Role Of NgR-specific SiRNA In Repairing The Brain Injury Of Premature Rats Caused By Intrauterine Infection

Objective: To investigate the effects of Nogo-66 receptor(NgR) specific silencing RNA(siRNA) expression on the brain injury of premature rats resulted from intrauterine infection as well as on the behavioral changes and the patholgocial findings, and preliminarily elucidate the potential mechanisms of NgR-RhoA singling pathway in reversing the brain injury.Methods: Forty SD rats(gestation in 18 days) were randomly selected, from which 35 were administered with LPS in dose of 0.25 mg/(kg·time), once a day for consecutive 2 days to induce the brain injury in premature rats. Then 212 preterm-born rats of either sexes were selected, and randomized into brain injury group(n=62), negative controls(n=50) and interference group(n=50). Another 50 were used as reference to determine the location for lateral ventricle injection. The remaining 5 pregnant rats were given abdominal subcutaneous injection with commensurable dose of RU486(0.15mg/kg·time), once a day for consecutive 2 days to induce the preterm. Then 62 newborns of either sexes were randomly included as premature group. Rats in the preterm group and brain injury group were free of any intervention, wherase those in the interference group and negative controls were respectively injected with NgR-specific siRNA and lentiviral vector on postnatal day 1(P1) in same dose of 250ul/kg via lateral ventricle. By the exmperimental protocol, 6 rats at their P1(excluding the interference group and negative controls), P3, P10 and P14 were randomly selected to undergo heart infusion for obtaining the brain tissues. Those tissues were used as frozen sections and homogenized. Histological changes in the brain were examined after HE staining, and activation of microglia was observed via immunofluorescent staining with CD11 b antibody. Differentiation of oligodendrocyte precursor cells(OPCs) was observed with immunofluorescence images showing O4+ antibody. NgR-mRNA expression was determined with QRT-PCR, and activity of the RhoA protein with Western blot. Special software was used to detect the animal behavior in remaining 20 preterm-born rats in each group.Results: 1)NgRmRNA:The relative expression level of NgR-mRNA in the brain injury group was higher in simple prematures by P1 and P3, the difference was significant(P<0.05), yet the difference was not significant by P10 between the two groups(P>0.05). NgR-mRNA was relatively expressed in lower level in the interference group by P3, as compared with the brain injury and negative groups(P<0.05), and the difference was not significant compared with the prematures, brain injury group and negative controls by P10 and P14(P>0.05).2)RhoA protein: Gray value of RhoA protein in the brain injuried rats was higher than that of prematures on P3(P<0.05), yet the difference was not significant on P10 between the two groups(P>0.05). Although the gray value of RhoA protein was significantly lower than that of brain injury group, negative controls and prematures by P3 and P10(P<0.05), the difference was not significant by P14(P>0.05).Relative expression level of NgR-mRNA was positively correlated with RhoA protein on P3, P10 and P14(r = 0.92, 0.86, 0.92, respectively).3)HE staining: No distinctly dissparsed white matter injury but OPCs in large quantity was observed in the preterm-born rats on P10 and P14, yet the condition was inverse in brain injuried rats.4.CD11 b immunofluorescent staining: The brain injury group had higher fluorescence intensity values of CD11 b on average on P1, P3 and P10 than the prematures(P<0.05), and the difference was not significant by P14(P>0.05). Mean intensity of fluorescence showing CD11 b antibody was lower in the interference group than that of the brain injury group and negative controls on P3 and P10(P<0.05). However, there was no statistical significance among the three groups(P>0.05).5)O4+immunofluorescent staining: The cells labeled with O4+ antibody demonstrated tripolar morphology in rats experienced 3 days of intrauterine infection and negative controls, and by P10 and P14, those cells were presented with diverse morphology except for the tripolar cells, and distributed in sattered manner. The cells showed meshy distribution in brain injury and interference groups besides tripolar morphology on P3, and additional patchy distribution by P10 and P14.6)Behavioral observation: Brain injury group scored higher on the experiment items of hanging, resisting arrest, open filed test and inclined plane test by P30 than the prematures(P<0.05). The difference was not significant among groups of interference, brain injury and negative controls(P>0.05).Conclusions: 1) In the brain injury of preterm-born rats due to intrauterine infection induced by LPS, NgR-RhoA signaling pathway may be one of the important roles in facilitating the injury.2) The potential role of NgR-RhoA signalling pathway in the brain injury of premature rats caused by intrauterine infection may be associated with consistent activation of this signaling pathway that leads to excessive activation of microglia, resulting in damaged OPCs and inhibited differentiation of OPCs.3)Lateral ventricle injection of NgR-specific siRNA can significantly improve the behaviror of rats with brain injury as a result of intrauterine infection. This findings suggest that interfering NgR gene expression and inhibiting NgR-RhoA activation can be protective effect in repairing the brain injury of premature rats caused by intrauterine infection.