The Effect Of Prematurity And Abnormal Oxygen Concentration On Bilirubin Neurotoxicity

PartⅠEVALUATION OF A NEW APPROACH OF INJECTINGBILIRUBIN SOLUTION INTO CEREBELLOMEDULLARY CISTERN TO ESTABLISH BILIRUBIN ENCEPHALOPATHY MODEL IN NEONATAL MOUSEObjective: To evluate the feasibility of animal model of bilirubin encephalopathy by injecting bilirubin solution into cerebellomedullary cistern of neonatal full-term mice.Methods: Thirty 7-day-old mice were randomly divided into 5 groups: control group, model group 1, model group2, model group3, and model group 4. 10mg/ml Bilirubin solution was injected into the cerebellomedullary cistern in model groups (bilirubin 20μg /g bodyweight), while equal volume of normal saline was injected into the cerebellomedullary cistern in control group. The mice in model groups were sacrificed at the time of 12hrs, 24hrs, 48hrs and 72hrs after injection, respectively, and those in control group were sacrificed at 24hrs after injection. The apoptosis and necrosis of neurocytes, the expressions of Bcl-2 and Bax proteins were detected by HE stain, immunohistochemical method and TUNEL in the hippocampus, respectively.Results:The mice of model groups appeared abnormal behaviors in different degree,such as proneness,tumble,limbs twitched,rolled and become less active. Under light microscope, the neurocytes in their hippocampus appear apoptosis and necrosis. The nissl body in pyramidal cell body and dendron decreased, even dissolved to disappear. TUNEL showed that the apoptosis rate in the hippocampus increased obviously at 12hr post-injection, reached the peak at 24hr post-injection(47.8±8.10). The positive rate of Bax protein in the hippocampus reached the peak at 24hr post-injection(6977.98±402.6), while the positive rate of Bcl-2 protein decreased to the lowest(3096.46±126.3).Conclusions:The method of injecting bilirubin solution into neonatal mice'cerebellomedullary cistern to set up the animal model of bilirubin encephalopathy is simple and feasible. The peak time of neurocyte apoptosis in hippocampus was 24hour post-injection. It's crucial to prevent bilirubin encephalopathy as early as possible. PartⅡTHE EFFECT AND POSSIBLE MECHANISM OF PREMATURITY AND ABNORMAL OXYGEN CONCENTRATION ON BILIRUBIN NEUROTOXICITYObjective: To observe effects of prematurity,hypoxia and hyperoxia on bilirubin neurotoxicity, and to investigate the potential pathogenetic mechanism.Methods: Senventy two 7-day-old mice in full-term group and premature group (32 in each group), were randomly divided into 6 groups: control group, bilirubin group, hypoxia grouop, hyperoxia group,hypoxia+bilirubin group, hyperoxia+bilirubin group. For each mouse, 0.02ml normal saline was injected into its cerebellomedullary cistern in control group, while 10mg/ml bilirubin solution(20μg bilirubin/g weight) was injected into the cerebellomedullary cistern in bilirubin groups. The mice in hypoxia group were exposured in 8% O2 for 3 hours,while the mice in hyperoxia group were exposed in 95% O2 for 3 hours. The mice in the hypoxia+bilirubin group were injected with the bilirubin solution after exposed in 8% O2 for 3 hours, and the mice in the hyperoxia+bilirubin group were injected with the bilirubin solution after exposed in 95% O2 for 3 hours. The mice in every group were sacrificed at 24hrs after injection. The apoptosis and necrosis of neurocytes, the expressions of iNOS and TNF-αwere detected by HE stain, TUNEL and immunohistochemical method in the hippocampus, respectively; and then, the expression levels of the neurocyte apoptosis and the inflammatory factor in the groups were compared with each other.Results::(1) The mice in the bilirubin group appeared to have different severities of abnormal behaviors , such as proneness, tumble, four limbs twitched, rolled and less action. The symptoms of premature mice seemed to be more serious. (2) HE stain showed that the phenomenon of apoptosis and necrosis of neurocytes in hypoxia+bilirubin group is more significant than in the other groups. (3) TUNEL showed that compared with the bilirubin group in full-term mice (23.33±4.89), the apoptosis rate in the hippocampus of bilirubin group in premature mice (30.17±3.76) was significantly increased (P<0.05); compared with the bilirubin groups of full-term and premature mice (23.33±4.89和30.17±3.76), the apoptosis rate of the hypoxia+bilirubin group in corresponding term (27.67±3.01 and 34.33±3.01) increased obviously (P<0.05); whereas the apoptosis rate of the hyperoxia+bilirubin groups (26.50±2.35和30.67±3.33) didn't not significantly increase (P>0.05); (4) Immunohistochemistry showed, compared with the mice of bilirubin group in full-term(0.388±0.020 and 0.253±0.010), the level of TNF-αin the hippocampus of premature mice in bilirubin group (0.425±0.014) increased significantly (P<0.05),while the expression of iNOS (0.253±0.009) was not significantly increased(P>0.05); however, the expression of iNOS (0.437±0.024) and TNF-α(0.440±0.001) in hypoxia+bilirubin group increased obviously (P<0.05). In contrast, in the hyperoxia+bilirubin group, the expression of iNOS (0.254±0.010) was not significant increased (P>0.05), the release of TNF-α(0.416±0.011) did significantly increase(P<0.05).Conclusions:Bilirubin can cause the damage of hippocampus neurocyte by inducing apoptosis and releasing TNF-αinflammatory factor. Prematurity can aggravate bilirubin-induced apoptosis and increase the release of TNF-α, but can't increase the expression of iNOS. Hypoxia can deteriorate bilirubin-induced apoptosis and elevate the release of TNF-αand the expression of iNOS. In contrast, 3-hour hyperoxia did not worsen bilirubin-induced apoptosis or increase the expression of iNOS, though it increased TNF-αlevel. Presumabley, neurotoxicity of bilirubin might become more severe when accompanied with prematurity and abnormal oxygen concentration . To control high risk factors, for example, inhibiting the neurocyte apoptosis and the release of inflammatory factors, plays a key role in prophylaxis of bilirubin encephalopathy.