Expression And Significance Of AIF And The Apoptosis Of Nervous Cells On Infectious Brain Injury Model Of Rats Induced By Pneumolysin

Background and ObjectiveStreptococcus pneumoniae is the major pathogen of purulent meningitis in children. Pneumococcal meningitis has a high morbidity and high mortality. Pneumolysin(PLY) is the most important toxin that released during bacterial growth and lysis, and belongs to the family of thiol-activated, cholesterol-binding cytolysins. PLY has numerous functions,such as destroy the brain micro vascular endothelium cells, activate the host complement, induce proinflammatory reactions in immune cells, induce apoptosis of cells, cause widespread direct cellular and tissue damage by virtue of its membrane pore forming properties and so on, and ultimately cause the brain serious injury.The apoptosis causes permanent loss of neurons in the dentate gyrus of the hippocampus, which reduces the capability of learning and memory and seriously influence the life of children. Recently the mechanism of brain injury induced by PLY receives much concern,but these research more limited in vitro,lack of direct objective study in vivo.Previous studies on brain injury focus on cell necrosis. But it is realized that brain injury contains both cell necrosis and cell apoptosis. Susin s experimental results showed that in the present of the broad-spectrum caspase inhibitor Z-VAD-FMK, rat liver cell mitochondrial intermembrane components were still have promoting apoptosis activity, can cause separate HeLa chromatin condensation and large-scale DNA fragmentation with approximately 50-kb fragments in the way of caspase- independent, and named the protein as apoptosis-inducing factors (apoptosis inducing factor,AIF).AIF plays important roles in the brain injury, but the understanding of the specific mechanism of brain injury is still less. Research has shown that PLY causes neuronal cell apoptosis through mitochondrial damage and releasing of AIF in vitro, and there is less experiment in vivo.The purpose of this study was to determine the role and mechanisms of brain cell apoptosis and AIF in the brain injury induced by Streptococcus pneumoniae in animal models of infectious brain damage which were established by injecting PLY through internal carotid artery in vivo, and provide better therapeutic strategy of Pneumococcal meningitis.Materials and MethodsA total of 80 infant rats were randomly divided into 2 groups:pneumolysin group (PLY groups, n=40),0.2mL(7μg)PLY was injected via the left internal carotid. nomal saline group(NS group, n=40), isopyknic nomal saline was given to each rat by the same methods as PLY group. Each group was devided into 4 subgroups:6h,12 h,24 h and 48 h,10 infant rats were prepared in every subgroup. Rats were decapitated at different time point and brain tissue samples were prepared. By measuring both wet and dry weight, the water content of brain tissue were recorded. The Evan's blue(EB) content of brain tissue was measured by the formamide method,the expression of NSE protein,GFAP protein and AIF were studied by immunohistochemistry. And apoptosis was examined by TUNEL technique. All the data were analyzed with SPSS 17.0 software, the significant level was assigned at a< 0.05.Results1 morphology: In PLY group, the left side of the brain was larger than the right side, the brain was swelling and the cerebral pia mater was adhesive, cerebrovascular hyperemia was clearly revealed. After the HE dyeing and observation by light-microscopy, the clearance around vessels became broader and inflammatory cells infiltrated out of vessels. Neurons and gliacytes became swelled, vacuolar degenerated and their cubage augmented than before. These changes did not discovered in NS group. 2 The BWC, brain EB content and the expression levels of NSE protein and GFAP protein were higher in PLY group at each time point comparing with those in corresponding NS group, the difference was significant(p<0.05).3 After injection of PLY, cell apoptosis can be found in the cerebral cortex and the hippocamp.The differences of AIF and the number of apoptosis between in PLY group and the control group had statistic significance at every time point (P<0.05).4 Relative correlation analysis: positive correlation was got between AIF protein and the number of apoptosis, and the r value was 0.879, P<0.05.Conclusions1 PLY can induce brain injury by the way of neurocyte apoptosis.2 PLY can induce neurocyte apoptosis through the releasing of AIF by the mitochondria.