The Study On The Role Of Nogo-A And Its Receptor NgR In Rat Visual Cortical Plasticity

Chapter 1 Dynamic expression of Nogo-A and NgR in normal development and monocular deprivation rats’ visual cortexObjective:To explore the expression tendency and significance of Nogo-A/NgR mRNA and protein in normal development and monocular deprivation rats’ visual cortex.Method:eighty-four neonatal rats were randomly allocated into 6 groups:postnatal 0 day normal group (NorPO), postnatal 14 day normal group (NorP14), postnatal 28 day normal group (NorP28), postnatal 60 day normal group (NorP60), postnatal 28 day monocular deprivation group (MDP28) and postnatal 60 day monocular deprivation group (MDP60). For the MD model, we sutured together the right eyelids of rats at P21. A subset of rats at each time points were killed, then the primary visual cortex in left brain were removed and frozen for detecting the expression pattern of Nogo-A/NgR by means of RT-PCR and western blot. In addition, we used the method of immunofluorescence histochemistry to observe a layer-specific expression of Nogo-A and NgR in rats’ visual cortex influenced by monocular deprivation.Results:Nogo-A/NgR mRNA and protein were detected in visual cortex of normal neonatal rats (NorPO). In general, Nogo-A mRNA and protein were significantly increased its expression at P28 and maintained a steady level between P28 to P60. Similarly, NgR mRNA was dramatically up-regulated at P14 and maintained a high level to P60; while NgR protein was obviously elevated at P28 and then remained highly expression up to P60. In response to MD, the mRNA and protein expression of Nogo-A/NgR decreased only minimally in primary visual cortex when compared to the age-matched normal controls (P>0.05). In addition, there was no significant difference in Nogo-A/NgR mRNA and protein expression between MDP28 group and MDP60 group (P>0.05). A few Nogo-A and NgR positive immunoreactive cells were detected in all layers of visual cortex in both NorP60 group and MDP60 group rats. Notably, the expression of Nogo-A and NgR positive cells decreased dramatically in layer II-III and layer IV of visual cortex contralateral to deprived eye after monocular deprivation.Conclusion:The mRNA and protein levels of Nogo-A and NgR increase steadily after birth and reach a plateau level into adulthood. After monocular deprivation, the protein expression of Nogo-A and NgR tends to decrease obviously in the layer II-III and layer IV of visual cortex. We speculate that Nogo-A/NgR mainly participated in the growth and guidance of axon during the early development stage; Meanwhile, it also regulated the termination of critical period and inhibited the plasticity of adult visual cortex. Chapter 2 Reactivation of visual cortical plasticity by NEP1-40 from monocular deprivation in adult ratsObjective:To observe the effect of visual cortical plasticity by NEP1-40 from monocular deprivation in adult rats and further elucidate the role of Nogo-A and NgR in regulating the rat visual cortical plasticity.Method:120 neonatal rats were randomly allocated into 6 groups: normal animals (Nor), MD animals without any treatment (MD), normal animals treated with PBS (Nor+PBS) or NEP1-40 (Nor+NEP), MD animals treated with PBS (MD+PBS) or NEP1-40 (MD+NEP). We subjected P21 rats to monocular deprivation model until P45. Then the deprived eyes of MD model rats were reopened and followed by NEP1-40 or PBS administration for 7 days. Dendritic spine densities, ultrastructral modifications of synaptic junctions and objective visual function were examined at P52 to determine the therapeutic effects of NEP1-40. In addition, Nissl-staining was used to confirm the location of lateral ventricles microinjection and the side-effects of NEP1-40 or PBS.Results:(1) The structure of lateral ventricle and visual cortex examined by Nissl-staining:The lateral ventricle of rats in MD group remained intact; while the rats received lateral ventricle microinjection in MD+PBS group or MD+NEP group displayed a damaged cerebral cortex and a needle trace into the lateral ventricle. However, there was no significant difference in the quantity and structure of neurons in visual cortex contralateral to deprived eyes among MD group, MD+PBS group and MD+NEP group.(2) The dendritic spine density of neuron in visual cortex examined by Golgi-staining:monocular deprivation decreased dendritic spine density in the visual cortex contralateral to the deprived eye (Nor vs. MD, P<0.05). After 7 days treatment, the average dendritic spine density of visual cortical neuron in MD+NEP group increased significantly and was on the verge of that of in Nor group, which were much more than those of in MD+PBS group (P<0.05) or MD group (P<0.05). However, there was no statistical difference in dendritic spine density among Nor group, Nor+PBS group and Nor+NEP group (P>0.05).(3) The structural modifications of synaptic junctions examined by electromicrographs:All of the structural parameters of the synaptic junction in visual cortex were altered by monocular deprivation in MD group compared with the Nor group, displaying an increased width of synaptic clefts, shortened synaptic active zone, decreased curvature of synaptic interface and decreased thickness of PSD. However, synaptic ultrastructural analysis showed that NEP1-40 treatment could recover all of the structural index in monocular deprivation rats (MD vs. MD+PBS vs. MD+NEP, P<0.05) but not normal rats (Nor vs. Nor+PBS vs. Nor+NEP, P>0.05). It was noteworthy that, although the width of synaptic clefts in MD+NEP group decreased remarkably in comparison with that of in MD group, it still had not reach the normal level (Nor vs. MD+NEP, P>0.05).(4) The objective visual function examined by F-VEP:At P45, F-VEP showed that compared with rats in Nor group, deprived eyes of rats in MD group, MD+PBS group and MD+NEP group displayed a longer latency(P<0.05) and a smaller amplitude(P<0.05), while the rats examined in Nor+PBS group and Nor+NEP group indicated no statistical difference in F-VEP in comparison with that of in Nor group (P>0.05); At P52, we could see that monocular deprivation prolonged the latency and reduced the amplitude of F-VEP in deprived eyes (Nor vs. MD, P<0.05). While comparing with the results in MD group and MD+PBS group, the latencies of F-VEP in MD+NEP group were shortened and the amplitudes were increased (P<0.05), which were similar with that of in Nor group (Nor vs. MD+NEP, P>0.05). However, there was no statistical difference in latency and amplitude of F-VEP among Nor group, Nor+PBS group and Nor+NEP group (P>0.05), indicating that NEP1-40 had no effect on normal animals without monocular deprivation.Conclusion:NgR antagonist peptide NEP1-40 could recover the dendritic spine density and structural modifications of synaptic junctions of neurons in visual cortex contralateral to deprived eye, as well as the objective visual function of deprived eye, which indicated a new role for NEP1-40 in reactivation of visual cortical plasticity from monocular deprivation in adult rats. In summary, our research gave a straight evidence of regulation role in visual cortical plasticity by Nogo-A/NgR system, and offered the theoretical foundation for curing adult patients with amblyopia in the clinic.