Expression Of NOS And CR In Rats Suffering From Primary Brain-stem Injury

Primary brain-stem injury is one of the most important causes of death in various kinds of craniocerebral injuries.Usually,pathological changes of brain-stem injury such as transaction,contusion,hemorrhage,diffuse axonal injury(DAI) and edema,etc,should occur after brain-stem injury.Beside that,the imcompatibility between morphology changes and malfunction can be observed in most cases.For example,hemorrhage,DAI and edema could result in death with unobvious changes.At present,the 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 immediatlly.Theoritically,death from craniocerebral injury is the outcame of functional disorder resulted from the damage to vital center in brain-stem.For these reasons,it is one of the urgent problem need to be solved in forensic mendicine to study the pathological changes in brain-stem failure,ande to establish objective standards for diagnosis of brain-stem injury.Nitric oxide(NO) has been described as one of the inflammatory mediators involved in the numerous pathophysiology including after a brain insult such as traumatic brain injury.NO plays a key role in the inflammation,however its role depends on the quantity of NO produced,the timing of its production,the milieu in which it is present,and the isoform of NO synthases(NOS) responsible for its synthesis.NO is produced from 1-arginine either by constitutive Ca2+-dependent NOS,including endothelial NOS(eNOS) and neuronal NOS(nNOS),or by inducible Ca2+-independent NOS(iNOS).NO has both beneficial and detrimental effects in cerebral ischemia.The nonselective NOS inhibitor N^G-nitro-1-arginine methylester either reduces infarction volumes in cerebral ischemia or increases ischemic injury.Identification of the three isoforms of NOS has increased the complexity of this issue.As we know,calcium overload is one of the important mechanism for neuron injury as selectivity and tardive,thereby,Calretinin(CR) could active the Ca²⁺/Mg²⁺-ATP enzyme to prevent Ca2+ accumulation and calcium overload.In the present study,we intend to establish a model of traumatic brain-stem injury in accordance with Deng Ping's model,and to study the pathologyical changes of neuron in reticular substance of medulla oblongata of SD rats,using routing HE staining,colorimetry and histochemistry.Finally to find out the criteria for early diagnosis and timing of brain-stem injury.This study was divided into three parts,the methods and results were as follows:1.Establishement of the model of traumatic brain-stem injury:The devise of this model was made by ourselves,which mainly consistedof iron stand,conduction pipe,percussion material and wooden board.Forty-five anaesthetized(100g/L chloral hydrate,300mg/Kg) male SD rats weighing 350～400g were used.In antemortem injury group,70 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 fifteen dead rats in 1 h as dead group caused by brain-stem contusion,those who didn't die were killed at 6h,12h, 24h and 48h after percussion as survival groups,another 10 rats were used as normal control without injury.The results showed that,the force of 65cm/450g/45°induced mild brain-stem injury without obvious injuries in cerebellum and cerebrum,only small focal hemorrhage could be found in brain-stem by gross examination in 1h dead group and the punctuate hemorrhage in brain-stem in survival group.The brain-stem mainly located on the line linking the posterior angle of fourth ventricle and interpeduncular fossa,or surrounding tissue,in which the respiratory center and cardiovascular center lies.2,The activity detection of NOS,iNOS,cNOS through colorimetryWe 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:both NOS and cNOS rised in 1h dead group and survival group compared with the normal control group(6h,12h,24h),in the other,iNOS was unchanged;meanwhile,iNOS were highten in 48h group and cNOS reverse back the normal.3,Experimental studies on nNOS and CR in reticular substance of medulla oblongata by immunohistochemistryIn normal control group,there was small nNOS positive cells in reticular substance of medulla oblongata,while a large number of nNOS positive cells can be seen in 1h dead group and survival group(6h,12h,24h).In normal control group,we can see a lot of CR positive cells,but the CR positive cells reduced distinctly in 1h dead group and survival group,furthermore,from 1h to 48h,the positive cells showed decrease continually.On the basis of above-mentioned results,tbllowing conclusions were obtained:1,The model of traumatic brain-stem injury could induce injuries mainly in brain-stem,and could be applied to study the pathogenesis,pathological changed and prognosis of brain-stem injury.The preliminary localization of lethal lesions in injured brain-stem indicated that,lesions in the neural structure concerning the respiratory center and cardiovascular center might be the cause of death,which were principally on or close to the line linking the posterior angle of fourth ventricle at the dorsal surface and interpeduncular fossa at the central side.This study provide the experimental basis for the identification of brain-stem injury.2,In a certain period(48h) as we know,NOS plays an important role in the primary brain-stem suffering from craniocerebral trauma,cNOS participates the primary injury and iNOS participates the secondary injury.3,The suffering of CR neuron lead to the rising of Ca²⁺ which active cNOS to catalyze a lot of NO.So we conclude that the disequilibration of Ca²⁺ and burst of NO may be an immporant mechanism of fast death suffering from primary brain-stem injury.4,Death caused by primary brain-stem injury is multiple factor process and cNOS plays an imoporant role in it.ln 24h,the expression of cNOS reach zenith,but the rats is survival,the reason for it is still to expore.5,We can observe that nNOS rise in the early period and decrease in 48h,in the comparison,CR present decrease straightly,from the results,we realize that CR is one of the protective factors of antagonizing calcium overload,so the next step we can study the mechanism of other protective factors.