| Part I:p-tau (Ser262) expression profile in epiletic rat hippocampus and its correlation with aberrant mossy fiber sprouting in hippocampusAbstractObjective:To explore the p-tau and total tau expression profile in rat hippocampus in temporal lobe epilepsy.Methods:150adult (6-8weeks of age), healthy male SD rats (180-220g) were divided randomly into PTZ group and control group, and divided randomly again into five time points:3d,1w,2w,4w, and6w. The chronic PTZ kindling model was induced by intraperitoneal injection of PTZ in PTZ group, while the controls were injected with equal dose of saline. After drug injection, experimental animals were monitored for a minimum of2hours to assess the severity and duration of the behavioral seizures and Camera for recording the behavioral activity of all animals were performed. Immunohistochemistry, Western blot and quantitative PCR were used to detect the expression of p-tau and total tau. Timm stain was used to detect mossy fiber sprouting.Results:1. The PTZ kindling model was successfully constructed and the samples used in experiment were gained. 2. Immunohistochemist showed the expression of p-tau (Ser262) significantly increased at1and2weeks, and reached its peak at4weeks, then reduced at6weeks (p<0.001). While the total tau show no significant difference at each time point between control and PTZ groups by immunohistochemist. Western blot showed a consistence with immunohisochemist in p-tau (Ser262) protein expression in PTZ group. qPCR demonstrated no significant difference in the expression of total tau mRNA between control and PTZ groups (p>0.05)3. Timm stain showed obvious mossy fiber sprouting at each time point except3days (p<0.05, p<0.01). With the increased times of PTZ injection, the degree of mossy fiber sprouting was correspondingly higher. At4weeks, mossy fiber sprouting reached its peak and maintained this level at6weeks. The parallel time course between p-tau (Ser262) expression pattern and the generation of aberrant mossy fiber sprouting alludes to the importance of p-tau (Ser262) in the formation of aberrant mossy fibers.Conclussions:Mossy fiber sprouting in hippocampus may be the cause of seizures in PTZ kindling model.p-tau (Ser262) involves in epileptogenesis of PTZ kindling model.p-tau (Ser262) may play important role in aberrant mossy fiber sprouting in epileptogenesis. Part Ⅱ The role of protein phosphatase2A in seizures of the PTZ Kindling modelObjective:To explore the role of protein phosphatase2A (PP2A), a regulator of tau phosphorylation, in seizures in the PTZ Kindling model.Methods:200adult (6-8weeks of age), healthy male SD rats (180-220g) were divided randomly into PTZ group, control group, sham-control group and sodium selenate group. The PTZ group and control group were constructed following the protocol used in Part Ⅰ Rats in both sham-control group and sodium selenate group were treated with normal drinking water and drinking water with sodium selenate for one week, respectively. Then all rats were treated with intraperitoneal injection of PTZ (30mg/kg). At the eighth day after the first PTZ injection, rats in sodium selenate group began to drink water with sodium selenate again for4days without stoping PTZ injection. After drug injection, experimental animals were monitored for a minimum of2hours to assess the severity and duration of the behavioral seizures. First, immunohistochemistry, Western blot and qPCR were performed to examin the expression pattern of PP2A、Methyl-PP2A and p-PP2A in PTZ model. Second, immunohistochemistry and Western blot were used to detect the expression of Methyl-PP2A and p-PP2A in hippocampus in sodium selenate group. Results:1. Rats treated with sodium selenate exhibited significantly reduced severity of PTZ-kindled seizures. The kindling rate was25%in sodium selenate group, while94%in sham-control group.2. Immunohistochemist showed a significant upregulation of p-PP2A expression at each time point except3days in PTZ group compared with control (p<0.05, p<0.01), while the expression of total PP2A protein showed no significant difference at each time point in PTZ group compared with control (p>0.05). Western blot verified the activity change of PP2A by examining the expression of p-PP2A and Methyl-PP2A in PTZ group. The PP2A expression in PTZ group was confirmed by qPCR.3. Sodium selenate can increase the expression of Methyl-PP2A significantly, reduce the expression of p-PP2A significantly at1wee、2weeks and4weeks in rat hippocampus in PTZ kindling model (p<0.05,)Conclussion:1. The decreased activity of PP2A plays important role in epileptogenesis in PTZ kindling model.2. Sodium selenate suppresses seizures in PTZ kindling model, suggesting that the changed activity of PP2A is related to severity of seizures in PTZ kindling model. Part Ⅲ The role of protein phosphatase2A in hippocampal mossy fiber sprouting of the PTZ Kindling modelObjective:To further explore the role of protein phosphatase2A (PP2A) in regulating expression of p-tau (Ser262) in hippocampus, and in hippocampal mossy fiber sprouting of the PTZ Kindling model.Methods:72adult (6-8weeks of age), healthy male SD rats (180-220g) were divided randomly into sham-control group and sodium selenate group, and divided randomly again into three time points:1week,2weeks,4weeks. The two models were constructed following the protocol used in Part Ⅱ. Immunohistochemist and Western blot were used to detect the expression of p-tau (Ser262). Timm stain was used to detect mossy fiber sprouting.Results:1. Both immunohistochemist and Western blot showed a significant decline of p-tau (Ser262) expression at each time point examined in sodium selenate group compared with sham-control group (p<0.05)2. Sodium selenate significantly lower the degree of aberrant mossy fiber sprouting at each time point in rat hippocampus compared with sham-control group (p<0.05, p<0.01)Conclussion:The decreased activity of PP2A promotes hippocampal mossy fiber sprouting in the PTZ pindling model by hyperphosphorylation of tau, which may be the cause of the relationship between PP2A and the severity of seizures of temple lobe epilepsy. |
PDF Full Text Request