The Dynamic Changes Of GSK-3β In The Temporal Lobe Epilepsy Hippocampus And The Potential Roles Of Its Inhibitor In Mossy Fiber Sprouting

Part One The dynamic changes of GSK-3β expression and activity in the pentylenetetrazole kindling rat hippocampusObjective To observe the dynamic changes of GSK-3β, P-GSK-3β (ser9), P-tau (ser396) protein expression in rat hippocampus during the pentylenetetrazole kindling Process, and to investigate the role of GSK-3β in the course of mossy fiber sprouting.Method Male healthy Sprague-Dawley (SD) rats (n=135, age,6-8weeks) were divided randomly into experimental group (n=90) and control group (n=45). The experimental group rats were intraperitoneally injected PTZ (30mg/kg) every24hours and stopped injection once were kindled. The control rats were intraperitoneally injected0.9%saline at the same time. The experimental and control group rats were equally divided into5subgroups at time points3day、1week、2weeks、4weeks and6weeks after the first PTZ injection, Each subgroup was subsequently divided into3panels (6rats in each panel) for the following tests respectively:(1) Timm staining and score;(2) The expression of GSK-3β, its inactive form P-GSK-3β (ser9) and downstream phosphorylated substrate P-tau (ser396) protein in rats’hippocampus by Western blot;(3)The Expression and distribution changes of the aforementioned three indicators were analyzed at different time in the dentate gyrus and the CA1, CA3regions of hippocampus by immunohist-ochemistry.Results1.94.7%rats in the experimental group were kindled, the mortality is2.1%. All the over kindled rats were developed spontaneous recurrent seizures. The control rats have not seizures. 2. There were no obvious mossy fiber sprouting in the dentate gyrus and CA3area of the control rats at different times. In the experimental group the Timm scores in the CA3region, which gradually increased from3d to6w, and maintained high level in the4w,6w groups (P>0.05). the Timm scores of the experimental group were significantly higher than that in controls at different times (P<0.05).There were no significantly differences in supragranular region between the PTZ treated groups and the control groups at different time points (P>0.05)3. Western blot results showed that:In the experimental group, the expression of GSK-3β protein gradually increased from3d after the first PTZ injection, reached the peak at2w and decreased to normal level at6w. the change trend of GSK-3β is the opposite of its inactive form P-GSK-3β (ser9) which was gradually decreased from3d, reached a minimum at2w and increased to normal level at6w. There were obvious difference in expression of GSK-3β and P-GSK-3β (ser9) between the experimental group and control group at all the time points (P<0.05) with the exception of6w(P>0.05). The expression of P-tau(ser396) protein gradually increased from3d after the first PTZ injection in the experimental group and reached the peak at6w. But there were no obvious difference between2w,4w and6w. the protein level at all the time points were higher than the control group (P<0.05)4. Immunohistochemical results showed that:The expression change trends of GSK-3(3, P-GSK-3β (ser9), P-tau (ser396) protein were consistent with the Western blot results. But the distribution changes of each indicator in various regions of the hippocampus is different, the biggest Changes difference was CA3(P＜0.05), followed by the DG (P<0.05), there was no obvious difference in CA1(P>0.05)between the experimental group and the control group.Conclusion1.GSK-3β may be involved in the progression of MFS through abnormal phosphorylation of tau protein. 2.The change of GSK-3β expression in CA3region might play a more important role in the onset of epilepsy than that in dentate gyrus.Part Two Effect of intervention in GSK-3β on mossy fiber sprouting and temporal lobe epilepsy formation and developmentObjective To investigate the effect of intervention in GSK-3(3activity on tau protein phosphorylation level and mossy fiber sprouting.Method Male healthy Sprague-Dawley (SD) rats (n=180; age,6-8weeks) were divided randomly into the PTZ+SB216763intervention group (n=90) and PTZ+DMSO experimental group (n=90). The two groups rats were intraperitoneally injected PTZ (30mg/kg) every24hours and stopped injection once were kindled. Rats in PTZ+SB216763intervention group received intracerebroventricular injection of Selective inhibitor of GSK-3(3SB216763on2d、4d、6d after the first PTZ injection; the PTZ+DMSO experimental group rats received intracerebroventricular injection of DMSO solvent was diluted with normal saline at the same time points.The two group rats were equally divided into5subgroups at time points3day、1week、2weeks、4weeks and6weeks after the first PTZ injection, Each subgroup was subsequently divided into3panels (6rats in each panel) for the following tests respectively:(1) Timm staining and score;(2) The expression of GSK-3β, its inactive form P-GSK-3β (ser9) and downstream phosphorylated substrate P-tau (ser396) protein in rats’hippocampus by Western blot;(3)The Expression and distribution changes of the aforementioned three indicators were analyzed at different time in the dentate gyrus and the CA1, CA3regions of hippocampus by immunohistochemistry.Results1. The latency to the first seizure induced by subconvulsive dose of PTZ in the intervention group was8days, but the latency in experimental group was5days. The experimental group rats were in accordance with the kindling criterion21.4±1.8days following PTZ injection. The intervention group rats were in accordance with the kindling criterion26.4±1.2days. In intervention group the successful rate of the model was82.6%, but91.4%in the experimental group. The above data are present statistically significant differences (P<0.01). In intervention group and experimental group the mortality rate were6.8%and7.9%, have no significant statistically differences (P>0.05). However, compared to the mortality2.1%of the PTZ treated rats on the first part, there were significant differences (P<0.01). All the over kindled rats in experimental group were developed spontaneous recurrent seizures at stage Ⅱ-Ⅳ. While the the over kindled rats in intervention group only50%rats were developed spontaneous recurrent seizures at stage Ⅱ-Ⅲ. There were significantly statistical differences (P<0.01). The were no Spontaneous seizures at stage V or status epilepticus in the two groups.2. There were Timm granules in the CA3region since3d in the experimental group, and gradually increased. while there no Timm granules in the CA3region on3d in the intervention group, only punctate Timm granules could be seen in1w, and gradually increased. Compared with the experimental group, the degree of sprouting in the intervention group was markedly slighter (P<0.05). There was no obvious difference in Timm scores in dentate gyrus between the two groups (P>0.05).3. The change trends of GSK-3β total expression in the two groups by western blot is accordance with immunohistochemical results. In the experimental group, it was markedly up-regulated on3d after the first PTZ injection, increased gradually reached the peak in2w, and then was down-regulated since4w, Compared with intervention group, there were no obvious differences at all the time points (P>0.05) except that markedly down-regulated in4w in the intervention group (P<0.05). Compared with experimental group, the expression of P-GSK-3β(ser9) in the intervention group was markedly up-regulated on3d (P<0.01), decreased gradually since1w, reached the lowest in2w, increased gradually since4w and reached the normal level in6w. There were obvious differences at all the time points(P<0.05) except for6w(P>0.05).The expression of p-tau (ser396) in the both group rats all showed increasing trends. But compared with experimental group, the degree of increasing trends in the intervention group was markedly slighter (P<0.05). The expression of p-tau (ser396) on3d even lower than normal level, there was obvious differences between the two groups at this time point (P<0.01).Conclusion1. Intervention in the activity of GSK-3β may delay the formation and development of temporal lobe epilepsy and decrease the severity of seizure.2. Intervention in the activity of GSK-3β can inhibit mossy fiber sprouting, possibly through down-regulating the tau protein phosphorylation levels.Part Three Effect of intervention in GSK-3β on related genes in hippocampus of temporal lobe epilepsyObjective To observe the gene expression change of GSK-30, Its upstream regulator PTEN, AKT1and downstream phosphorylated substrate MAPT in the pentylenetetrazole kindling rat hippocampus, and to investigate the role of GSK-3β signaling pathway in the onset of temporal lobe epilepsy.Method Male healthy Sprague-Dawley (SD) rats (n=50; age,6-8weeks) were divided randomly into3groups:the PTZ+SB216763intervention group(n=20), the PTZ+DMSO experimental group (n=20) and the DMSO+SB216763control group (n=10). The three group rats were equally divided into5subgroups at time points3day、1wee、2weeks、4weeks and6weeks after the first PTZ injection,4rats in each subgroups of experimental and intervention group,2rats in each subgroups of control group. Rats in experimental and intervention group rats were treated by intraperitoneal injection of PTZ (30mg/kg) every24hours and stopped injection once were kindled. The control group rats were intraperitoneally injected0.9%saline at the same time. Rats in intervention and control group received intracerebroventricular injection of Selective inhibitor of GSK-3(3SB216763on2d、4d、6d after the first PTZ injection; the experimental group rats received intracerebroventricular injection of DMSO solvent was diluted with normal saline at the same time points. To observe the gene expression change of GSK-3β,ts upstream regulator PTEN, AKTl and downstream phosphorylated substrate MAPT in the pentylenetetrazole kindling rat hippocampus at different time points by RT-PCR.Results1. Compared with DMSO+SB216763control group, the expression of GSK-3β in the PTZ+DMSO experimental group was markedly up-regulated on3d, reached the peak in1w, decreased gradually since4w and reached the normal level in6w, there were obvious differences at all the time points(P<0.05) except for6w(P>0.05). Compared with PTZ+DMSO experimental group, the expression of GSK-3β in the PTZ+SB216763intervention group was markedly down-regulated in4w(P<0.05), there were no obvious differences at the other time points(P>0.05).2. Compared with DMSO+SB216763control group, the expression of AKT1(upstream positive regulatory factor of GSK-3β) in the PTZ+DMSO experimental group was markedly up-regulated on3d,1w,6w(P<0.05). there were no obvious differences in2w and4w(P>0.05). Compared with PTZ+DMSO experimental group, the expression of GSK-3β in the PTZ+SB216763intervention group was markedly down-regulated in6w(P<0.05), there were no obvious differences at the other time points(P>0.05).3. Compared with DMSO+SB216763control group, the expression of PTEN(upstream negative regulatory factor of GSK-3β) in the PTZ+DMSO experimental group was markedly up-regulated on3d, reached the peak in lw, decreased gradually since2w and reached the lowest in6w even lower than normal level1there were obvious differences between the two groups(P<0.05) except for4w(P>0.05). Compared with PTZ+DMSO experimental group, the expression of GSK-3β in the PTZ+SB216763intervention group was markedly down-regulated in6w(P<0.05), there were no obvious differences between the experimental group and the intervention group at all the time points(P>0.05).4. Compared with DMSO+SB216763control group, the expression of MAPT(downstream phosphorylated substrate of GSK-3(3) in the PTZ+DMSO experimental group was up-regulated on3d, gradually increased and reached the peak in4w, decreasing reached the normal level in6w. there were obvious differences between the two groups(P<0.05) except for6w(P>0.05). Compared with PTZ+DMSO experimental group, the expression of MAPT in the PTZ+SB216763intervention group also showed increasing trends. But the degree of increasing trends in the intervention group was markedly slighter(P<0.05). there were obvious differences between the two groups at the all time points (P＜0.05) except for6w (P>0.05).Conclusion1. The gene expression changes of PTEN, AKT in temporal lobe epilepsy, in which GSK-3β signaling pathway may involve.2. Intervention in the activity of GSK-3β during the process of mossy fiber sprouting, can inhibit the gene expression of Tau and down-regulating its protein phosphorylation levels.