Effect Of Different Time Phase-regulated VEGF On Epileptogenesis After Status Convulsion In Immature Rats

PART Ⅰ DYNAMIC CHANGES OF VEGF AND RECEPTOR IN THE IMMATURE BRAIN AFTER STATUS CONVULSIONObjective: To explore dynamic changes of vascular endothelial growth factor(VEGF)and vascular endothelial growth factor receptor 2(VEGFR2)in hippocampus of immature rats in the process of epileptogenesis after status convulsion(SC).Methods: 144 20-day-old male Sprague-Dawley(SD)rats were randomly divided into the control group and the SC model group.The SC model was induced by intraperitoneal injection of lithium chloridepilocarpine.Animals were sacrificed at various time points 1d,3d,7d,14 d,28d and 56 d after SC to collect data on VEGF and VEGFR2 mRNA and protein levels using Q-PCR,Western-blot and ELISA.The distribution of VEGF and VEGFR2 protein expression in the hippocampus was tested by immunofluorescence.Results:(1)PCR results showed that the expression of VEGF and VEGFR2 mRNA in the hippocampus of immature brain SC was consistent.The expression of VEGF mRNA in hippocampus was higher than that in the normal control group at each time point after SC and it was up to the peak and continued to be highly expressed on the first day after SC.Compared with the control group,VEGF mRNA increased significantly at 1 day and 3 days(P<0.05);it began to decrease from 7 days after SC,and the expression was lowest at 14 days after SC,and re-emerged at 28 days after SC.The expression of VEGFR2 mRNA in hippocampus was significantly higher than that in the control group after SC,especially at 3 and 7 days after SC,the difference was statistically significant(p<0.01),peaked at 7 days after SC.The expression showed a gradual decrease trend from latent to chronic phase after SC and the expression was the lowest at 14 days after SC.However,the expression of VEGFR2 at 56 days after SC had an increasing trend compared with the control group.(2)The Western-blot and ELISA results showed that the expression of VEGF protein in hippocampus of immature brain increased gradually from 1 day after SC,reached the peak at 7 days,and the difference was statistically significant(p<0.05)compared with the control group at 1d,3d and 7d after SC.The expression of VEGFR2 protein was increased after immature brain SC,and reached the peak 3 days after SC.The difference was statistically significant(P<0.01)compared with the control group.Then gradually decreased and the lowest was at 14 days after SC.There is a trend of re-emergence from 28 days to 56 days after SC.The results of Q-PCR,Western-blot and ELISA showed that the expression of VEGF and VEGFR2 mRNA and protein in hippocampus of SC was basically consistent.(3)Immunofluorescence results showed that VEGF and VEGFR2 were mainly expressed in the hippocampal dentate gyrus(DG)in the control group,while in the SC group,it was found in the subgranular zone(SGZ)and even the Molecular layer of DG(MoDG),CA1 and CA3 pyramidal cell layers were expressed.Conclusion: The expression of VEGF and VEGFR2 in the immature brain after SC was fluctuating,showing an up-regulation in the acute phase,a gradual decrease in the latency and a re-emergence in the chronic phase.It is speculated that VEGF may be involved in the process of epileptogenesis,especially in the proliferative of the neural stem cells and the behavior of pyramidal neurons in the hippocampus after SC might play a certain role.PART Ⅱ EFFECTS OF VEGF ON HIPPOCAMPAL NEURAL STEM CELLS AFTER IMMATURE BRAIN STATUS CONVULSIONObjective:To explore the effects of VEGF on hippocampal neurogenesis in the acute phase in immature rats following SC.Methods: 20-day-old male SD rats received a single intracerebroventricular injection of human recombinant VEGF165(20 ng,40 ng or 60 ng per rat)and VEGFR2 blocker SU5416 to regulate VEGF expression,12 h before the induction of SC.The cell proliferation marker Brd U was injected intraperitoneally 2 h after SC,once every 8 h,6 times,and the animals were sacrificed 24 h after the last injection;The amount of Ki67 and Brd U/Dcx positive cells changes in DG area of hippocampus was detected by immunofluorescence and to determine the proliferative effect of VEGF on NSCs in acute phase after SC.Nerve staining was used to observe the damage of neurons in hippocampus after SC,and whether VEGF could inhibit hippocampal nerve loss caused by SC.Results:(1)The expression of Ki67 and Brdu/DCX positive cells in the SGZ region of hippocampus was observed under normal conditions.The number of Ki67 positive and Brdu/Dcx positive cells of SC group was significantly higher than that of normal control group(P < 0.05).There was no significant difference in the number of Brdu/Dcx positive cells between SC and SP groups,and the distribution area was also almost the same.The number of Ki67 and Brdu/Dcx positive cells in VEGF groups was significantly higher than that in PBS control group(P<0.01),and it was kind of dose dependent.The distribution area of Brdu/Dcx positive cells is wider than the SP group,not only in the SGZ,but also in the Po DG area and the Mo DG area.The number of Brdu/Dcx positive cells in the SU5416 group was significantly lower than that in the PBS control group(P<0.05).(2)Nissl staining shows that the hippocampal formation was destroyed after SC and neuron cells were reduced in the hippocampal CA1 and CA3 areas.Nissl bodies were significantly reduced,visible scattered nuclear dissolution.Promoting the expression of VEGF,which can effectively alleviate the structural damage of hippocampus after SC.In the VEGF intervention groups,the loss of neurons in the CA1 and CA3 areas was reduced,the arrangement was tight,the cell morphology was normal,and the nucleus and Nissl bodies were clearly visible,which was not significantly different from the normal control group.After inhibiting the expression of VEGF,the hippocampal formation was severely damaged after SC,similar to the hippocampal formation in the SC group.Conclusion: VEGF could promote the proliferation of hippocampal NSCs in the acute stage of immature brain SC,save the loss of neurons in hippocampal CA1 and CA3 areas caused by SC,improve the pathological damage of hippocampus after SC,and play a neuroprotective role.The VEGF/VEGFR2 pathway might be a promising effective intervention target.PART Ⅲ EFFECTS OF VEGF ON NEUROGENESIS AND VASCULAR REGENERATION IN DIFFERENT STAGES IN THE IMMATURE BARIN AFTER SCObjective: To observe the correlation between VEGF and neurogenesis and vascular regeneration in the process of epilepsy after immature brain SC and the effects of regulating VEGF expression in different periods after SC on hippocampal NSCs and microvessels and its possible molecular mechanism.Methods:The first part: 20-day-old male SD rats were randomly divided into control group and model group.lithium-pilocarpine was used to induce epilepsy model in immature SD rats.All subjects were sacrificed at 7d,14 d and 28 d after SC.Dcx and CD31 were analyzed to investigate the changes in neurogenesis and vascular regenesis after SC by immunofluorescence and immunohistochemical examination method.The spatial relationship between neovascularization and NSCs was detected by immuneconfocal of CD31/PSA-NACM.The second part:18-day-old male SD rats after birth were randomly divided into the VEGF intervention group at acute phase of SC(SV0),the SU5416 intervention group at acute phase of SC(SU0),the VEGF intervention group at latency of SC(SV5),and SU5416 intervention group at latency of SC(SU5).The lateral ventricle was buried on the 18 th day after birth and induced SC After 21 days of birth.The model rats were administered,VEGF 40 ng/d,SU5416 5 m M/ d,continuously at a rate of 0.5 ul/min from the lateral ventricle catheter at 2 h or 5 d after continuous SC.The expression of Dcx in hippocampus was detected by immunofluorescence 7 days after SC and the expression of CD31 was examined by immunohistochemistry 28 days after SC.Western-blot was used to detect downstream protein VEGFR2,AKT,P-akt,ERK and P-erk after continuous administration.Results:(1)In the normal control group,almost all Dcx positive cells in the hippocampus of rats were distributed in the SGZ area of the hippocampus.During the development of immature brain,the fluorescence intensity of Dcx showed a trend of decreasing with age.The fluorescence intensity of Dcx in SC group increased at different time points compared with the control group,and the difference of Dcx fluorescence intensity at 7 days after SC was statistically significant(P<0.01).Dcx positive cells were distributed in the dentate gyrus(GCL)7 days after SC and showed a tendency to migrate through GCL to Po DG.(2)Promoting VEGF expression in the acute phase after SC increased the expression of Dcx(P<0.01),and the distribution sites were still mainly SGZ and GCL regions.Promoting VEGF expression in the latent phase after SC did not significantly increase the expression of Dcx in hippocampus.However,the distribution of Dcx positive cells was not limited to SGZ or GCL,and more Dcx positive cells were scattered in Po DG or Mo DG.Inhibiting VEGF expression in the acute phase or latent period after SC significantly reduced the expression of Dcx in the hippocampus(P<0.01),and the distribution site was mainly SGZ region.(3)The CD31 expression and age was no obvious correlation in hippocampus of normal control group.The expression of CD31 in hippocampal DG area of SC group increased from 7 days to 28 days after SC,compared with normal control group.The microvessel density was significantly increased(P<0.01).(4)Promoting VEGF or inhibiting VEGF expression at acute phase after SC,the expression of CD31 and microvessel density in hippocampus were not significantly different from those in SC group.However,Promoting VEGF expression at latent period after SC significantly promoted the expression of CD31 and increased the microvessel density in the hippocampus of chronic phase after SC(P<0.01).Inhibiting VEGF expression at latent period after SC significantly reduced the expression of CD31 in hippocampus of chronic phase of SC(P<0.01).(5)The CD31 accompanied with PSA-NACM in the hippocampal DG area following the SC.(6)The expression of protein kinase B(PKB/AKT)and extracellular signal-regulated kinase(ERK)downstream of VEGFR2 was promoted after SC.Promoting VEGF expression in the acute phase and latent period after SC increased the expression of VEGFR2 protein.P-akt protein and P-erk protein in the hippocampus.Inhibiting VEGF expression in the acute phase of SC decreased expression of VEGFR2(P<0.01)and P-erk protein(P<0.01);Inhibiting VEGF expression at latent period after SC decreased P-erk protein(P<0.01)and P-akt protein expression(P<0.05).Conclusion:Hippocampal neurogenesis may be closely related to microvascular remodeling after SC and NSC might chain-migrated along the neovascular.Intervention of VEGF signaling pathway at different stages after SC may have different effects on neurogenensis and vascular remodeling in hippocampus of the immature rat brain.The molecular mechanism of neurogenensis and vascular remodeling after SC may be coordinated with through the EKR and the AKT signaling pathway.PART Ⅳ EFFECTS OF PHASE-REGULATED OF VEGF ON SEIZURES AND SUSCEPTIBILITY TO CONVULSIONS IN IMMATURE BRAIN AFTER SCObjective: To observe the effect of intervention of VEGF on seizures and susceptibility to convulsions in different periods after immature brain SC,and to explore the timing of treatment in the process of epilepsy.Methods: 18-day-old male SD rats were randomly divided into normal control group(control),model group(SC),the VEGF intervention group at acute phase of SC(SV0),the SU5416 intervention group at acute phase of SC(SU0),the VEGF intervention group at latency of SC(SV5),and SU5416 intervention group at latency of SC(SU5).Methods used to induce SC Model and treatment were the same as described in part III.The changes of hippocampal volume in chronic phase were detected by stereology.The ultrastructural changes of synapses in hippocampus were detected by electron microscopy and the morphology of hippocampus was observed by Nissl staining.EEG recordings began 10 days after SC and were recorded for 2 hours per day for 14 consecutive days to observe the characteristics of spontaneous seizures in rats.The susceptibility to convulsions was detected combined with in vivo convulsion reburning experiments and in vitro brain slices without magnesium induced spontaneous discharge model in the chronic phase of SC.Results: SC was accompanied by pathological damage such as smaller hippocampal volume,synaptic ultrastructural damage and hippocampal morphological changes.Promoting VEGF expression in the acute phase after SC reduced hippocampal neurons loss and alleviated the lack of niche bodies at SC chronic.Inhibiting VEGF expression at latent period after SC effectively reduced the hippocampal volume reduction after SC(P<0.01),reduce the length of presynaptic membrane activity band after SC(P <0.05),and reduce the width of synaptic gap after SC(P <0.05),to alleviate the pathological damage of the hippocampal formation after SC.(2)Inhibiting VEGF expression at latent period after SC effectively reduced the severity of SRS epileptic power generation.Compared with SC group,the instantaneous frequency of SU5 group decreased(p<0.05),the duration of seizure shortened(p<0.01),the number of episodes Decrease(p<0.05).(3)Inhibiting VEGF expression at latent period after SC prolonged the convulsion latency(P<0.01),brain slice discharge latency(P<0.05),and reduced the susceptibility to convulsion.Conclusion: In conclusion,our study suggests that the latent period after SC is a key period affecting the process of epilepsy.Promoting VEGF expression in the acute phase and inhibition VEGF in the latent period might improve the pathological damage of hippocampus and prevent the epileptogenesis after SC.