| Objective Axonal regeneration failure in the adult mammalian CNS is attributed in part to the inhibitory nature of CNS myelin. RGMa is one of the three homologues of repulsive guidance molecule (RGM),which is not only an axon guidance molecule but also a novel axonal growth inhibitor preventing any regenerative growth of injured nerve fibres in the central nervous system. Ras homologous A (RhoA) is a key intermediary factor in the signal transduction of the axon growth inhibition after central nervous system injury and plays a key role in the axon regeneration process. The study focused on the expression changes of RGMa and RhoA in the infarcted cortex and hippocampus after cerebral ischemia-reperfusion in rats from each group. In addition, we detected the dynamic changes of RGMa expressions in the cortex and hippocampus after chronic cerebral ischemic (CCI) in rats. Therefore,we explored RGMa expression in axon growth and regeneration after acute and chronic ischemic brain injury,also the effect and significance of electrostimulation of fastigial nucleus on RGMa and RhoA expression and axonal regeneration. Methods The first part: The focal middle cerebral artery occlusion(MCAO) /reperfusion model was induced by ligation with nylon monofilament in rats in the study. One hundred and twenty eight male Sprague-Dawley rats were selected and randomly divided into 8 groups including the sham group ,12h,24h,48h,1w,and 2w group after cerebral ischemia-reperfusion,and the ischemia-reperfusion group with a FNS treatment (FNS intervention group, divided into 24 h and 2 w group). The rats after ischemia reperfusion 2h were electrostimulated immediately in cerebellar fastigial nucleus lasted 1 h.Then the histological features of nerve cells were identified by HE staining. RT-PCR was used to determine the changes of RGMa and RhoA mRNA expression in ischemia cortex and hippocampus. And immunohistochemistry was used to detect the expression of RGMa protein and the state of axonal regeneration in the corresponding time at the same site.The second part:Forty SD rats were randomly divided into 5 groups including the sham group,2w,4w,6w,and 8w after persistent bilateral carotid artery ligation. Morris water maze was used to study the learning and memory ability of action, RT-PCR and Western blotting were respectivelyused to determine the changes of RGMa mRNA and RGMa protein expressions.Results The first part:A slight increasing of RGMa and RhoA mRNA expression in the infarcted cortex and hippocampus were observed at 12h and gradually up to the peak at 48h ( p<0.01 ) after cerebral ischemia-reperfusion and remained for 2w(p<0.01).The expressions of RGMa and RhoA mRNA in the ischemia cortex were more than in the hippocampus respectively. In control brains, RGM immunoreactivity was detected on cortex, the perikarya of cerebellum,choroid plexus, brainstem neurons and perivascular tissues . The levels of RGMa and RhoA in the infarcted cortex and hippocampus were significantly decreased(p<0.01) and neurologic deficit was obviously improved (p<0.05)in FNS treatment group in contrast to single ischemia-reperfusion group. The axons were damaged most seriously at 24h after ischemic reperfusion(p<0.05), axons expression then gradually increase(p<0.05), the mean optical density of axons was higher in FNS treatment group than those in ischemia group (P<0.05).The second part:Morris water maze showed learning and memory impairment at 4w after persistent bilateral carotid artery ligation in rats(p<0.01), learning and memory capacity remains reduced at 8w(p<0.05). Compared with sham group, RGMa mRNA and protein in 2w began to increase(p<0.01) in the cortex and hippocampus and showed an upward trend(p<0.01) .Conclusions The expression of RGMa and the key factor RhoA of its downstream of Rho-RhoA kinase signaling pathway in the infarcted cortex and hippocampus were increased. The motor dysfunction in MCAO/reperfusion rats were improved, and the levels of RGMa and RhoA were downregulated after FNS treatment,then FNS treatment reduced the damage to the axons of ischemic injury and promoted CNS axonal regeneration. In addition, the higher expression of RGMa after chronic cerebral ischemia may play an important role in the nerve regeneration in rats.
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