| PART ? CHANGES OF DENDRITES AND SYNAPTIC INJURY OF HIPPOCAMPUS AFTER STATUS CONVULSIONObjective:To determine the success of intraperitoneal injection of kainic acid?KA?to establish status convulsion?SC?and epilepsy model.To evaluate the synaptic injury and changes of dendrites and dendritic spines?DS?in hippocampus of adult and infant mice on the 7th,14th,and28th day after SC.Methods:Adult male C57BL/6J mice aged 8 weeks and infant male C57BL/6J mice aged 14 days were selected and divided into normal control group?NCG?and SC group?SCG?respectively.The SCG was injected with KA to induce SC,and NCG was injected with an equal dose of 0.9%sodium chloride solution.EEG monitoring was performed on the adult mice to verify the epilepsy model,the seizures and spontaneous recurrent seizures?SRS?was observed.Western blot was used to analyze the expression of synaptic vesicle protein?SYP?,post-synaptic dense protein-95?PSD-95?,and growth-associated protein-43?GAP-43?of hippocampus at different time points after SC.Golgi-Cox staining was used to stain brain tissues and the dendritic complexity of granule cells in the DG subregion,pyramidal cells in CA1 and CA3 subregions,and the number of different types of DS were counted and analyzed.Results:?1?15-30 minutes after intraperitoneal injection of KA,adult and infant mice showed typical behavioral changes.15 minutes after the injection of KA,the EEG of experimental group of adult mice began to show multiple high-amplitude spikes.Furthermore,the frequency of the spike wave gradually increased until SC was developed after about 30minutes,and the EEG showed a high-frequency and high-amplitude spike wave.Until 28 days after SC,EEG still showed repeated spines and sharp waves,consistent with the SRS and,repeated seizures.The EEG of the control group showed no epileptic discharge.?2?On the 7th day after SC,the expression of SYP,GAP-43 and PAD-95 in the mature hippocampus of the SCG were 0.71±0.28,0.49±0.17,and 0.68±0.19,which are significantly reduced compared with the NCG?1.43±0.37,1.21±0.05,and1.36±0.39 respectively;p=0.004,0.003,and<0.001 respectively?,but gradually returned to the NCG level on the 14th and 28th day after SC.However,in the immature brain,on the 7th,14th and 28th day after SC,the SYP expression was increased significantly?1.08±0.10 vs 0.34±0.16,p=0.012;1.08±0.25 vs 0.44±0.15,p=0.026;1.06±0.05 vs 0.50±0.36,p=0.026?,and levels of PSD-95 and GAP-43 were not significantly different from those of the NCG.?3?In the adult brain,on the 7th day after SC,the average length of dendrites in DG,CA1,and CA3 subregions of hippocampus were significantly lower than that of the NCG?p=0.012,<0.000,and 0.000 respectively?.On the 14th day after SC,only the average length in the CA3 subregion was significantly reduced?942.0±193.2?m vs1212.0±291?m,p=0.000?.On the 28th day after SC,the average length in CA1 and CA3 subregions were significantly lower than those in NCG.In the infant brain,on the 7th day after SC,the average length in DG,CA1,and CA3 subregions were significantly increased compared with the NCG?869.7±28.9?m vs 636.5±36.7?m,p=0.000;941.2±39.3?m vs833.9±39.8?m,p=0.009;863.2±46.1?m vs 846.8±65.8?m,p=0.001?,on the 14th day,the average length in the DG subregion was decreased significantly?738.4±45.5?m vs 821.4±47.6?m,p=0.045?.?4?At each time point after SC,the average intersection points of the dendrites and concentric circles in DG,CA1,and CA3 subregions of the mature hippocampus were significantly reduced compared with the NCG?p<0.000?.In contrast,in the immature hippocampus,on the 7th and 14th day after SC,the average number of intersections in the DG subregion was increased significantly?3.53±3.97 vs 2.12±3.22,p<0.000;4.78±5.36 vs2.40±2.94,p<0.000?,and decreased significantly on the 28th day after SC?1.75±2.20 vs 4.24±4.46,p<0.000?.And the numbers of intersections in CA1 and CA3 subregions were significantly lower than those in the NCG at each stage after SC?CA1:p=0.033,<0.000,and<0.000;CA3:p<0.000?.?5?In the mature hippocampus,the total numbers of DS,CA1,and CA3 subregions were significantly reduced compared with the NCG on the 7th day after SC?p<0.000,<0.000,and=0.001?,mainly due to the reduction of thin and mushroom DS,while the numbers of stubby and filopodia DS were significantly increased.However,the total numbers of DS in the DG,CA1,and CA3 subregions of immature hippocampus were increased significantly?p=0.000,0.002,and<0.000?,mainly due to the increase of thin DS.On the 14th day after SC,stubby and filopodia DS in the DG and CA3 subregions were increased significantly?p<0.000 and p=0.001?,while the mushroom DS were decreased significantly?p<0.000?.The stubby DS in the CA3 subregion was also increased significantly,and the number of mushroom DS was significantly decreased.And the mushroom DS in the DG subregion were decreased significantly?p<0.000?.The total number of DS in the CA1 subregion was decreased significantly,and mainly due to the reduction of thin and mushroom DS,while the stubby DS were increased significantly.In the infant mice,the total number of DS in the CA3 subregion was decreased significantly?p=0.000?,due to the reduction of thin and mushroom DS.On the 28th day after SC,the total number of DS in each subregion was decreased significantly again compared with the NCG?p<0.000,p<0.000,and p=0.001?,mainly due to the reduction of thin and mushroom DS.In CA1 and CA3 subregions,the stubby DS were increased significantly.In the infant mice,the total numbers of DS in DG and CA1 subregions were significantly decreased than those in the NCG?p=0.003,and 0.001respectively?,mainly due to the reduction of mushroom DS in the DG subregion,and the reduction thin,mushroom,and branched DS in the CA1subregion.Conclusion:Intraperitoneal injection of KA can induce chronic epilepsy.In the acute stage after SC,the synapse of the mature hippocampus was obviously injured,and the expression of synaptic proteins gradually returned to normal levels.However,the expression of SYP was increased significantly at the acute and transitional stages,and the levels of PSD-95 and GAP-43 proteins are not significantly different from the normal control levels.Compared with the mature hippocampus,the immature one is resistant to the injury induced by SC.The dendrites and DS of hippocampus changed dynamically at different stages after SC.The mature brain mainly presented the reduction of dendritic complexity and the loss of DS.However,the immature brain showed reactive increase of DS and dendrites in the acute phase after SC.Gradually,the complexity decreased and DS were lost.PART ? ALTERATION OF MICROGLIA AND THEIR SURFACE MARKERS IN THE HIPPOCAMPUS AFTER STATUS CONVULSIONObjective: To explore the dynamic alteration of microglial phenotypes and their surface markers in mature and immature brain tissues on the 7th,14 th and 28 th day after KA-induced SC.Methods: Adult male C57BL/6J mice aged 8 weeks and infant male C57BL/6J mice aged 14 days were selected and divided into NCG and SCG respectively.The SCG was injected with KA to induce SC,and NCG was injected with an equal dose of 0.9% sodium chloride solution.Flow cytometry?FCM?was used to calculate the proportion of M1 and M2 microglia?MG?and the surface markers C3 R,CX3CR1,CXCR3,CD200 R and CD68 on the 7th,14 th,and 28 th day after SC.Results:?1?The proportion of M1 MG of mature brain tissues was increased significantly on the 7th day after SC?85.80±4.08% vs 78.15±2.83%,p=0.001?,the proportion of C3R+M1 and CX3CR1+M1 MG were significantly increased and the proportion of CD200R+M1 MG was significantly decreased.On the 14 th day after SC,the proportion of M1 MG was decreased to the NCG level?81.82±3.80% vs 78.15±2.83%,p=0.880?,and the proportion of M2 MG was significantly increased?6.07±1.90% vs 3.38±0.14%,p=0.037?.The proportion of CD200R+M1,CD68+ M1 and CXCR3+M2 MG was increased significantly.On the 28 th day after SC,the proportion of M1 MG was increased significantly again?84.33±3.07% vs 78.15±2.83%,p=0.001?,and the proportion of C3 R and CX3CR1+ were significantly increased and the proportion of CD200R+M1 MG was significantly decreased.?2?In the immature brain,on the 7th day after SC,the proportion of both M1 and M2 MG was significantly increased?91.65±3.58% vs 66.13±11.88%,p=0.002;2.63±0.91% vs 0.72±0.37%,p=0.002?.the proportion of C3R+M1,CXCR3+M1,CD200R+M2,CX3CR1+M2,CXCR3+ M2 and CD68+M2 MG was increased significantly.On the 14 th day after SC,the proportion of M1 MG returned to the NCG level?90.53±2.91% vs 86.12±5.05%,p=0.177?,while the proportion of M2 MG was decreased significantly?1.44±0.34% vs 3.52±1.39%,p=0.020?.The proportion of CX3CR1+M1 MG was increased significantly,and the proportion of CD200R+M1,CD68+M2 and CXCR3+M2 MG was decreased significantly.On the 28 th day after SC,the proportion of M1 and M2 MG showed no significant difference from the NCG?91.37±1.92% vs 90.63±4.33%,p=0.713;2.15±0.65% vs 2.13±0.29%,p=0.945?,and the proportion of CD200R+M1 was significantly decreased compared with the NCG.Conclusion: The phenotypes and surface markers of MG were altered dynamically after SC,indicating that they were involved in the process of epilepsy,and the dynamic alteration of MG in the mature and immature brain tissues were different.PART ? EFFECT OF MINOCYCLINE ON MICROGLIA AND DENDRITES IN THE HIPPOCAMPUS OF IMMATURE BRAIN AFTER STATUS CONVULSIONObjective: To explore the changes of hippocampal synaptic proteins,alteration of dendrites and DS in DG,CA1 and CA3 subregions of hippocampus,and the proportion of microglia phenotypes and surface markers in brain tissues of immature brain on the 7th,14 th,and 28 th day after KA-induced SC and intervention by intraperitoneal injection of Minocycline?MINO?.Methods: Infant male C57BL/6J mice aged 14 days were selected and KA was used to induce SC.MINO was intraperitoneally injected at a dose of 10 mg/kg on the second day after successful SC induction,once a day for three consecutive days.On the 7th,14 th,and 28 th day after SC,western blot was used to detect the expression of hippocampal SYP,PSD-95 and GAP-43,FCM was used to count the proportion of M1 and M2 MG and their surface markers CX3CR1,CXCR3,CD68 and CD200 R,furthermore,Golgi-Cox staining was used to stain brain tissue and the dendritic complexity of the granulosa cells in the DG subregion and the pyramidal cells in the CA1 and CA3 subregions,and the number of different types of DS were counted and analyzed.Results:?1?After the intervention of MINO,on the 7th,14 th and 28 th day after SC,comparing with the SCG,the expression level of SYP was significantly decreased,but was not significantly changed compared with the NCG?0.7±0.28 vs 0.34±0.16 vs 0.55±0.11,p=0.001;1.08±0.25 vs 0.44±0.15 vs 0.73±0.19,p=0.062;1.06±0.05 vs 0.50±0.36 vs 0.93±0.18,p=0.093?.The expression of GAP-43 was significantly increased at each stage after SC compared with the SCG and NCG0.49±0.17 vs 0.22±0.04 vs 1.04±0.58,p=0.028;0.20±0.07 vs 0.22±0.08 vs 1.13±0.22,p=0.000;0.10±0.01 vs 0.10±0.01 vs 1.26±0.28,p<0.000).In addition,on the 14 th and 28 th day after SC,the expression of PSD-95 was increased significantly compared with the SCG and NCG?0.13±0.02 vs 0.15±0.07 vs 0.63±0.30,p=0.023;0.12±0.03 vs 0.22±0.09 vs 0.43±0.04,p=0.006?.?2?After the intervention of MINO,on the 7th day after SC,the proportion of M1 MG and M2 MG were decreased and increased significantly compared with the SCG respectively,and comparing with the NCG,the proportion of M1 MG was not significantly changed and the proportion of M2 MG was significantly increased?M1: 91.65±3.58 vs 66.13±11.88 vs 66.35±2.77,p<0.000;M2: 2.63±0.91 vs 0.72±0.37 vs 5.49±1.50,p<0.000?.Comparing with the SCG,the proportion of C3R+M2,CX3CR1+M2 and CD200R+M2 MG was significantly increased,and the proportion of CXCR3+M2,CD68+M2 were decreased significantly;but comparing with the NCG,the proportion of C3R+M2,CX3CR1+M2 and CD200R+M2 were significantly increased,but the proportion of CXCR3+M2 and CD68+M2 were not significantly changed.Accompanying with the reduce of M1 MG and C3R+M1 MG,the proportion of CD200R+M1 MG was increased significantly,and the proportion of CX3CR1+M1,CXCR3+M1 and CD68+M1 MG were significantly decreased comparing with the SCG and NCG.On the 14 th day after SC,the proportion of M1 and M2 MG was significantly reduced M1: 90.53±2.91 vs 86.12±5.05 vs 51.73±1.93,p< 0.000;M2: 1.44±0.34 vs 3.52±1.39 vs 0.14±0.04,p<0.000).Comparing with the NCG,the proportion of CD200R+M1 MG was significantly increased.On the 28 th day after SC,the proportion of M1 MG decreased significantly?91.37±1.92 vs 90.63±4.33 vs 66.13±11.88,p<0.000?,and the proportion of M2 MG was not significantly changed.The proportion of CXCR3+M2,CX3CR1+M2,CD68+M2,C3R+M1,CX3CR1+M1,CD68+M1,and CXCR3+M1 MG were significantly decreased,and the proportion of CD200R+M1 MG was significantly increased.?3?After the intervention of MINO,on the 7th day after SC,although the total dendritic length in DG subregion was still increased significantly compared with the NCG,but has a decreasing trend compared with the SCG?SC: 869.77±48.8?m vs SC+MINO: 636.5±28.9?m vs Ctl: 312.1236.7?m,p=0.023?.And the number of dendritic intersections of concentric circles in was not different compared with the SCG?SC: 3.53±3.97 vs SC+MINO: 2.35±3.20 vs Ctl: 2.12±3.22,p<0.000?.The total dendritic length in the CA1 and CA3 subregions were not changed significantly compared with the NCG?SC: 941.2±39.3?m vs SC+MINO:1106.0±282.6?m vs Ctl 833.9±39.8 ?m,p=0.152;SC : 863.2±46.1?m vs SC+MINO: 666.3±213.2?m vs Ctl: 706.0±10.7?m,p=0.115?.And the number of intersections in the CA1 subregion was increased significantly compared with the SCG,but not different with the NCG.The number of intersections in the CA3 subregion was still significantly decreased?1.94±3.06 vs 3.34±4.97,p<0.000?.On the 14 th day,the intersections in the DG and CA1 subregions were not different compared with the NCG,and the intersections in the CA3 subregion were still significantly decreased?2.03±3.51 vs 6.95±4.66,p < 0.000?.On the 28 th day,the intersections in DG,CA1,and CA3 subregions were significantly decreased compared with the NCG,but the intersections in the DG subregion had a decreasing trend compared with the SCG?DG: 1.94±2.62 vs 4.24±4.46,p<0.000;CA1: 4.83±4.58 vs 7.44±4.32,p<0.000;CA3: 2.48±3.21 vs 5.93±4.62,p<0.000?.?4?On the 7th and 14 th day.comparing with the NCG,there was no significant difference in the total number of DS?SC: 31.14±5.48 vs SC+MINO: 25.36±3.78 vs Ctl: 24.53±4.98,p<0.000;SC:27.19±6.10 vs SC+MINO: 27.58±4.06 vs Ctl: 29.50±3.57,p=0.493?in the DG subregion,and all types of DS were not significantly changed.On the 28 th day after SC,the total number of DS in the DG was significantly decreased compared with the CNG,and the thin and mushroom DC were mainly decreased,however the number had an increasing trend compared with the SC group?SC : 22.76±3.56 vs SC+MINO: 25.25±4.16 vs Ctl: 28.13±2.48,p=0.002?.In the CA1 subregion,the number of filopodia DS was significantly decreased?p=0.024?.On the 7th day after SC,the total number of DS in the CA3 subregion was significantly increased compared with the NCG,mainly due to the increase of thin DS,but the total number showed a decreasing trend compared to the SCG?SC: 28.32±3.76 vs SC+MINO:27.09±3.07 vs Ctl:20.97±4.46,p<0.000?.On the 14 th day,the total number of DS and was significantly decreased compared with the NCG,mainly due to the reduction of thin DS,and the total number had an increasing trend compared to the SCG?SC:19.11±5.16 vs SC+MINO:20.97±4.46 vs Ctl:25.31±3.77,p<0.000?.On the 28 th day,the total number of DS was not significantly changed,but the numbers of mushroom DS and stubby DS were significantly increased and decreased respectively.Conclusion: After the intervention with MINO,the activity of MG was inhibited,especially the inhibition of M1 MG activity.The proliferation of hippocampal dendrites and DS in the early stage and the loss in the chronic stage were corrected. |
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