Expression Changes And Mechanism Of Microtubules At Postsynaptic Density In Rats With Temporal Lobe Epilepsy

Part One The down-regulation of microtubules at postsynaptic density in rats with temporal lobe epilepsyObjective To investigate the expression change of proteinsα-tubulin andβ-tubulin at postsynaptic density(PSD) in rats with temporal lobe epilepsy(TLE).Method 200 male Sprague-Dawley(SD) rats were randomly divided into control group and TLE group.The lithium-pilocarpine model of TLE was established.,The whole brain was collected to isolate the PSD at 1d,3d,7d,15d and 30d after SE.First,the expression of proteinsβ-actin,β-tubulin and GAPDH were detected using Western Blot to define the internal control of Western Blot analysis of proteins at PSD. Then,Western Blot and immuno-electron microscope analysis were used to detect the expression of microtubule proteinsα-tubulin andβ-tubulin at PSD.Results 1.The lithium-pilocarpine model of TLE:SE was induced at the achievement ratio of 91.1%and total mortality of 20.6%.2.Purity of PSD:The proteins NR1 and PSD-95 were all expressed in P2 fraction,synaptosome and PSD fraction,but Synphy protein was absent in PSD fraction.3.Definition of internal control in western blot analysis of rat PSD with TLE:The proteinsβ-actin,β-tubulin and GAPDH were all expressed in control group.β-actin andβ-tubulin were decreased from 1d after SE, and were slowest on 7d after SE(P＜0.01).Compared to on 7d after SE, the two proteins were increased on 15d and 30d after SE,but still less than control group(P＜0.01).The expression of GAPDH in TLE group was no significant difference compared to control group(P＞0.05).After then, we randomly chose two PSD samples from control group and 15-day SE group.Each sample were loaded on six lanes.Our results showed that the expression ofβ-actin andβ-tubulin were significantly lower in six lanes of the SE group than in the control group(P＜0.01).Whereas the expression of GAPDH had no difference in two samples(P＞0.05).It was indicated that the variation ofβ-actin andβ-tubulin expression was a result of TLE instead of loading error.4.The expression ofα-tubulin andβ-tubulin at PSD①the expression ofα-tubulin andβ-tubulin were markly decreased from the time point of 1 day after SE,and were lowest on 7-day after SE(P＜0.01). The protein levels on 15-day and 30-day after SE were higher than on 7-day after SE,but still largely lower than in control group(P＜0.01).②Immuno-electron microscopy showed that in CA1 part of hippocampus in control group,β-tubulin was expressed in cytoplasm and synapse including presynapse,synaptic cleft and PSD.Our study emphasized on the change of expression ofβ-tubulin at PSD.Immunogold labeled particles ofβ-tubulin were markly decreased from 3-day after SE,and decreased lowest on 7-day after SE(P＜0.01).And then the expression was increased from 15-day after SE compared to on 7-day after SE,but still less than in control group(P＜0.01).Conclusion 1.GAPDH was more suitable for the internal control in western blot analysis of rat PSD with TLE.2.Microtubulesα-tubulin andβ-tubulin may be one of the mechanism in the formation of temporal lobe epilepsy. Part Two Effect of microtubules at postsynaptic density on spontaneous seizure of rat with temporal lobe epilepsyObjective To investigate the effect of microtubulesα-tubulin andβ-tubulin at postsynaptic density(PSD) on recurrent spontaneous seizure of rat with temporal lobe epilepsy(TLE).Method 236 male SD rats were randomly used to set up lithium-pilocarpine model of TLE and decided into control group,TLE with recurrent spontaneous seizure(SRS) group and TLE with no recurrent spontaneous seizure(NSRS) group.The whole brain was removes to isolate the PSD.The expression ofα-tubulin andβ-tubulin was detected with western blot and immuno-eletron microscopic analysis.Results 1.The lithium-pilocarpine model of TLE SE was induced at the achievement ratio of 91.1%,total mortality of 20.6%and spontaneous seizure ration of 50.5%.EEGs of rats in TLE-SRS group were abnormal,presenting moreθwave,single or multiple spikes and groups of spikes.EEGs of rats in TLE-NSRS group were normal or mild abnormal,with sporadic spikes and sharp waves.2.Niss1 staining Compared with the control group,the numbers of surviving neurons in regions of CA1,CA3 and dentate gyms were markedly decreased in TLE-SRS group(p＜0.01).We found that the neurons with disorder,cell swelling or shrinkage,irregular border and decrease of Nissl bodies in cytoplasm.The surviving neurons in TLE-NSRS group were markly decreased compared to control group (p＜0.01),but more than in TLE-SRS group(p＜0.01).3.The expressions ofα-tubulin andβ-tubulin at PSD①Compared to control group,the expressions ofα-tubulin andβ-tubulin at PSD in TLE-SRS group and TLE-NSRS group were largely decreased(p＜0.01). And the expressions of these proteins in TLE-SRS group were also lower than in TLE-NSRS group(p＜0.01).②Immuno-eletron microscopy analysis The immunogold labeled particles ofβ-tubulin at PSD were significantly decreased in TLE-SRS and TLE-NSRS group compared to in control group(p＜0.01).And the particles in TLE-SRS group were also less than in TLE-NSRS group(p＜0.01).Conclusion expression of microtubules may be negatively related to recurrent spontaneous seizures of temporal lobe epilepsy. Part Three Effect of microtubule stability on temporal lobe epilepsyObjective To observe the effect of intervention in microtubule stability on temporal lobe epilepsy.Method①75 male SD rats were randomly used to make stereotactic infusion of hippocampus with different doses of cochicine and paclitaxol respectively and divided into control group, cochicine-treated group and paclitaxel-treated group.Nissl staining and detection ofβ-tubulin with immnohistochemistry were made to define the suitable infusion dose of the two drug.②60 male SD rats were randomly set up lithium-pilocarpine model of TLE.At the time point of 7-day after SE,the suitable dose of cochincine and paclitaxel were stereostacticly infused hippocampally respectively,which were divided into TLE-control group,TLE-cochincine group and TLE-paclitaxel group.We observed rats in three group behaviourally for 30 days,and 30-day after SE we made EEG of rats.Then each group was divided into spontaneous recurrent seizure(SRS)group and no spontaneous recurrent seizure (NSRS)group again according to whether or not the rats had recurrent spontaneous seizures.Immuno-electron microscopy was used to detect the expression ofβ-tubulin.Results 1.The definition of suitable infusion dose of cochicine①Nissl staining:compared to control group,the surviving neurons were no significant difference in cochicine-treated group with the infusion dose of 0.01,0.03,0.1,0.3ug(p＞0.05),and were markly decreased with the dose of 1.0 and 2.0ug(p＜0.01).②Immunohistochemistry:β-tubulin was largely expressed in cytoplasm in control group.The expression ofβ-tubulin was no difference in cochicine-treated group with the dose of 0.01,0.03,0.1ug compared to control group and was markly decreased from the dose of 0.3ug(p＜0.01).③Our results indicated that the suitable infusion dose of cochicine was 0.3ug,which induced microtubule depolymerization but did not affect the survival of neurons.2.The definition of suitable infusion dose of paclitaxol①Nissl staining:compared to control group,the surviving neurons were no significant difference in paclitaxel-treated group with the infusion dose of 0.1,0.5,1.0ug(p＞0.05),and were markly decreased with the dose of 1.5ug(p＜0.01).②Immunohistochemistry:Compared to control group,The expression ofβ-tubulin was no difference in paclitaxel-treated group with the dose of 0.1,0.5ug and was increased in the dose of 1.0ug(p＜0.01),but markly decreased in the dose of 1.5ug(p＜0.01).③Our results indicated that the suitable infusion dose of paclitaxel was 1.0ug,which induced microtubule polymerization but did not affect the survival of neurons.3.In cochicine 0.3ug -treated group and paclitaxel 1.0ug-treated group,the behavior and EEG recording of rats were no difference compared to control group,indicating that these drugs had no epileptogenesis.4.The recurrent spontaneous seizures ratio of TLE-control group was 75%,TLE-cochicine group 90%and TLE-paclitaxel group 40%. EEGs of rats in TLE-SRS group were abnormal,presenting moreθwave,single or multiple spikes and groups of spikes.EEGs of rats in TLE-NSRS group were normal or mild abnormal,with sporadic spikes and sharp waves.Compares to TLE-SRS group,EEG of rats in TLE-cochicine SRS group were no difference and EEG in TLE-paclitaxel SRS group had less spike and sharp waves,of which frequency and amplitude were decreased.There were no significant difference in TLE-cochicine NSRS group or TLE-paclitaxel NSRS group compared to TLE-NSRS group. 5.The expression ofβ-tubulin at PSD Compared to TLE-SRS group,the immunogold labeled particles ofβ-tubulin at PSD in TLE-cochicine SRS group were no difference(p＞0.05) and particles in TLE-paclitaxel SRS group largely increased(p＜0.01).Compared to TLE-NSRS group,the expression ofβ-tubulin in TLE-cochicine NSRS and TLE-paclitaxel NSRS group was no significant difference(p＞0.05).Conclusion 1.Abnormality of microtubule stability is the predisposing factor of temporal lobe epilepsy.2.microtubule polymerized agent may prevent temporal lobe epilepsy.