The Effect Of Nogo-A Recepter Antagonists In Neurite Outgrowth In Newborn Rats With Hypoxic Ischemic Brain Damage

Objective: The hypoxic-ischemic encephalopathy (HIE) caused by hypoxia in peripartum is a serious disease in newborn infants, and is one of the main cause of cerebral palsy, mental retardation and epilepsy. The lack of regenerative ability in central nervous system after injury is considered as the fundamental cause. In recent years lots of studies have revealed that there were at least three myelin-associated neurite outgrowth inhibitory factors that limit elongation of central nerve fibers, neural regeneration an plasticity, including Nogo-A,myelin-associated glycoprotein(MAG) and oligodendrocyte-myelin glycoprotein(OMgp). These three factors exert inhibiting nerve regeneration through the same inhibitor Nogo receptor (NgR). 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 NEP1-40 and GM-1 as the comparative agent,we adopted immunochemical staining to detect the expression of Nogo-A protein in the cerebral cortex and of each group at different stage, and observed the possible changes of neurons and axons through transmission electron microscopy,thus to explore the significance of Nogo-A and the possible neuroprotective effect of NEP1-40 and GM-1 on HIBD.Methods: 128 healthy wistar rats at the age of 7 days were purchased from Shandong University Laboratory Animal Center, male and female open. They were randomly divided into 4 groups on average: the control group, HIBD group, NEP1-40 group and GM-1 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 GM-1 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 GM-1 group were administrated with NEP1-40 12.5μg/d, GM-1 10 mg/(kg.d) for continuous 3 or 7 days on request in each group,respectively, were executed at 6 h, 24 h, 72 h or 7 days after the injection. Brain tissues were fixed in sugar solution containing 4% paraformaldehyde , when the brain tissue sunk ,paraffin wax fixed them.Then the brain tissue were produced with thickness of 20μm. One section of every 20 was choosed for the test of immunochemical staining to detect Nogo-A 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.Results:①For rats of the control group, the expression of Nogo-A 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.).②With the administration of NEPl-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.).③Nogo-A positive cells of GM-1 group upregulated at 6h, reached the peak at 24h, then decreased, as were significantly different from that of HIBD 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. 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 were serious.Irregular shape of neurons of GM-1 group at 6h and 24h 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, while not much neurite outgrowth was observed.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.Conclusion 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. Nogo-A exerted inhibitory effect on nerve regeneration of CNS after hypoxia ischemia. While NEP1-40 could neutralize 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. GM-1 inhibited in part the expression of Nogo-A after HIBD.