The Effects Of Rgma-specific RNAI On RGMA Expression, Axonal Growth And Neural Function After Middle Cerebral Artery Occlusion/reperfusion In Rats

Objective Ischemic stroke is a great threat to human health and life safety and the related problem referring neurological rehabilitation has received extensive attentions. Regeneration of neurons is extremely difficult in adult central nervous system (CNS) after injury, due to the effections of numerous neurite growth inhibitors. But in some cases, axons damaged by mechanical factors or deprivation of blood and oxygen, could regrow and promote functional recovery as a result of special intervention. Repulsive guidance molecule a (RGMa) is a kind of axonal guidance molecule, one of the most potential reported neurite growth inhibitors to this day. Intrathecal administration of a neutralizing antibody to block function of RGMa could significantly enhance axonal growth and promote neurologic function recovery after spinal cord injury in rats. We planned to adopt a type of specific intervention approach, RNA interference (RNAi) mediated by recombinant adenovirus rAd5-shRNA-RGMa, to cure ischemic brain tissues, including cortex and hippocampus, of rats after middle cerebral artery occlusion (MCAO)/reperfusion. Then the important role of RGMa in all neurite inhibitors could be demonstrated by axonal growth and functional recovery due to the changes of RGMa expression brought by RNAi. It would provide an important clue for designing therapeutic strategies for clinical cerebral ischemia.Methods 1. Recombinant adenoviruses rAd5-shRNA-RGMa and rAd5-HK were amplified through HEK-293 cells culture and purified by Sartorious Vivapure Adeno PACK 20. 50% tissue culture infective dose (TCID50) was used to estimate their titers. Three different titers of rAd5-shRNA-RGMa and PBS (negative control) were injected via microinjection needle into the cortex and hippocampus of ischemic side in rats (n=32) after MCAO/reperfusion by stereotactic surgery. Toxicity was detected by histopathology and immunohistochemistry approaches. Proportions of green fluorescent protein (GFP) positive cells in the recombinant adenovirus injected tissues were calculated by staining total nucleus by DAPI.2. 66 heathy male adult Sprague Dawley (SD) rats were randomly divided into 11 groups, including normal group, sham 2d and 7d group, model (MCAO/reperfusion) 2 d and 7 d group, blank control (PBS) 2 d and 7 d group, negative control (rAd5-HK) 2 d and 7 d group, RNAi intervention (rAd5-shRNA-RGMa) 2 d and 7 d group. The mRNA and protein expressions of RGMa in the cortex and hippocampus of ischemic side were detected by reverse transcription polymerase chain reaction (RT-PCR) and immunehistochemistry respectively. And the axonal growth in corresponding regions was also estimated by immunehistochemistry.3. Normal group, MCAO/reperfusion group, negative group and RNAi intervention group , divided from 42 SD rats, were set up to identify ischemic regions and newborn neurite branches by TTC staining and BDA neuronal tracing. Behavior tests were adopted to evaluate neurologic function recovery of the rats after focal cerebral ischemic injury and correlative interventions.Results 1. Titers of purified rAd5-shRNA-RGMa and rAd5-HK were respectively 5.01×1010 pfu/ml and 3.16×1010 pfu/ml. All the three titers of rAd5-shRNA-RGMa could transfect cortex and hippocampus of ischemic side in rats'brain. Transfection efficacies of medium and high titer groups were similar (p >0.05), obviously better than low titer group. The phenomenon of perivascular inflammatory cell infiltration was only observed around the injection site in brain tissues of high titer group. The expression levels of IL-1βin brain tissues of ischemic side in this goup were also higher than other groups on 7 d post-operation (p <0.01).2. Compared to normal and sham groups, both mRNA and protein expression levels of RGMa in cortex and hippocampus of ischemic side in other groups were significantly increased (p <0.01). The increase degrees of MCAO/reperfusion groups, blank control groups and negative groups were especially high, along with obviously reduction of axon numbers. Meanwhile the expression levels of RGMa were down-regulated and axonal growth was improved simultaneously in RNAi intervention groups (p <0.01).3. The cerebral infarction volumes of rats in model group,negative group and RNAi intervention group were similar (p >0.05). Behavior test scores of rats in RNAi intervention group after 6 weeks were significantly better than other groups (p <0.01). And numbers of axonal sprouting of corticorubral tract from healthy side to opposite side in this group were also obviously increased (p <0.01).Conclusions Purified RGMa specific RNAi recombinant adenovirus rAd5-shRNA-RGMa, which could transfect cortex and hippocampus of ischemic side in rats'brain with high stability, high efficacy and low toxicity,is a very effective tool in gene therapeutic research of cerebral ischemia. RGMa mRNA and protein expressions were persistently induced in cortex and hippocampus of ischemic side in rats after middle cerebral artery occlusion/reperfusion,which could be prominently reduced by RGMa-specific RNAi intervention and lead to improved axonal growth and neural anatomy plasticity, as well as neural function recovery.