The Expression Of HMGB1Around Hematoma And Its Relation To Neuronal Cells Apotosis After Intracerebral Hmorrhage In The Rats

Objectives To make a stable and identical model of intracerebral hemorrhage in rat and to studythe expression of HMGB1around hematoma and its relation to neuronal cells apotosis.Methods Seventy Sprague-Dawley rats(weight，200to250g)were randomly individed into twogroup，trial group(n=36)and sham-operation group(n=36）.Bacterial collagenase IV(0.4U) wasinfused by using a microinfusion pump into the right caudate nucleus of rats.The sham-operationgroup received an equivalent dose of physiological saline with the same method.The behavior of ratswith intracerebral hemorrhage was evaluated using4tests in each rat at6h，12h，1d，3d，5d and7dafter operation．The specific4tests included Neurological Severity Scores，beam-walking test，net-screen test and rotating-rod walking.At6h，12h，1d，3d，5d and7d after the motor-behavior testswere done，each rat was killed by beheading．The brain was quickly removed and placed on a cooledsurface.The expression of HMGB1was measured by Immunohistochemistery and RT-PCR.Theexpression of IL-6and TNF-α were measured by ELISA.Terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate-bioti in situ nick end-labeling (TUNEL) was used todetect deoxyribonucleic acid (DNA) fragmentation.Results The results showed that the Neurological Severity Scores and the motor-behaviortests(beam-walking tes, net-screen test,and rotating-rod walking)were the highest at1d afteroperation in the four tests. The scores of the intracerebral hemorrhage group was falling at3d untilall the rats recovered and the scores fell to the lowest at7d.At all time points，the NeurologicalSeverity Scores and the motor-behavior tests Scores of the Sham-operated rats were lower than thatof the intracerebral hemorrhage rats and significant differences existed between the two groups (P＜0.05).HMGB1-positive cells appeared around the periphery of the hematoma.The immuno-reactivescore of HMGB1-positive cells was zero in the sham-operation group. The immuno-reactive score ofHMGB1-positive cells was markedly increased from6h and reach the first peak at24h after intracerebral hemorrhage in trial group,then it was reduced to a lower level at3d after intracerebralhemorrhage,but,interestingly,it was increased again at5d and reach the second peak at7d afterintracerebral hemorrhage.There was a significant difference compared with sham-operation group(P＜0.01).RT-PCR of expression for HMGB1mRNA showed same changes compared withImmunohistochemistery results.Concentration of IL-6and TNF-α around the periphery of the hematoma was increased from6hafter intracerebral hemorrhage and reach the peak in trial group, then it was falling until7d afterintracerebral hemorrhage and reduced to a lower level,but there was no difference between trial andsham-operation group.The TUNEL-positive cells appeared in the periphery of the hematoma.The number ofTUNEL-positive cells was nearly zero in the sham-operation group，but it was significantly increasedfrom6h and reach the peak at24h after intracerebral hemorrhage in trial group,after that it wasreduced to a lower level. There was a significant difference in two groups (P＜0.01).There was positive correlation between the expression of HMGB1protein and the number ofTUNEL-positive cells by Speaman’S correlation coefficient(r=0.320，P＜0.01)．Conclusions This study demonstrates that the expression of perihematomal HMGB1isupregulated with bimodal trends after intracerebral hemorrhage in rats. The results suggested that theup-regulation of expression of HMGB1may be related to neuronal cells apotosis during the acutephase after intracerebral hemorrhage.