The Impact On Newborn Rat Neurite Outgrowth Of NgR Specific Sirna After Hypoxic-ischemic Brain Damage

Objective:1.To study the expression of NgR mRNA and protein inhibited by the chemical synthetic siRNA cognate to NgR the cultured PC12 cells,a commonly used in vitro system for studying neurite growth.2.To study the effect of NgR siRNA on promoting axonal growth and functional recovery after neonatal rat hypoxic-ischemic brain damage (HIBD) .And provide a theoretical basis for the further application of long-acting express with carrier siRNA treatment.Methods :1. PC12 cell,a rat's phaeochromocytoma cell line,was cultured and induced by nerve growth factor(NGF).A specific siRNA which was designed against NgR gene was transfected into pc12 cells with lipofectamine. Then use real-time RT-PCR and Western blot methods to detect NgR in pc12 cells.2.60 SD newborn rat were randomly divided into HIBD set,false surgical operation set,NS-injection set and siNgR-injection set.We ligated the rat's left common carotid artery and hypoxemia to set up the rat's HIBD model.Drugs were intracerebroventricular microinjected into the rat's brain for three consecutive days after the injury .Then the expression of NgR mRNA was measured by RT-PCR and the the levels of protein in inplasm were measured by immunohistochemistry 72h later.Two weeks later, used water maze to observe the behavior variety;used immunohistochemistry to measure GAP-43 to observe the condition of nerve regeneration.Results:1.The specific NgR siRNA could inhibit the RNA replication.The results of real time PCR and Western blot showed that the mRNA and protein level of NgR decreased.There was a significant difference compared with control set.2.We ligated the rat's left common carotid artery and hypoxemia to set up the animal's HIBD model,after the injury we dealed the rat with siRNA(30nnol) ventricle injection for 3 consecutive days.And 72h later,the mRNA expression of NgR was silenced by the siNgR compared with rats injected by NS,and NgR protein was suppressed also which was demonstrated by immunohistochemistry analysis rusults.3.Two weeks later,we measured the rat's mean escape time by water maze expriment.The results showed that the difference between HIBD set rats and false surgical operation set rats had statistics significance;And the difference between siNgR-injection set rats and false surgical operation set rats had no statistics significance;But there was obvious difference compared wih NS-injection set rats.4.The levels of GAP-43 in plasm were measured by immunohistochemistry and the results showed that the difference between HIBD set rats and false surgical operation set rats had statistics significance;The difference between siNgR-injection set rats and and false surgical operation set rats had no statistics significance;But there was obvious difference when compared wih NS-injection set rats.Conclusions:We demonstrate that the specific siRNA can effectively inhibit NgR replication in cultured pc12 cells.In newborn rat HIBD model,the level of NgR mRNA and protein was silenced by the specific siNgR.siNgR could promote neurologic functional recovery and axon regeneration of rats after SCI,which indicates that siNgR may have a potential therapeutic properties in the management of HIBD of neonate rats.One possible of mechanism siNgR therapeutic effect after HIBD is that siNgR blocks the combintion of NgR with it's three ligands(Nogo,MAG and OMgp)which results in promoting the regeneration of injured axon at the lesion site after HIBD.