The Effect Of Adenovirus-Delivered RNAi On NgR In Ischemia/Reperfusion Model Of Hippocampus Slice Cultures

Objective: Axonal regeneration failure in the adult mammalian CNS is attributed in part to the inhibitory nature of CNS myelin. To date, three inhibitors have been identified in myelin: Myelin-Associated Glycoprotein (MAG), Nogo-A, and Oligodendrocyte-Myelin glycoprotein (OMgp). Neuronal Nogo receptor (NgR) binds to each of the three inhibitors and has been proposed to mediate their inhibitory signals by complexing with a signal-transducing coreceptor, the neurotrophin receptor p75NTR, LINGO-1 or TROY. In this study, in order to provide a therapeutic means to promote recovery from ischemic brain injury, we will assess the contribution of NgR to mediating myelin inhibitory signals and regeneration failure by Adenovirus-mediated RNAi in ischemia-reperfusion model of hippocampal slice cultures.Methods: 1.Prepared organotypic hippocampal slice cultures in SD rats,then established ischemia-reperfusion model of hippocampal slice cultures;2.Constructed the recombinant adenovirus vector AD-shRNA-NgR targeted to the rat Nogo-66 receptor gene; Transfected adenovirus vector into hippocampal slice cultures and selected one sequence shRNA that is the highest efficient of gene silencing by reverse transcription-polymerase chain reaction (RT-PCR); 3. The hippocampal slice cultures after transfected by AD-shRNA-NgR for 48h were subjected to ischemia by oxygen-glucose deprivation for 30 min and then placed into the normal culture condition for 24 h,72h and 5d.The NgR mRNA level were detected by RT-PCR to determine silencing efficiency of RNAi and the changes of silencing efficiency.At the same time,the level of NgR mRNA in ischemia/reperfusion group was tested by RT-PCR to investigate the changes of mRNA expression. 4. Hippocampal slice cultures were randomly divided into 3 groups including the control group, ischemia-reperfusion group and RNAi treated group .Western blotting and immunohistochemistry were used to determine the expression of NgR protein at 48h after ischemia-reperfusion. Axonal regeneration was detected by gold chlorid staining.Results: 1. The rate of infection reached above 90% , and NgR mRNA expression in hippocampal slice cultures was decreased remarkably by 69% at 48h after transfection ; 2. The phasic expression in ischemia-reperfusion group: The level of NgR mRNA was greatly increased compared with control group(p﹤0.05); NgR mRNA reached maximal level at 24h after ischemia-reperfusion,and decreased subsequently at 72h. At 5d ,the level of NgR mRNA bounced back to a higher level,but it was lower than 24h (compared with 24h after ischemia-reperfusion, p﹤0.05); The expression of NgR mRNA is greatly decreased in RNAi treated group compared with ischemia-reperfusion group(p﹤0.01), The efficiency of gene silencing was induced at 5d after ischemia-reperfusion (p﹤0.05),but the general level was maintained a higher level;In ischemia-reperfusion group ,the expression of NgR protein was higher detected by Western blotting and immunohistochemistry(p﹤0.01), and decreased remarkably in RNAi treated group (compared with ischemia-reperfusion group,p﹤0.01),but not recover to the nomal level(p﹤0.05); The length of axons were decreased remarkably in ischemia-reperfusion group by gold chlorid staining (p﹤0.01), and obviously increased in RNAi treated group(p﹤0.01). A negative correlation in NgR protein expression and the length of axons were noted(r=-0.798,p﹤0.01).Conclusion:1. In our study, organotypic hippocampal slice cultures can remain a normal morphosis in incubation period; Ischemia-reperfusion modle of hippocampal slice cultures was successfully and stably constructed, and can effectively simulate ischemic brain injury in vitro.2. We have successfully constructed recombinant adenovirus Adeno-U6-shRNA targeted against the rat NgR gene and successfully selected effective interference fragment.3. The expression of NgR mRNA in hippocampal slice cultures after ischemia-reperfusion was increased remarkably, and reached maximal level at 24h after ischemia-reperfusion. The phasic expression displayed a curve change.4. The efficiency of AD-shRNA-NgR transfected into hippocampal slice cultures is relatively high. The level of NgR mRNA and protein was efficiently and stably silenced by RNAi.5. Knock-down of NgR by Adenovirus-mediated RNAi may offer an effective means to support axonal regeneration in CNS ischemic injury.