The Effects Of Nogo-A,Nogo Receptor And Their Pathway Inhibitors On CNS Regeneration Of The Newborn Rats With Hypoxic Ischemic Brain Damage

Objective:The hypoxic-ischemic encephalopathy (HIE) caused by asphyxia in peripartum is a serious disease in newborn infants, with a high disability and mortality rate.Lack of regenerative ability in central nervous system after injury is considered as the fundamental cause.The traditional symptomatic intervention can not effectively improve the prognosis.However, in recent years many studies have revealed that there are at least three myelin-associated neurite outgrowth inhibitory factors that exert inhibiting effect of the elongation of central nerve fibers, neural regeneration and plasticity through the Nogo receptor(NgR), including Nogo-A,myelin-associated glycoprotein(MAG) and oligodendrocyte-myelin glycoprotein(OMgp). NEP1-40 is the competitive antagonist of NgR for sequences of Nogo-66 amino, thus prevents the combination of central nervous system inhibitor with NgR, contributes to nerve regeneration. In this study,by neonatal rats with HIBD injected with NEP1-40 and fasudil, we adopted in situ hybridization testing to detect the expression of Nogo-A in the cerebral cortex and hippocampus of each group at different stage, and immunochemical staining for NgR, and observed the possible changes of neurons and axons through transmission electron microscopy(TEM), thus to explore the significance of Nogo-A and NgR,and the possible neuroprotective effect of NEP1-40 and fasudil in newborn rats with HIBD. We hope the results may provide a revolutionary way for the treatment of neonatal hypoxic ischemic encephalopathy.Methods:128 healthy wistar rats with the age of 7 days were purchased from Shandong University Laboratory Animal Center, weighing 12-18g, male and female open. They were randomly divided into 4 groups on average:the control group, HIBD group, NEP1-40 group and fasudil group, respectively. In addition, rats of each group were randomly devided into 4 groups on average by observation time of 6h, 24h,72h and 7days. All the rats of HIBD model, NEP1-40 and fasudil group were performed strictly according to Rice procedure. Immediately after the model production, rats of the control group and HIBD model group were injected with saline (0.25 mL/kg) by intraperitoneal injection, while NEP1-40 group and fasudil group were administrated with NEP1-40 12.5μg/d, fasudil 10 mg/(kg.d) for continuous 3 or 7 days on request in each group, respectively, then executed at 6h,24h,72h or 7 days after the injection. Brain tissues were fixed in sugar solution containing 4% paraformaldehyde and consecutive frozen sections with thickness of 20μm were produced when the brain tissue sunk. One section of every 20 was choosed for the test of in situ hybridization and immunohistochemical staining to detect Nogo-A or NgR positive cells, and some for HE staining to observe pathological changes.In addittion, part of the brain tissues were reserved for observation of histological changes through transmission electron microscopy. The SPSS statistical software package for Windows, version 10.0, was used to run Chi-Square tests and least significance difference (LSD-t) on the data presented, and P value of less than 0.05 regarded as statistically significant.Results:For rats of the control group,the expression of Nogo-A and NgR got slightly increased with the rise of age,but no significant differences were found among the 4 time points, respectively,(All P values were more than 0.05.). Elevated levels of Nogo-A in HIBD group were detected even at early stage of 6h and with the tendency of up-regulation (All P values were less than 0.01.). However, Down-regulated expression of NgR was observed at time point of 6h,24h and 72h after hypoxia ischemia (All P values were less than 0.05).With the administration of NEP1-40, Nogo-A positive cells decreased greatly at every time point, which had statistical difference from that of HIBD group, respectively (All P values were less than 0.01.).In addition, the duration of down-regulated level of NgR was prolonged to time point of 7days (P value was less than 0.05).Nogo-A positive cells of fasudil group upregulated at 6h, reached the peak at 72h, then decreased slightly, as were significantly different from that of the control and NEP1-40 groups, respectively (All P values were less than 0.01.).Under transmission electron microscopy, regular shape of neurons, amount of nucleus, well-developed Golgi complexes and mitochondria were all observed in the control group, as well as normal ultrastructure of axon.Of HIBD group, the shape of neurons was irrugelar.Few cytoplasm, nucler condensation, chromatin margination and dissolution. The mitochondrin was swelling and mitochondrial cristae was fragmented or disappeared, some mitochondrina showed vacuolar degeneration and plenty of apoptosis bodies. Damage and dissolution of axons was serious.Irregular shape of neurons of Fasudil group at 6h and 24 h was also found.The cell bodies were swelling and nuclear shape in normal rule with visible nucleolus.Additionally, nuclear contents were observed dissolved, together with many apoptosis bodies. Whereas no axonal regeneration was achieved. Then for those of 72 hours and 7 days, the swelling of neurons attenuated gradually and reactive hyperplasia of glial cells was apparent, together with neurite outgrowth.The results turned for much better of the NEP1-40 group. At 6 hour stage, similar changes were also found of such ultrastructure as cell bodies, nucleolus, nuclear contents and apoptosis bodies. The cell bodies were swelling and nuclear shape in normal rule with visible nucleolus at 24h and nuclear contents were observed dissolved, together with many apoptosis bodies.While the damage of axonal form mitigated with no obvious regeneration since then.When it came to 72 hours and 7 days, the predominant sign was for the axons.axonal regeneration was revealed with axon elongation and fasciculation, expansion in amount budding and synapse formation. Apoptosis bodies were seldom observed.Conclusions The expression of Nogo-A increased obviously in brain tissues of newborn rats with HIBD,even at early stage of 6h and with the tendency of upregulation.While down-regulated level of NgR could to some extent neutralize exerted inhibiting effect of Nogo-A on nerve regeneration of CNS after hypoxia ischemia. NEP1-40 and Fasudil could antagonize the inhibitory effect of Nogo-A.It reduced cerebral edema and enhanced regional cerebral blood flow, thus played the vital role in the nerve regeneration after HIBD.