| Part one Changes of GPER1 expression and synaptic plasticity during the development of epilepsy induced by lithium chloride and pilocarpine in ratsOBJECTIVE To observe the changes of G protein-coupled estrogen receptor 1(GPER1)expression in hippocampal neurons and synaptic plasticity of temporal epilepsy rats.METHODS(1)Grouping: 56 SD male rats were randomly divided into Control group(Control),epilepsy 1d group(2d group),epilepsy 3 group(3d group),epilepsy 7d group(7d group),Epilepsy 14 d group(14d group),epilepsy 28 d group(28d group),8 rats in each group;(2)Lithium-pilocarpine was injected intraperitoneally to make rat temporal lobe epilepsy,TLE)model,the model was evaluated by the Racine epileptic seizure scoring standard;(3)Morris water maze was used to detect changes in learning and memory ability of rats;(4)Use Nissl staining to observe the morphological changes of hippocampal neurons;Immunohistochemistry and Western Blot techniques were used to observe the expression characteristics and changes of GPER1,PSD95,Synapsin ?,and Gephyrin in hippocampal neurons;(6)Timm staining was used to observe the mossy fiber sprouting of hippocampus;Golgi staining was used to observe the axon and dendritic mutation of hippocampal neurons.RESULTS(1)The results of the water maze showed that compared with the Control group,the rats who made the model for 14 days had longer time to find the platform within 60 s,and the number of times of crossing the platform decreased(P <0.05);(2)Nissl staining results showed that the cells in the Control group Tightly arranged and uniformly stained,the cell arrangement of rats at 1 d was slightly loose,and the staining of Nisslite became lighter after 2 d,and after 3 d,the cells atrophied,the number decreased,the cell spacing increased,and the Nissl body stained became deeper,and the cell volume decreased at 7 d.After 14 d,the cells began to return to normal,and the Nissl body staining was still light.After 28 d,the cell arrangement basically returned to normal.(3)Immunohistochemical results showed that the immunopositive expression in the Control group was located in the cell membrane.Compared with the Control group,the expression of GPER1 was increased at 2 days after modeling(P <0.05);the positive expression was stained in the cytoplasm at 3 days after modeling;the expression began to decrease after 14 days(P> 0.05).PSD95 positive staining was distributed in the hippocampal CA3 area of each group in a patch-like manner.Compared with the control group,the expression of PSD95 decreased on the 1st day of the model(P <0.05)and slightly increased on the 2nd day of the model(P <0.05).The expression returned to Control level at 7d and 14 d,and decreased again at 28d(P <0.05).The positive expression of Synapsin I in the hippocampal CA3 region of the rats on the 1st day of modeling was significantly reduced(P <0.01),and the expression began to rise on the 2nd day of the modeling(P <0.01).The model approached the Control group at the 14 th day of the modeling(P> 0.05).At 28 d,the expression level decreased again(P <0.01).Gephyrin immunopositive staining was mainly expressed in the cytoplasm.Gephyrin positive expression was significantly increased at 1d(P <0.01),positive staining was slightly decreased at 2d(P <0.01),and positive expression was decreased at 3d(P < 0.05),showing cell membrane staining.The expression increased slightly on the 7th day of the model(P <0.05).The expression continued to increase on the 14 th day of the model(P <0.01),and remained almost the same on the 28 th day of the model(P <0.01).(4)The results of Timm staining showed that Timm particles were not seen at 1d-2d(acute phase),and Timm particles near continuous distribution began to appear at 3d(P <0.01).After 7d,band-like Timm particles were observed(P <0.01),at 28 d,it appears as a continuous continuous distribution of Timm silver-stained particles(P <0.05).(5)Golgi staining results showed that in the CA1 region,hippocampal neuron axonal mutations were short at 1d,dendrites decreased and axons shortened at 3d,there were only a few dendrites at 7d,axons grew at 14 d,and axons gradually extended at 28 d.Dendrites increased slightly..In the CA3 area,the neurons did not change significantly at 1d,the dendrites began to decrease at 3d,the dendrites decreased significantly at 7d,and the axon mutation was short.In the DG area,the neuronal morphological changes were not obvious at 1d,3d,7d,and 14 d,and a slight reduction of dendrites was observed at 28 d.(6)Western Blot results were basically consistent with immunohistochemical results.CONCLUSION(1)GPER1 increases with neuron damage during the development of epilepsy;(2)Epilepsy-induced hippocampal neuron axis and dendrite density decrease significantly at 7d,with CA3 area being the most Significantly;mossy fiber sprouting was seen in the granular cells and CA3 pyramidal cells of the dentate gyrus in the chronic phase of epilepsy(14d,28d).(3)Repeated seizures,in the acute phase,down-regulated the expression of the presynaptic membrane protein Synapsin I,the excitatory post-synaptic membrane protein PSD95,and the inhibitory synaptic membrane protein Gephyrin.Part two The effects of GPER1 on synaptic plasticity of hippocampal neurons in temporal lobe epilepsy ratsOBJECTIVE To investigate the possible regulation of GPER1 on synaptic plasticity of hippocampal neurons during the development of temporal lobe epilepsy in rats.METHODS(1)Grouping: 72 male SD rats were randomly divided into a DMSO epilepsy 2d control group(DMSO 2d group),a 2d epilepsy G1 intervention group(G1 2d group),and a 2d epilepsy G15 intervention group(G15 2d group),DMSO7 d control group(DMSO 7d group),7d epilepsy G1 intervention group(G1 7d group),7d epilepsy G15 intervention group(G15 7d group),DMSO epilepsy 28 d control group(DMSO 28 d group),28d epilepsy G 1 intervention group(G1 28 d group),28 d epilepsy G15 intervention group(G15 28 d group),8 in each group;(2)Intraperitoneal injection of lithium chloride-pirocarpine to make rat temporal lobe epilepsy model,Racine Epilepsy scoring criteria were used to evaluate the model.The lateral ventricle was implanted with a cannula and a small amount of drug was administered.(3)Morris water maze test was used to detect changes in the learning and memory ability of rats in each intervention group.(4)Nissl staining was used to observe the rats in each intervention group.Changes of hippocampal neuron cell morphology;(5)Immunohistochemical and Western Blot techniques were used to observe the expression characteristics and changes of GPER1,PSD95,Gephyrin,and Synapsin ? in hippocampal neurons of rats in each group;(6)Timm staining was used to observe the abnormal sprouting of mossy fibers in hippocampus of each intervention group;(7)Golgi neuron fiber staining experiment was used to observe the changes of axons and dendrites in each intervention group.RESULTS(1)Water maze: Compared with the DMSO 28 d group,the time required to find a platform in the G1 28 d group was significantly reduced in 60 s(P <0.01),and the G15 28 d group was prolonged(P <0.05);In the G1 28 d group,the number of times of crossing the platform area within 60 s increased significantly(P <0.05),while the G15 28 d group decreased.(2)Nissl staining: The number of cells in the CA1 area of the DMSO 2d group is small and the arrangement is loose,the number of cells in the CA3 area is reduced,and some of the cells are deeply stained.A small number of densely stained cells are also observed in the DG area;Fullness,uniform staining of Nissl bodies,densely arranged pyramidal cell layers in the CA3 region,similar to the Control group,densely packed granular cell layers in the DG region,and the staining approached normal.Cell damage in the three regions of the G15 2d group was more severe than in the DMSO 2d group.The cell changes in the three regions of the DMSO 7d group are similar to those of the Nd stained cells in the model 7d.The CA3 region is more serious.The number of cells in the three regions of the G1 7d group increases and is closely packed.The number of cells in the three regions of the G15 7d group decreases sharply.The arrangement is disordered,and the staining of Nissl body becomes lighter than that of G1 7d group.In the DMSO 28 d group,the cell layers in the three areas became thinner and deeper stained.In the G1 28 d group,the cells in the three areas were arranged neatly,while in the G15 28 d group,the cells in the CA1 area became lighter and loosely arranged.The cell damage in the CA3 and DG areas was slightly less than that in the CA1 area.(3)Immunohistochemical results: Compared with the DMSO group,the expression of GPER1-positive cells in the G1 intervention group increased significantly at the three time points of epilepsy at 2d,7d,and 28d(P < 0.05),while the expression in the G15 intervention group decreased(P < 0.05)No transfer of GPER1 positive expression to cytoplasm was observed.Compared with DMSO group at each time point,the expression level of PSD95 in hippocampus of G1 intervention group decreased(P <0.05).Similarly,compared with the time in the DMSO group,the expression of Synapsin ? in the hippocampus of the G1 intervention group at three time points was reduced(P <0.01).Compared with the DMSO group,the expression of Gephyrin in the hippocampus of the G1 intervention group was significantly reduced at 2 days(P <0.05);it was significantly increased at 7 days(P <0.05);and decreased again at 28 days.(4)Timm staining results: Compared with the DMSO group,the Timm score of the G1 intervention group at three time points was significantly reduced(P <0.05),and the Timm score of the G15 group was significantly increased(P <0.01).(5)Western Blot results: Compared with the DMSO group,GPER1 expression in the G1 group increased at three time points(P <0.05),and the G15 group decreased(P <0.05);the trend of ER? was consistent with the GPER1,but the change in the 28 d group No statistical significance;PSD95: Compared with the DMSO group,the expression of the G1 2d group decreased,the G1 7d group continued to decline(P <0.05),the G1 28 d group slightly increased expression(P <0.05),the G15 2d group decreased expression,and G15 7d expression decreased significantly(P <0.05),G15 28 d group expression increased;Synapsin ?: Compared with DMSO group,G1 2d group expression decreased(P < 0.05),G1 7d group slightly increased(P < 0.05),G1 28 d group Continue to rise(P <0.05),G15 2d group expression decreased(P <0.05),G15 d group slightly increased(P <0.05),G15 8d group was flat;Gephyrin: Compared with DMSO group,G1 2d group slightly decreased,G1 The 7d group increased significantly(P <0.01),the G1 28 d group decreased again(P <0.05),the expression of G15 2d group was the same as that of DMSO group,the G15 7d group decreased significantly(P <0.01),and the expression of G15 8d group increased.(6)Golgi staining results: In the CA1 area,the DMSO 28 d epilepsy control group had sparse neuron density,short axon mutations,and reduced dendritic branches.The G1 28 d group had denser neurons,longer axons,and dense and orderly dendritic nets,while the G15 28 d group There are fewer dendritic nets.In the CA3 area,dendrites in the DMSO 28 d group were significantly reduced,the axons in the G1 28 d group were longer,the dendritic network recovered,and the density increased,while the G15 28 d group was sparse and shortened.In the DG area,the changes were not obvious in each group.CONCLUSION(1)GPER1 selective agonist G1 can significantly improve learning and memory impairment in epilepsy rats;(2)G1 can significantly improve hippocampal neuron damage in rats caused by epilepsy,G15 can offset the protective effect of G1 on hippocampal neurons;(3)G1 can improve epilepsy The formation of abnormal mossy fibers in the hippocampus of rats;(4)G1 may participate in the repair of synaptic plasticity by reducing Synapsin I and increasing the expression level of Gephyrin,and inhibitory synaptic Gephyrin may play a significant role. |
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