| Part one The culture and identification of cortical neurons from ICR neonatal mice and the establishment of cell epilepsy modelObjective: To isolate,culture and identify the cortical neurons of ICR neonatal mice,and optimize the induced concentration of ROCK inhibitor Y-27632 and pilocarpine to establish the neuron epilepsy model.Methods:1.The cortical neurons of ICR neonatal mice were isolated by using the method of papaya protease digestion and identified by immunocytochemistry staining using microtubule associated protein MAP2 antibody on the 8th day;2.The neurons cultured for 8d were divided into control and epilepsy groups.The neurons in control group were cultured in normol neurobasal-A medium,and the neurons of epilepsy groups were cultured in medium with final concentrations of 1,2,3,and 4mmol·L-1 of pilocarpine respectively was replaced by normol neurobasal-A medium after 24 h.3.Recording cell growth morphology at 1,3 and 7d.The neurons of mice in various groups were fixed at1,3 and 7d after modeling,then fluorescence staining were performed.4.the neurons of inhibitor groups were cultured in medium with final concentrations of 3mmol·L-1 of pilocarpine firstly,replaced by medium with final concentrations of 5,10 and 15μmol·L-1 of inhibitors Y-27632 after 24 h,and then replaced by normol neurobasal-A medium after 24 h.5.Recording the action protential of epilepsy model neuron and inhibitor groups neuron by using the technology of whole cell patch clamp.Results: 1.The cortical neurons of ICR neonatal mice were isolated and cultured successfully,and the mature neurons(cultured 15d)were full and extensions,with a clear structure and a dense network;2.Immunocytochemical staining showed that the positive staining of MAP2 was brown and the purity of neurons was 90%;3.The results of F-acitn fluorescence staining showed that the neurons in 2 mmol·L-1 pilocarpine group had no obviously changes after modeling compared with control group;1d after modeling,the fluorescence intensity of F-actin in the 3 mmol·L-1 and 4mmol·L-1 pilocarpine group decreased obviously,cell processes shorten and disappeared,and cell network connection collapsed;at 3d and 7d,in 3mmol·L-1 pilocarpine group,the neurites and intercellular network gradually restored and neuron state is improved,and the F-actin fluorescence intensity is enhanced;the F-actin fluorescence intensity of 4mmol·L-1 pilocarpine group is weaker,and the basic structure of neuron is disintegrated,a large amount of cells died.4.The results of cell morphological observation used by phase contrast microscope showed that the neurons of pilocarpine+Y-27632(5μM)group recovered well,not only the cell body gradually full and three-dimensional with showing luster,but also re-establishing the neural network;there are a large number of cell death in pilocarpine+Y-27632(10μM)group and pilocarpine+Y-27632(15μM)group on the 7th day.5.The resluts of whole cell patch clamp action potential detection showed that the spontaneous discharge is occasionally or single in neurons of control group,and low frequency discharge;after induced by 3mmol·L-1 pilocarpine,the excitability of neurons increased significantly,the frequency of discharge was higher,and the spontaneous epileptiform discharges were presented in a short time;the concentrations of 5μmol·L-1 of Y-27632 can inhibit epileptiform discharges induced by pilocarpine,and the excitability,discharge frequency and amplitude are all decreased.Conclusion: 3mmol·L-1 pilocarpine can induced neuronal epileptiform discharge and the establishment of cell epilepsy model is successfully,and the optimal concentration of ROCK inhibitor Y-27632 was 5μmol·L-1Part two The change of the expression level and density of dendritic spines in epileptic neurons induced by pilocarpineObjective: To investigate the change of the dendritic morphology,dendritic spine density and spinophilin expression level i n neurons after pilocarpine induction and ROCK2 inhibitor Y-27632 treatment.Methods: 1.The neurons cultured for 8d were divided into control,epilepsy and inhibitor groups.The neurons in control group were cultured in normol neurobasal-A medium;the neurons of epilepsy groups were cultured in medium with final concentrations of 3mmol·L-1 of pilocarpine was replaced by normol neurobasal-A medium after 24h;and the neurons of inhibitor groups were cultured in medium with final concentrations of 3mmol·L-1 of pilocarpine firstly,replaced by medium with final concentrations of 5μmol·L-1 of inhibitors Y-27632 after 24 h,and then replaced by normol neurobasal-A medium after 24 h.2.Each experimental group performed cell growth morphology recording,F-actin fluorescence staining,the number of dendritic spines per unit length of experimental counting,spinophilin immunocytochemical staining and Western blot in 1,3 and 7 days respectively after the treatment of inhibitors Y-27632.Results: 1.The results of F-actin fluorescence staining and the number of dendritic spines per unit length showed that the dendritic spines were located on the dendrites of neurons and showed a small protrusion.Compared with the control group,in 1d and 3d,the number of dendritic spines in epileptic model group neurons decreased obviously and the fluorescence intensity also decreased;while on the neuronal dendrites of the inhibitor group appeared a large number of new dendritic spines precursor,lower than the control group but significantly higher than that of epilepsy group,and the number gradually increased with the culture time was positively correlated.At 7d,the number of dendritic spines in these two groups was almost recovered to the control level.(n=30,*P<0.05 vs Con,**P<0.01 vs Con;#P<0.05 vs Pilo,##P<0.01 vs Pilo).2.The results of immunocytochemical staining showed that: in the control group,the spinophilin are less expression and mainly distributed in the cytoplasm in 1d;with the extension of the culture time,the expression increased gradually,and the spinophilin distributed in the dendritic segment widely at 7d.In the epilepsy model group,the positive staining in the cytoplasm was stronger than that in the control group,but weaker in dendritic spines.The positive expression of the inhibitor group was stronger than that of the control group and epilepsy group,and distributed in cytoplasm and dendrites widely.3.The results of western blot showed that: compared with the control group,the protein expression levels of spinophilin in epileptic model group is decreased significantly,although it’s recovered gradually with the increase of time,but still lower than the control group.these two groups have significant difference;compared with the epilepsy model group,the expression level of spinophilin was significantly increased in inhibitor group,significant difference.(n=30,*P<0.05 vs Con,**P<0.01 vs Con;#P<0.05 vs Pilo,##P<0.01 vs Pilo).Conclusion: In the epileptic neurons induced by pilocarpine,the depolymerization of F-actin leads to a decrease in dendritic spine density,while the ROCK2 inhibitor Y-27632 can alleviate this phenomenon.It’s suggesting that Rho/ROCK2 signal were activated in the epileptic neurons induced by pilocarpine and damaged neurons,while Y-27632 can protect neuron.Part three The change of the protein distribution and expression of Calponin3,ROCK2 and pho-ROCK2 in epileptic neuron induced by pilocarpineObjective: To observe and analysis the influence on the change of protein distribution and expression in Calponin 3,ROCK2 and phosphorylated ROCK2(pho-ROCK2)induced by pilocarpine and ROCK2 inhibitor Y-27632,and investigate the relationship between dendritic spine remodeling,F-actin rearrangement,Rho/ROCK2 signaling pathway and Calponin 3Methods: 1.The neurons cultured for 8d were divided into control,epilepsy and inhibitor groups.The neurons in control group were cultured in normol neurobasal-A medium;the neurons of epilepsy groups were cultured in medium with final concentrations of 3mmol·L-1 of pilocarpine was replaced by normol neurobasal-A medium after 24h;and the neurons of inhibit or groups were cultured in medium with final concentrations of 3mmol·L-1 of pilocarpine firstly,replaced by medium with final concentrations of 5μmol·L-1 of inhibitors Y-27632 after 24 h,and then replaced by normol neurobasal-A medium after 24 h.2.Each experimental group performed immunocytochemical staining and Western blot in 1,3 and 7 days respectively after the treatment of inhibitors Y-27632.Results: 1.The result of immunocytochemical staining showed that in the control group,the distribution of protein ROCK2 and Calponin 3 is wildly and mainly dispersed in the cytoplasm and dendritic spines,the positive staining is shallow and the line is blurring;but in the epileptic model group,the distribution of protein ROCK2 and Calponin 3 is mainly gathered in the cell membrane and the expression level increased with time,the line becomes clear and sharp,and the Beaded lesions structure of dendrites comes out;in the inhibitor group,the positive staining of ROCK2 weaker than that in epileptic model group,the line is not clear partly and the Beaded lesions structure of dendrites gets smaller.The positive staining of Calponin 3 was significantly weaker in 7d2.Western blot analysis showed that compared with the control group,the protein enpression levels of ROCK2 and pho-ROCK2 were significantly increased in epilepsy model group in 1d and 3d,and the protein level increased with the prolongation of culture time.The expression level of ROCK2 protein in the inhibitor group was higher than that in the control group at 1d and 3d,and lower at 7d;at 1d,the protein expression of pho-ROCK2 in the inhibitor group was significantly higher than that in the control group,but the expression level decreased gradually with the prolongation of culture time,and was significan tly lower than that in the control group at 7d;at the same time,the ROCK2 protein level in the inhibitor group was significantly lower than that in the epilepsy model group.Compared with the control group,the protein expression level of Calponin 3 was significantly increased in the epilepsy model group and inhibitor group at 1d and 3d;until the 3rd day,in inhibitor group,and then decreased to the levels of the control;but in the epilepsy model group,the expression of Calponin 3 reached the peak until the 7th day..(n=30,*P<0.05 vs Con,**P<0.01 vs Con;#P<0.05 vs Pilo,##P<0.01 vs Pilo).Conclusion: After the seizure induced by pilocarpine,the Rho/ROCK2 signal is activated and the neurons will start a negative fee dback mechanism of self repair.The expression of F-actin binding protein Calponin3 increased,and may also be phosphorylated by pho-ROCK2,recognize and bind to F-acitn,promote the F-acitn structure stability and rearrangement,promote dendritic spine growth and repair injured neurons. |
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