Phosphorylation Of CRMP-2 On Neural Regeneration After Ischemia/Reperfusion Brain Injury In Rats

Objective: RGMa, as one of inhibitiors of axonal regeneration, is highly expressed in the injured central nervous system, with the function of axon rejection and induction of growth cone collapse. After growth cone collapse, the structure of axon is severely damaged, inevitably leading to an interruption of links among neurites, blockage of information transmission between cells, ultimately affecting the operation of neural function. In cerebral ischemia/reperfusion injury model, its inhibition of axonal regeneration and nerve function recovery is observed. It is also found in the adult brain slides after focal ischemia, that RGMa immunopositive cells are mainly gathered in the infarcted white matter, hemorrhage area, infarct center and peripheral area. Therefore, by specific RNA interference, to inhibit RGMa gene transcription, thereby affecting protein expression, may contribute to axon regeneration and neurological function recovery. In addition, intracellular signaling pathway of RGMa is not clear, possibly related to Rho-kinase downstream CRMP-2. In order to find a more appropriate intervention target, so that neural regeneration after ischemic injury becomes easier and neurological function recovery more complete, it is very important to explore the mechanism of RGMa. The results of this study will provide a good theoretical basis for conversion from basic experiments to clinical application.Methods1. One hundred and thirty-two adult male SD rats were randomly divided into: normal group, sham group, MCAO/reperfusion group, PBS injection group, rAd-HK group, rAd-shRGMa group. 2 days and 7 days after surgery were set as observation time points. MCAO/reperfusion model was established by "suture method", RGMa-specific adenovirus rAd-shRGMa and empty vector were stereotactically injected to ischemic cortex, RGMa and CRMP-2 mRNA and protein expression in ischemic cortex of rats were detected by RT-PCR or immunohistochemistry/ fluorescence methods;2. Sixty-six adult male SD rats were grouped as above, MCAO/ reperfusion model established, adenovirus injected, the expression of NF200 in ischemic cortex of rats detected by immunohistochemistry;3. The neurological scores of rats were evaluated in each group;4. Forty-eight adult male SD rats were randomly divided into: normal group, MCAO/reperfusion group, rAd-HK group, rAd-shRGMa group. By Western blot, RGMa, CRMP-2 and pCRMP-2 protein expression was investigated. The relationships between them were analyzed by statistical method of the correlation analysis; 5. Primary cortical neurons of neonatal rats were cultured; identification of neurons and the purity calculation were done by immunofluorescence. Neurons were induced by recombinant RGMa in vitro, observed under light microscope for neurite morphology and length changes, detected by Western blot for its pCRMP-2 variation with time;6. Primary cortical neurons of neonatal rat were cultured; before RGMa induction in vitro, cells were preconditioned with Rho-kinase and GSK-3βspecific inhibitors, then pCRMP-2 expression was detected by Western blot.Results1. After MCAO/reperfusion, RGMa mRNA and protein expression in ischemic cortex of rats were significantly increased (p<0.01), while CRMP-2 expression were significantly decreased (p<0.01); The RGMa level was significantly reduced (p<0.01) by RNAi 2 days after MCAO/reperfusion, and CRMP-2 were significantly up-regulated (p<0.01), to 7 days close to the normal level.2. pCRMP-2 protein in ischemic cortex of rats was significantly increased (p<0.01) after MCAO/reperfusion, axons was damaged most severely, NF200 expression was significantly decreased (p<0.01), and neurological deficit in rats was obvious (p<0.01). In RNAi treatment group, pCRMP-2 protein level in rats was significantly reduced (p<0.01), part of the axons were preserved, no significant neurological improvement was observed (p>0.05). To 7 days, pCRMP-2 level was only slightly higher than normal (p<0.01), NF200 expression was significantly increased (p<0.01), no obvious neurological deficits were found (p<0.01).3. pCRMP-2 protein expression in ischemic cortex of rats was positively correlated with RGMa (r=0.994), and negatively correlated with NF200 (r=-0.895).4. Cultured primary cortical neurons in vitro survived in good condition, with plump cell bodies and normal developing neurites. The purity was more than 90% .5. After the incubation of recombinant RGMa, neurite retraction in each well was significant (p<0.01), most neurons were seen with cell bodies only, whose light transmission was poor. Incubated by Y-27632 or GSK-3βinhibitor plus RGMa, the phenomenon of neurites contraction was improved than those of incubated by RGMa only (p<0.01).6. With RGMa induction time, intracellular pCRMP-2 level was gradually increased, higher than normal level to 6 h (p<0.01), up to a peak till 24 h (p<0.01), then declined, the level was found no significant difference between 48 h and 6 h (p>0.05). Neurons pretreated by Rho kinase and GSK-3βspecific inhibitors, showed resistance to the induction of RGMa, whose pCRMP-2 level was not significantly different with normal (p>0.05).ConclusionsAfter cerebral ischemia/reperfusion, cortical RGMa and pCRMP-2 levels of rats were significantly increased; nevertheless adenovirus -mediated RGMa-specific RNAi could significantly reduce RGMa overexpression and inhibit phosphorylation of CRMP-2, thereby promoting axonal regeneration and neural function recovery. RGMa may regulate the phosphorylation of CRMP-2 through activated Rho-kinase and GSK-3βsignal pathway and mediate axonal retraction.