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DYNAMIC CHANGES OF HIPPOCAMPAL NEUROGENESIS AND NOTCH SIGNALING IN IMMATURE BRAIN AT DIFFERENT TIME AFTER STATUS CONVULSIONObjective: To explore the dynamic changes of neural stem cells(NSCs)in the hippocampus at different time after the status convulsions(SC)in immature brain,and to clarify features of Notch signaling after long-term convulsions.Methods: 120 healthy Sprague-Dawley(SD)males rats aged 20 days were selected and randomly divided into SC model groups(n=60)and normal control groups(n=60).The SC model was induced by intraperitoneal injection of lithium chloride and pilocarpine.We conducted the experiment as follows:(1)The cell proliferation status was marked with Ki67.Immunofluorescence staining was used to detect cell proliferation in the hippocampal dentate gyrus(dentategyrus,DG)at 1d,3d,and 60 d after SC.(2)After the SC model was established,BrdU was injected intraperitoneally to label NSCs,and the distribution of BrdU was detected by immunofluorescence staining 7 days after SC to analyze the migration of NSCs.(3)The dual-label BrdU and GFAP or dual-label BrdU and NeuN were stained 28 days after SC to detect NSCs differentiation into astrocytes and neurons.(4)The expression of NeuN and GFAP protein in hippocampus 7d,28 d and 60 d after SC was detected by Western blot.(5)Nissl staining was used to analyze hippocampal neuron loss at 28 days after SC.(6)Q-PCR was used to detect the differences of Notch1 and Hes1 mRNA expression in hippocampus at 1d,3d and 60 d after SC.(7)Immunohistochemical staining was used to observe the expression and distribution of Notch1 protein at 1d,3d and 60 d after SC.The expressions of Notch1,NICD1,Numb1,the major proteins related to Notch signaling pathway at 1d,3d,and 60 d after SC were analyzed by Western blot.Results:(1)Compared with the normal control group,Ki67-positive cells increased in the hippocampal DG area 1day after SC(t=6.147,p= 0.0003),and the expression was higheron the 3rd day(t=6.534,p= 0.0002).The number of Ki67-positive cells in the SC group decreased rapidly and was less than that in the normal control group at 60 days after SC(t=3.158,p=0.0134).The number of Ki67 positive cells in the SC group was statistically different at different periods(F=33.21,P <0.0001).(2)7 days after SC,the BrdU-positive cells in the normal group were mostly distributed in the lower layer of granulocytes,and a small part started to migrate to the granulocyte layer,and there were very few BrdU-positive cells in the CA1 and CA3 regions.However,in SC group,most of the BrdU-positive cells located under the granular cells of the DG have migrated.In addition to the normal migration to the granular cell layer,there are more newborn cells in the hilus area,and some cells migrate to inner molecular layer.In addition to the DG,BrdU positive cells were found in the CA1 and CA3 regions of the SC group.(3)The ratio of BrdU / NeuN double positive cells to the BrdU positive cellsin the DG,CA1,and CA3 areas of the SC group were 57.29%,46.29%,and 53.84%,which were significantly lower than those of the normal control group(68.24%,80.23%,and 76.37%)(p<0.05).The ratio of BrdU / GFAP double positive cells to the BrdU positive cells in the DG,CA1,and CA3 areas of the SC group were 38.24%,41.27%,and 43.72%,which were significantly higher than the normal control group(20.49%,10.59%,and 19.37%,p<0.05,0.001,0.05 respectively).(4)The protein expression of GFAP and NeuN in hippocampus of SC group and control group of the same age was detected by western blot.There was no statistically significant difference in GAFP and NeuN protein expression in the hippocampus of the two groups at 7 days after SC.At 28 days after SC,the expression of GFAP in SC group increased compared with the control group(t=3.679,p= 0.0062),the expression of NeuN decreased(t=3.621,p=0.0068),and the difference still exists 60 days after SC.(5)Nissl staining revealed that the hippocampal structure was severely damaged at 28 days after SC.The neurons were lost,and scattered nucleus was dissolved.The Nissl stained cell counts in hippocampal DG,CA1 and CA3 region of SC group were significantly reduced compared with normal control group(p<0.05).(6)The expression of Notch1 mRNA in hippocampus 1d after SC was higher than that in the control group,but the difference was not statistically significant(t=2.291,p=0.0512).The expression of Notch1 mRNA 3d after SC was significantly higher than that of the control group(t=3.555,p=0.0074),while it was significantly reduced after 60d(t=4.942,p= 0.0011).The difference within the SC group at different periods was statistically significant(p=0.0001).The changes of Hes1 mRNA in hippocampal area after SC are similar to Notch1,that is,the expression of Hes1 mRNA in hippocampal area on day 1 and 3 after SC is increased compared with the control group of the same age(t1=3.020,p1=0.0166;t3 = 4.657,p3 =0.0016),which is decreased 60 days after SC(t =3.879,p = 0.0047).The difference within the SC group at different periods was statistically significant(p <0.0001).(7)Immunohistochemical method was used to detect the expression of Notch1 protein in hippocampal DG region 1 day,3 days and 60 days after SC.Notch1 is expressed in the entire DG region.Western blot detection shows that at 1 day after SC,the expression of Notch1 protein began to increase compared with the control group(t=3.030,p=0.0163),and the increase was more significant 3 days after SC(t =4.833,p=0.0013).The expression of Notch1 protein at 60 days was lower than that of the control group(t = 4.463,p=0.0017).NICD1 protein had the same change trend,and Numb1 had the opposite change to Notch1 after SC.Conclusion:(1)Neurogenesis increased significantly in the hippocampus during the acute phase after SC in juvenile rats and decreased during the chronic phase.(2)After SC,hippocampal NSCs had obvious ectopic migration.More newborn cells migrated to the hilus area,and some newborn cells migrated to the inner molecular layer,CA1 and CA3 areas.In addition.(3)The proportion of hippocampal NSCs differentiation is unbalanced after SC,neuronal damage increases,and astrocytes proliferate,leading to the destruction of hippocampal structure.(4)Notch signaling pathway is activated in the hippocampus during the acute phase after SC in immature brain,and it is inhibited during the chronic phase.The dynamic change of Notch signal pathway is consistent with the dynamic change of hippocampal neurogenesis after SC.PART ? EFFECT OF INHIBITION OF NOTCH SIGNALING PATHWAY IN THE ACUTE PHASE AFTER SC ON HIPPOCAMPAL NEUROGENESIS IN IMMATURE BRAINObjective: To explore the changes in the proliferation and differentiation of hippocampal neural stem cellsafter inhibiting the Notch signal pathway in the acute phase after SC,and to observe its effects on SC-induced glial proliferation and hippocampal neuron loss.Methods: 60 SD rats of 20 days old were selected.After the SC model was established by intraperitonealinjection of Lithium chloride-pirocapine.?-secretase inhibitor DAPT was injected into the lateral ventricle to inhibitthe activation of Notch signaling pathway.The subjects were randomly divided into SC model group,DAPT groupand DMSO group.We conducted the experiment as follows:(1)Western blot was used to detect Notch1 protein expression 3 days after SC to verify the effect of DAPT on Notch signaling pathway.(2)NSCs were labeled by Bru U.Immunofluorescence method was used to detect the proliferation of NSCs in hippocampal DG area 3 days after SC.(3)Double-labeled Brd U and GFAP or double-labeled Brd U and Neu N were detected by immunofluorescence to show the differentiation of NSCs into astrocytes and neurons in the DG region at 28 days after SC.(4)The protein expressions of GFAP and Neu N in hippocampal tissues were detected by western blot at 28 and 60 days after SC.(5)Nissl staining was used to observe the loss of hippocampal neurons at 28 days after SC.Results:(1)The expression of Notch1 protein in the inhibitor DAPT group was lower than that in the SC group(t=3.194,P=0.0127)3 days after SC,and also decreased in the DMSO group(t=2.910,P=0.0196).The Notch1 protein expression in the DMSO group was not significantly different fromthat in the SC group.(2)At 3 days after SC,Brd U-positive cells in the hippocampal DG area were decreased significantly after injection of DAPT compared with SC group(t=4.923,P=0.0012).The proliferation of hippocampal NSCs in the vehicle DMSO group was not significantly different from the SC group.(3)At 28 days after SC,the average number of Brd U,Brd U / GFAP,and Brd U/ Neu Ncells in the DAPT group was 27.29,7.36,and 18.01,which were significantly less than those in the SC group(68.24,25.93,and 38.89 respectively)and the DMSO group(65.33,24.17,37.45 respectively).The difference was statistically significant(compared with SC group,p <0.05,0.01,0.05 respectively;compared with DMSO group,p <0.05).The ratio of Brd U / GFAP double positive cells in the DAPT group was 27.0%,which was lower than 38.0% in the SC group and 37.0% in the DMSO group(p<0.01).The ratio of Brd U / Neu N double positive cells in DAPT group was 66.0%,which was higher than 57.0% in SC group and 57.3% in DMSO group(P <0.05).(4)GFAP expression in hippocampus of DAPT group was reduced compared with SC group and DMSO group at 28 days after SC(p <0.05),and the difference between SC and DAPT group was more significant at 60 days after SC(t=4.325,p=0.0025).There was no statistically significant difference in GFAP protein expression between SC group and DMSO group.The expression of Neu N in hippocampus of DAPT group was increased at 28 days after SC compared with SC group(t=2.464,p=0.0390),and the difference was more obvious at 60 days after SC(t=3.363,p=0.0099).Neu N protein expression in SC group and DMSO group was not statistically different at 28 days after SC,but it increased at 60 days after SC in the DAPT group(t = 2.779,p = 0.0240).(5)Nissl staining showed that the cell arrangement in the CA3 region of the DMSO group was relatively neat and dense compared with the SC group,and nuclear lysis was reduced.However,in the CA1 area,significant nuclear lysis was still seen in the DMSO group,and the cell arrangement was disordered.In the DAPT group,the neuron loss in the CA3 and CA1 areas was reduced compared with the SC group.It can be seen that the neurons are arranged more neatly and tightly,the cell hierarchy is clear,and the Nissl bodies are clearly visible.Conclusion: DAPT can effectively inhibit the activation level of Notch pathway in the acute phase after SC in immature brain.Injecting DAPT into the lateral ventricle in the acute phase after SC can significantly inhibit the proliferation of hippocampal NSCs,inhibit the differentiation of hippocampal neural stem cells into glial cells,increase the proportion of neural stem cells differentiated into neurons,reduce neuronal loss after SC,and help repair the hippocampus structure.PART ? EFFECT OF INHIBITION OF NOTCH SIGNALING PATHWAY IN THE ACUTE PHASE AFTER SC ON EPILEPTOGENESIS IN IMMATURE BRAINObjective: To investigate the effect of DAPT on the mechanism of epileptogenesis in immature brain by inhibition of Notch signaling pathway in the acute phase.Methods: 72 SD rats of 20 days old were selected and randomly divide into normal groups(n=24),SC group(n=24)and DAPT group(n=24).Among them,5 in the normal group and 10 in the quasi-modeling group were equipped with EEG electrodes 3 days before the SC model was established.The SC model was established after the wounds of the rats healed.DAPT was injected into the lateral ventricle after the SC model was established to inhibit Notch pathway activity.Long-term EEG monitoring was used to observe the spontaneous recurrent seizures(SRS)in the chronic phase.Brain tissue samples were taken 28 days after SC,and the differences in synaptic structures of hippocampus were observed by transmission electron microscopy.Brain slice discharge induced by magnesium-free solution in vitro was used to analyze the changes in convulsive susceptibility of hippocampal slices 28 days after SC.Timm staining was used to analyze the sprouting of moss fibers in hippocampus at 28 days after SC.Results:(1)Transmission electron microscopy showed that the length of the presynaptic membrane active band in the SC group was reduced compared with the normal control group(t=3.406,p=0.0067),the synaptic gap was widened(t=3.441,p=0.0063).The post-synaptic density was blurred in SC group,and the thickness of the post-synapticdensitydecreased(t=3.213,p=0.0093).In the DAPT group,the length of the presynaptic active band increased compared with the SC group(t =2.242,p=0.0489),the thickness of the post-synaptic dense increased(t = 2.267,p = 0.0468).The width of the synaptic gap was not significantly different from that in the SC group.(2)EEG monitoring showed that SRS was also found in the chronic phase after SC in the DAPT group,but the average duration of each episode(t =2.326,p=0.0423),and the frequency of discharge(t=3.181,p=0.0098)and the average number of attacks per day(t=2.256,p=0.0477)were significantly reduced compared with the SC group.The severityof seizures behavior of the rats in the DAPT group was significantly reduced compared with the SC group,mainly spontaneous binocular gaze,facial muscle twitches,and less unilateral or bilateral forelimb clonics.(3)Spontaneous discharge of hippocampal brain slices after induction with a magnesium-free recording solution was recorded using patch-clamp technology.The average latency of the DAPT group was 25.64 minutes,which was longer than that of the SC model group by 14.16 minutes.The difference was significant(t=3.364,p=0.0099).The duration of spontaneous discharge was mostlyless than 5 minutesin SC group and DAPT group.The average times in DAPT group was 4.10,which was less than 6.46 in SC group(t=3.119,p=0.0143).In the DAPT group,0.80 episodes occurred over 30 minutes,which was less than 1.86 in the SC group(t=3.027,p=0.0164).(4)Moss fiber staining revealed that a small number of fine moss particles were seen in the CA3 and DG areas of the normal rats.At 28 days after SC,moss fiber particles in CA3 and DG areas increased significantly,Timm scores increased(t=4.189,p=0.0030),and moss fiber density detection values also increased(t=4.444,p=0.0022).After DAPT intervention,the Timm scores and moss fiber particle density in CA3 and DG region were not significantly different from those in non-intervention SC group.Conclusion: The use of DAPT to inhibit Notch signal pathway in the acute phase after SC may repair synaptic ultrastructure damage to a certain extent,reduce the convulsive susceptibility of immature brain,and effectively reduce the severity of epilepsy discharge in the SRS phase,suggesting that the inhibition of Notch signal pathway in the acute phase after SC is helpful to prevent theepileptogenesis.And its mechanism may not be significantly related to the moss fibers sprouting in hippocampus after SC. |
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