Role of Tumor Necrosis Factor-α in Pathogenesis of Normobaric Hyperoxia-Induced Brain Cell Apoptosis in Newborn Rats

Objective To establish normobaric hyperoxia-induced brain injury animal models,and explore the effects of TNF-α on the pathogenesis of hyperoxia-induced neural cell apoptosis in the newborn rats.Methods One hundred and twelve 7-day-old SD rats weighting 12-18 g were randomly assigned into 2 groups:hyperoxia group(further divided into 2,6,12,24 hours subgroups according to the exposure time,n=14) and air control group.For hyperoxic group,the pups were kept in a chamber containing[(800±50) mL/L of oxygen(3 L/min)] with their dams for 2-24 h.The oxygen concentration was monitored with digital oxygen monitor.The rats in air control group were placed in air environment with 210 mL/L oxygen exposure,and then 10 rats were killed in each group at 24 h after the beginning of oxygen/air exposure.The brains were dehydrated,embedded and sliced for terminal deoxynucleotide transferase-mediated dUTP nick end labeling(TUNEL) staining to calculate the apoptotic index(AI).Immunohistochemistry staining was performed to detect expression of TNF-α,Caspase-3 of the brains.Four rats were decapitated in each group at 2,6,12,24 h after the beginning of oxygen/air exposure.The total cellular RNA of the left hemispheres was isolated and the expression of TNF-α mRNA was measured by reverse transcription polymerase chain reaction(RT-PCR).The relative ratio of optical density in hyperoxia group was calculated with β-actin as internal control and compared with that of normoxic group.All the data were analyzed with SPSS 10.0 software.Results The AI in 2 h hyperoxia group was significantly higher than that in air control group(F=88.39 P0.01).The AI in hyperoxia group was increased gradually with the prolongation of exposure to hyperoxia and reached the highest level after exposure to oxygen for 12,24 h(F=119.57,153.48 Pa0.01).TNF-α mRNA expression began increase at 2 h(P0.05) and maintained at high levels until 24 h(P0.01) after oxygen exposure.Protein expressions of TNF-α,Caspase-3 were significantly increased after oxygen exposure compared with those of air control group.TNF-α mRNA expression level in the hyperoxic group was positively correlated with the degree of neural cell apoptosis and the expression level of Caspase-3(r=0.986,0.982 Pa0.01).Conclusions Normobaric-hyperoxia can induce the neural cell apoptosis and over-expression of TNF-α in the newborn rats.Over-expression of TNF-α might result in neural cell apoptosis via activation of Caspase-3.TNF-α playes a role in the pathogenesis of hyperoxia-induced neural cell apoptosis.