Establish The Rat Model Of Refractory Epilepsy And Investigate The Rule Of P-glycoprotein Expression

ObjectiveTo establish and research the rat model of refractory epilepsy and investigate the rule of P-glycoprotein expression to make the mechanism of drug resistance clear. MethodsFifty-four male Wistar rats were divided into control group (group I), kainic acid epileptogenesis group (group II) and AEDs-treating group (group III). The EEG of all rats were observed by embedding electrodes. In group II, the rats were sacrificed at 2h, 6h, 24h, 3d, 1w, 2w 和 1m after KA injection, respectively. In group III, all rats were injected valproate intraperitoneally for 2 weeks after estabablishing the epilepsy model, three of which were chosen randomly for continuing valproate treating and behavior and EEG observing, the rest of which were sacrificed at the set time. All the rat brain samples were examined by HE, Nissl dyeing and the expression of GFAP and P-gp were inspected semi-quantitively by using immumohistochemistry with integrated optical density. Results1 The temporal complex partial epilepsy model was caused by KA injection, which was proved by behavior, EEG, pathology and immumohistochemistry studies. ①Automatism and complex partial epilepsy exhibition were seen after KA injection and developed into tonic-clonic seizure subsequently. After lh, the persistent status epilepticus (SE) was put down by intramuscularly injecting diazepam. The rat developed into silent period after lor 2 days and then into spontaneous recurrent seizures (SRS) after 2 weeks. In EEG recording, we found that spikes emerged more and more obviously as compared with background in ipsilateral CA3 about 5-20 min after KA injection, meantimes or subsequently the spikes spreadin other limbic system, cortexes and contralateral regions and then the epileptiform activity changed into rhythmic slow wave activity. During SE, periodic paroxysmal activity was observed and injecting diazepam inhibited the epileptiform activity. After 1 or 2 days, EEG activity came into silent period until after 2 weeks or so, paroxysmal spikes occurred frequently. When EEG information was analysed using linear dynamics, the overall and each frequency power spectrum all increased obviously immediately after KA injection (p<0.05), in which we could not found significancy from 0.5h to 6h(p>0.05) and among different seizure levels (p>0.05). During SE, the overall and each frequency power spectrum arised periodically as with each seizure (p<0.05), while it declined during silent period (p<0.05 ) until the coming of SRS when it had a tendency of going up again (p>0.05). When EEG information was analysed using nonlinear dynamics, the sample entropy had a tendency of decreasing immediately after KA injection(p>0.05 ), in which we could not found significancy from 0.5h to 6h (p>0.05) and among different seizure levels(p>0.05). During SE, the sample entropy of seizure came down periodically as compared with after-seizure (p<0.05) and had a tendency of dropping as compared with before-seizure (p>0.05), while it arised during silent period (p<0.05 ) until SRS when it dropped again (p<0.05). (3) HE dyeing showed that obvious histopathological changes were seen in ipsilateral CA3, which exhibited severe cell degeneration and necrosis, while in CA1, DGand contralaterl, the changes were much less. After acute seizure, the CA3 displayed the loss of its layers gradually and presented reactive gliosis, and ultimately showed hippocampal sclerosis. No histopathological changes were seen in control group. Nissl dyeing displayed that the number of the neuron cell in ipsilateral CA3 declined gradually. ?The astrocyte showed hyperplasia after KA injection by using IHC. weclassified group III as RE model when it eventually presented with spikes in EEG. In control group, epileptiform behavior and EEG activity did not emerge.2 Immunohistochemistry presented that the weak P-gp reactive positive cell appeared about 2h in bilateral hippocampus and thalamencephalon after KA injection, and the degree of immunohistochemistry reactive positive intensified obviously in 6h and 24h in ipsilateral CA3 but has no significance between them(p>0.05), both of which were stronger than that of 2h (p<0.05), while it disappeared in 3d and did not appear in SRS. The degree of immunohistochemistry reactive positive in microvascular endothelial cell between group II and group III was with significant difference (p<0.05 ). Conclusion(1) The rat model of refractory epilepsy could be established successfully by injecting KA in the ventra hippocampus CA3.(2) SE could strongly induce P-gp expression in HP pyramidal cell while valproate can do the same in the microvascular endothelial cell.(3) Combing power spectrum and sample entropy can describe the developing of rat refractory epilepsy and analyse the EEG in different state quantificationally, which is seldom reported.