The Effect And Potential Mechanism Of Repulsive Guidance Molecule A Involved In Epilepsy

Part one: The expression of repulsive guidance molecule a in patientswith temporal lobe epilepsy and experimental animalsObjective: A chronic pathological feature of temporal lobe epilepsy ismossy fiber sprouting, which results in synaptic reorganization and playsimportant roles in the pathogenesis of epilepsy. Repulsive guidancemolecule a (RGMa) is an axon guidance molecule with repulsive activityinvolved in inhibition of axonal outgrowth in the central nervous system.Here we investigated the expression of RGMa in brain tissues of patientswith temporal lobe epilepsy (TLE) and experimental animals in order toexplore the probable relationship between the RGMa expression and TLE.Method:1. The expression of RGMa was assessed in twenty three humantemporal lobes derived from patients undergoing operation for intractableTLE and was also detected in ten brain tissues from controls.2. Adult male Sprague-Dawly (SD) rats were used in this study,including control rats (n=9) and lithium-pilocarpine-treated rats (n=54) on 6hours,72hours, and days7,14,30,60.3. Expression of RGMa was assessed by double immunolabeling,immunohistochemistry, and immunoblotting.Result:1. RGMa protein was mainly expressed in membrane and cytoplasmof neurons in the temporal neocortex from TLE patients and non-epilepticcontrols by immunohistochemical staining. The mean optical density (OD)value of RGMa protein in the temporal neocortex of patients with TLE wassignificantly lower than that in the non-epileptic controls (p<0.05). Doubleimmunolabeling showed that RGMa expressed exclusively in neurons oftemporal neocortex samples from non-epileptic controls and patients withTLE, but not expressed in astrocytes. Analysis of immunoblotting showedthe RGMa expression was strong in control subjects, whereas it wasrelatively weak in the subjects with intractable TLE.2. Immunohistochemical positive signals of RGMa were extensivelydetected in the membrane and cytoplasm of neurons of rat hippocampusand adjacent cortex, and the intense staining was observed in the dentategyrus, CA1and CA3regions of hippocampus. Compared with control, themean OD value of each time point after pilocarpine induced seizure wasdecreased. Double immunolabeling showed that RGMa was expressed inneurons of the hippocampus and adjacent cortex from epileptic rats andcontrols, whereas RGMa positive cells were not colocalized with GFAP+ astrocytes. Immunoblotting analysis showed that RGMa expression inepileptic rats decreased in the latent period and in the chronic periodcompared with control. The difference of the mean OD ratio in the RGMaexpression level in hippocampus between the each time point group and thecontrol group was significant (p<0.05).Conclusion: The expression of RGMa was decreased in brain tissuesin TLE patients and lithium-pilocarpine-treated rats. Our results suggestthat RGMa may play an important role in the development of TLE.Part two: The effect of RGMa overexpression mediated by lentiviruson epileptic seizuresObjective: To further study whether overexpression of RGMa couldsuppress seizures, we investigated the behavioral changes of epileptic ratsperformed by lentivirus-mediated overexpression of RGMa using bothLiCl-PILO Model and PTZ kindling Model.Method:1. The following three groups were included, namely group not treated(control), group with injection of vehicle virus (LV-empty) and group withinjection of lentivirus-RGMa (LV-RGMa). Using immunoblotting andimmunofluoresence, we investigated the efficiency and stability of ourlentivirus-mediated transfer.2. Behavioral testing for LiCl-PILO induced seizures: we performedbehavioral investigation7days after virus infusion. We observed the behavioral changes of rats within2hours after pilocarpine injection. Racinescore every30minutes and seizure latency were recorded.3. Behavioral observation for spontaneous recurrent seizures in thechronic period after pilocarpine-induced SE. Three days after SE, theselesioned animals were randomly assigned to three groups: one group wasinjected with the vector expressing RGMa (LV-RGMa); the second groupwas injected with the control vector (LV-empty); and the third group wasnot treated at all (control). Seizures were assessed by video-monitoring ofthe animals. We recorded seizure frequency and Racine score24h/day for28consecutive days, beginning7days after SE.4. Behavioral testing for Pentylenetetrazol (PTZ) kindling seizures.Rats in LV-RGMa group and LV-empty group were injected PTZ every day3days after virus infusion. After PTZ injection, the behavior of animalswas observed for30min, seizure score and kindling latency were recored.Relut:1. Immunoblotting analysis showed that the mean OD ratio of RGMasignificantly increased in the LV-RGMa group than that in LV-empty atdays3,7,35respectively after infusion (p<0.05). The immunofluoresenceshowed that EGFP was localized in hippocampus, especially in dentategyrus area.2. With time after pilocarpine injection, the mean racine scoregradually increased, the seizures in LV-RGMa group were less severe than that in LV-empty group every30minutes (p<0.05). RGMa overexpressionsignificantly delayed seizure latency compared with LV-empty group(P<0.05).3. In the time window between day21and day35after SE, ratsinjected with LV-RGMa exhibited significantly less frequent and lesssevere SRSs compared with LV-empty group (p<0.05), but there was nodifference between the two control groups (p>0.05).4. During PTZ kindling, rats injected with LV-RGMa exhibitedsignificantly less severe seizures compared with LV-empty group (p<0.05);RGMa overexpression significantly delayed kindling latency comparedwith LV-empty group (p<0.05).Conclusion:1. The RGMa expression in hippocampus is successfully increasedafter lentivirus-RGMa infusion, and the efficiency of transfer for RGMalasts for a long time from day3to day35after infusion.2. Overexpression of RGMa attenuates pilocarpine-induced seizureseverity and susceptibility.3. Overexpression of RGMa mediated by lentivirus reducesspontaneous seizures up to5weeks after pilocarpine-induced statusepilepticus.4. lentivirus-mediated overexpression of RGMa delays the latency toPTZ kindling and reduces seizure severity. Part three: The potential mechanism of RGMa involved inepileptogenesisObective: To explore the probable underlying mechanisms of RGMainvolved in spontaneous recurrent seizure, we investigated mossy fibersprouting using silver staining and detected the expressions of RhoA,Cdc42, Rac1which is involved in axonal outgrowth at day35after SE.Method:1. Three days after pilocarpine-induced SE, rats were randomlyassigned to three groups: one group was injected with the vector expressingRGMa (EP+LV-RGMa); the second group was injected with the controlvector (EP+LV-empty); and the third group was not treated (EP). Ratsinjected with saline instead of pilocarpine and not treated with vector wereused as normal control (control).2. Timm staining was used to investigate mossy fiber sprouting (MFS),and the severity of MFS was assessed according to Timm score.3. Immunoblotting was used for analysis of RhoA, Cdc42and Rac1inthe epileptic rats at day35.Relult:1. Prominent Timm granules in the supragranular region that form aconfluent dense laminar band between tips and crest of hippocampus inLV-epmty-treated rats. Comparison of the mean timm score indicatedsignificantly higher timm score in EP+LV-empty group than that in control group and significantly lower score in EP+LV-RGMa group than that inEP+LV-empty group (p<0.05).3. The mean OD value of immunoblotting for RhoA in control groupwas significantly lower than that in EP group (p<0.05), and the mean ODvalue in LV-RGMa group was significantly higher than that in LV-emptygroup (p<0.05), but there is no difference between EP group and LV-emptygroup (p>0.05).4. Both Cdc42and Rac1protein levels were significantly higher in EPgroup than those in control group (p<0.05), and the expressions inLV-RGMa group were significantly lower than those in LV-empty group(p<0.05), but there is no difference between EP group and LV-empty group(p>0.05).Conclusion:1. RGMa overexpression mediated by lentivirus attenuates mossyfiber sprouting in epileptic rats.2. Lentivirus-mediated overexpression of RGMa changes theexpression of RhoA, Cdc42and Rac1in hippocampus, which may beassociated with mossy fiber sprouting.Part four: The effect of RGMa overexpression on neuronal excitabilityin hippocampusObjective: To further explore the probable underlying mechanisms ofRGMa involved in seizures, we investigated the electrophysiological changes in hippocampal slices induced by0Mg2+ACSF.Method:1. Rats were divided into three groups at random: one group wasinjected with the vector expressing RGMa (LV-RGMa); the second groupwas injected with the control vector (LV-empty); and the third group wasnot treated (control). Animals were killed for brain slices preparation at day7after vector injection.2. Whole-cell recordings of pyramidal neueons in CA1region of brainslices were performed, the action potential (AP), miniature excitatorypostsynaptic currents (mEPSCs) and evoked excitatory postsynapticcurrents (eEPSCs) were recorded.Result:1. The frequency of0Mg2+ACSF induced AP in LV-RGMa groupwas significantly lower than that in LV-empty group or in controlgroup(p<0.05). Compared with LV-empty group or control group, themean amplitude and frequency of mEPSC in LV-RGMa group weresignificantly reduced (p<0.05).2. A significant reduction in NMDAR/AMPAR ratio was observed inLV-RGMa group compared with control group or LV-empty group(p<0.05). Compared with LV-empty group or control group, LV-RGMagroup showed a significant decrease in the mean amplitude ofNMDAR-mediated EPSCs (p<0.05), but not in AMPAR-mediated EPSCs (p>0.05).Conclusion:1. Overexpression of RGMa in hippocampus of rats inhibitshyperexcitability in hippocampal slices induced by0Mg2+ACSF.2. Overexpression of RGMa in hippocampus of rats reducesNMDAR-mediated synaptic currents in hippocampal slices.