, using routing HE staining, Gless staining, real time PCR and immunohistochemical. Finally to find out the S100B and GFAP for early diagnosis and timing of brain-stem injury.This study was divided into three parts, the methods and results were as follows: 1. Establishment of the model of traumatic brain-stem injury and pathomorphologyThe devise of this model was made by us, which mainly consisted of iron stand, conduction pipe, percussion material and wooden board.85 anaesthetized (100g/L chloral hydrate,300mg/Kg) male SD rats weighing 180-220g were used. In antemortem injury group, every rats were hitted once on the occipital protuberance, with force of 65cm/450g/45°,the changes of respiratory rate and palpitation following hitting were observed. There were five rats removed in the groups because of suddenly death. As a result, we get 10 dead rats in 30min as head group caused by brain-stem contusion, those who didn't die were killed at 2h,6h,12h,24h,48h and 72h after percussion as survival groups, another 10 rats were used as normal control without injury.The results showed that, subarachnoid hemorrhage,brain-stem edema and hemorrhage could be found in all of rats who were hitted. And the 30min group hemorrhages are more than survival group, and more widespread. Gless staining showed, the morphologic changes of axon in brain-stem were showed as twisted, broken and swollen at 30min, and the myelin sheaths broken could be observed, the retraction ball was found at 12h and reached to peak up to 24h, then released at 72h.2. S100B-mRNA through Real time PCRWe chose the rats from the above groups randomly and distributed five in every group. The operation was followed by the kit directions seriously.The result showed below:30min group no significant difference compared with the normal, S100B-mRNA reduced in 2h group compared with the normal control group. Afterward, expression increased gradually with time, and peaked up to 24h group, 48h and 72h group reverse back the normal.3.Experimental studies on S100B and GFAP in brain-stem by immunohistochemistry In normal control group, S100B positive cells in astrocyte bodies and ecphyma, while a large number of S100B positive cells can be seen in 30min dead group and survival group(2h,12h,24h,48h).72h group reverse back the normal. In normal group, we can see GFAP side been small and the dendrite been minute. From 30min to 48h, the GFAP positive cells showed rise continually, and 72h group started to decrease.On the basis of above-mentioned results, following conclusions were obtained:1. The model of traumatic brain-stem injury made the mechanical to attack skull directly and could induce injuries mainly in brain-stem, and could be applied to study the pathogenesis, pathological changed and prognosis of brain-stem injury.2. After brain-stem injury, the expression of the S100B-mRNA changed immediately and plays an important role in the stress when neurons suffered from damage. The expression of the S100B-mRNA varied regularly and specificity. S100B can be used as a supplement index to estimate the primary brain-stem injury.3. The expression of GFAP and S100B play an important role in neurons repairing. And the expression of GFAP and S100B is correlated with post traumatic intervals after brain-stem injury in rats, suggesting that the expression of GFAP and S100B served as the makers for timing of brain-stem injury in forensic practice.