Objective:To investigate the regulatory role and mechanism of synaptic vesicle protein 2A (SV2A) in apoptosis of drug-resistant epilepsy neurons.Methods:One hundred and fifty specific pathogen-free (SPF) Sprague-Dawley (SD) rats were randomly numbered; 20 rats were selected as a normal control group (NC group), and the remaining 130 rats were subjected to chronic epilepsy models of lithium-pilocarpine. Phenobarbital and phenytoin sodium for 2 weeks were given to these rats after modeling; fanally, 94 rats with chronic epilepsy were divided into a drug-resistant group (phenobarbital and phenytoin sodium-resistant epilepsy [PRE] group, reduction in episodes<50%, n= 55) and a drug-sensitive group (phenobarbital and phenytoin sodium-sensitive epilepsy [PSE] group, reduction in episodes by≥50%, n=38). Rats in the PRE group were further randomly divided into 5 groups, namely a up-regulated-SV2A PRE group (UPRE group), a down-regulated-SV2A PRE group (DPRE group), a up-regulated-SV2A control group (UPRC group), a down-regulated-SV2A control group (DPRC group), and a non-transfected PRE group; different types of lentivirus were given to the first 4 groups via stereotactic brain injection. Ten days after lentiviral transfection, virus detection was performed; and 2 weeks after lentiviral transfection, occurrence of epileptic seizures was observed, and after that, rats were sacrificed and the hippocampal tissues were collected for subsequent experiments. Western blotting was used to detect the expressions of SV2A, cyclophilin A (CyPA), apoptosis-inducing factor (AIF), and superoxide dismutase 2 (SOD2) in the cell nucleus or mitochondria; coimmunoprecipitation experiment was performed to observe the interaction among SV2A, CyPA and AIF proteins; immunofluorescent co-staining was used to observe the CyPA/AIF localization; mitochondrial damage was detected by electron microscopy; ATP content in the hippocampal tissues was detected by luciferase method, 8-hydroxy-2-deoxyguanosine (8-OHDG) expression was detected by immunofluorescent staining, and neuronal apoptosis rate was calculated by double staining with neuron-specific nuclear protein (NeuN) and TUNEL. Results:(1) Confocal microscopy revealed that the hippocampal tissues in the 4 transfected groups showed green fluorescence inherent to the lentivirus, indicating successful viral infection. Compared with the UPRC group, the UPRE group had significantly reduced frequency of epileptic seizures and seizure duration ( P<0.05). (2) Western blotting showed that, in mitochondria, the UPRE group had significantly higher expressions of SV2A (0.475± 0.105 vs. 0.136±0.043), CyPA (0.473±0.041 vs. 0.175±0.047), AIF (0.443±0.058 vs. 0.131±0.037), and SOD2 (0.457±0.037 vs. 0.152±0.038) compared with the UPRC group ( P<0.05); the DPRE group had significantly decreased expressions of SV2A (0.038±0.013 vs. 0.184±0.047), CyPA (0.041±0.010 vs. 0.214±0.040), AIF (0.040±0.019 vs. 0.175±0.046), and SOD2 (0.043±0.017 vs. 0.187±0.039) compared with the DPRC group ( P<0.05). In the cell nucleus, the UPRE group had significantly lower expressions of AIF (0.336±0.084 vs. 0.649±0.209) and CyPA (0.331±0.086 vs. 0.620±0.162) compared with the UPRC group ( P<0.05); the DPRE group had statistically higher expressions of AIF (0.771± 0.180 vs. 0.519±0.144) and CyPA (0.738±0.223 vs. 0.488±0.091) compared with the DPRC group ( P< 0.05). (3) Co-immunoprecipitation experiment results showed that the bidirectional precipitation results of SV2A and AIF, SV2A and CyPA, and AIF and CyPA were all positive, suggesting existence of interactions. (4) Immunofluorescent co-staining showed that the fluorescence changes of CyPA and AIF were consistent. (5) Electron microscopy showed that mitochondria in the NC group had intact structure; mitochondria in the UPRE group, UPRC group, DPRC group and DPRE group showed swelling and cristae fragmentation; among them, injury in the UPRE group was relatively less severe than that in the UPRC group, while that in the DPRC group was more severe than that in the DPRE group. (6) Compared with the UPRC group, the UPRE group had statistically higher ATP content ( P<0.05); compared with the DPRC group, the DPRE group had significantly lower ATP content ( P<0.05).(7) Immunofluorescent staining results showed that the UPRE group had significantly lower 8-OHDG expression than the UPRC group ( P<0.05); compared with the DPRC group, the DPRE group had statistically higher 8-OHDG expression ( P<0.05). (8) The UPRE group had significantly lower NeuN-TUNEL double staining positive rate than the UPRC group ( P<0.05); compared with the DPRC group, the DPRE group had significantly higher NeuN-TUNEL double staining positive rate ( P<0.05). Conclusion:SV2A can play a role in the apoptosis of hippocampal neurons in rats with drug-resistant epilepsy by regulating the nuclear translocation of AIF/CyPA and mitochondrial damage.

Objective:To investigate the regulatory role and mechanism of synaptic vesicle protein 2A (SV2A) in apoptosis of drug-resistant epilepsy neurons.Methods:One hundred and fifty specific pathogen-free (SPF) Sprague-Dawley (SD) rats were randomly numbered; 20 rats were selected as a normal control group (NC group), and the remaining 130 rats were subjected to chronic epilepsy models of lithium-pilocarpine. Phenobarbital and phenytoin sodium for 2 weeks were given to these rats after modeling; fanally, 94 rats with chronic epilepsy were divided into a drug-resistant group (phenobarbital and phenytoin sodium-resistant epilepsy [PRE] group, reduction in episodes<50%, n= 55) and a drug-sensitive group (phenobarbital and phenytoin sodium-sensitive epilepsy [PSE] group, reduction in episodes by≥50%, n=38). Rats in the PRE group were further randomly divided into 5 groups, namely a up-regulated-SV2A PRE group (UPRE group), a down-regulated-SV2A PRE group (DPRE group), a up-regulated-SV2A control group (UPRC group), a down-regulated-SV2A control group (DPRC group), and a non-transfected PRE group; different types of lentivirus were given to the first 4 groups via stereotactic brain injection. Ten days after lentiviral transfection, virus detection was performed; and 2 weeks after lentiviral transfection, occurrence of epileptic seizures was observed, and after that, rats were sacrificed and the hippocampal tissues were collected for subsequent experiments. Western blotting was used to detect the expressions of SV2A, cyclophilin A (CyPA), apoptosis-inducing factor (AIF), and superoxide dismutase 2 (SOD2) in the cell nucleus or mitochondria; coimmunoprecipitation experiment was performed to observe the interaction among SV2A, CyPA and AIF proteins; immunofluorescent co-staining was used to observe the CyPA/AIF localization; mitochondrial damage was detected by electron microscopy; ATP content in the hippocampal tissues was detected by luciferase method, 8-hydroxy-2-deoxyguanosine (8-OHDG) expression was detected by immunofluorescent staining, and neuronal apoptosis rate was calculated by double staining with neuron-specific nuclear protein (NeuN) and TUNEL. Results:(1) Confocal microscopy revealed that the hippocampal tissues in the 4 transfected groups showed green fluorescence inherent to the lentivirus, indicating successful viral infection. Compared with the UPRC group, the UPRE group had significantly reduced frequency of epileptic seizures and seizure duration ( P<0.05). (2) Western blotting showed that, in mitochondria, the UPRE group had significantly higher expressions of SV2A (0.475± 0.105 vs. 0.136±0.043), CyPA (0.473±0.041 vs. 0.175±0.047), AIF (0.443±0.058 vs. 0.131±0.037), and SOD2 (0.457±0.037 vs. 0.152±0.038) compared with the UPRC group ( P<0.05); the DPRE group had significantly decreased expressions of SV2A (0.038±0.013 vs. 0.184±0.047), CyPA (0.041±0.010 vs. 0.214±0.040), AIF (0.040±0.019 vs. 0.175±0.046), and SOD2 (0.043±0.017 vs. 0.187±0.039) compared with the DPRC group ( P<0.05). In the cell nucleus, the UPRE group had significantly lower expressions of AIF (0.336±0.084 vs. 0.649±0.209) and CyPA (0.331±0.086 vs. 0.620±0.162) compared with the UPRC group ( P<0.05); the DPRE group had statistically higher expressions of AIF (0.771± 0.180 vs. 0.519±0.144) and CyPA (0.738±0.223 vs. 0.488±0.091) compared with the DPRC group ( P< 0.05). (3) Co-immunoprecipitation experiment results showed that the bidirectional precipitation results of SV2A and AIF, SV2A and CyPA, and AIF and CyPA were all positive, suggesting existence of interactions. (4) Immunofluorescent co-staining showed that the fluorescence changes of CyPA and AIF were consistent. (5) Electron microscopy showed that mitochondria in the NC group had intact structure; mitochondria in the UPRE group, UPRC group, DPRC group and DPRE group showed swelling and cristae fragmentation; among them, injury in the UPRE group was relatively less severe than that in the UPRC group, while that in the DPRC group was more severe than that in the DPRE group. (6) Compared with the UPRC group, the UPRE group had statistically higher ATP content ( P<0.05); compared with the DPRC group, the DPRE group had significantly lower ATP content ( P<0.05).(7) Immunofluorescent staining results showed that the UPRE group had significantly lower 8-OHDG expression than the UPRC group ( P<0.05); compared with the DPRC group, the DPRE group had statistically higher 8-OHDG expression ( P<0.05). (8) The UPRE group had significantly lower NeuN-TUNEL double staining positive rate than the UPRC group ( P<0.05); compared with the DPRC group, the DPRE group had significantly higher NeuN-TUNEL double staining positive rate ( P<0.05). Conclusion:SV2A can play a role in the apoptosis of hippocampal neurons in rats with drug-resistant epilepsy by regulating the nuclear translocation of AIF/CyPA and mitochondrial damage.

Objective:To investigate the behavioral, electroencephalographic, and cognitive functional differences in drug-resistant epileptic rat models of cognitive impairment prepared by intraperitoneal injection of lithium chloride-pilocarpine followed by intracranial injection of pilocarpine or carbamylcholine.Methods:One hundred and sixty adult male SD rats were randomly divided into normal control group ( n=10), lithium chloride-pilocarpine group (establishing epileptic rat models by intraperitoneal injection of lithium chloride-pilocarpine, n=50), pilocarpine-pilocarpine group (intracranial injection of pilocarpine after intraperitoneal injection of lithium chloride-pilocarpine, n=50)and pilocarpine-carbamylcholine group (intracranial injection of carbamylcholine after intraperitoneal injection of lithium chloride-pilocarpine, n=50). Frequency and duration of spontaneously recurrent seizures (SRSs) were observed by video monitoring system, and 2 weeks after that, phenobarbital and phenytoin sodium were injected intraperitoneally to screen drug-resistant models. Frequency and amplitude of the epileptic waves in EEG were recorded by BL-420 Bio-signal Acquisition and Processing System. Novel object recognition experiment was used to detect the novel exploration, Y-maze free exploration experiment and new and different arm experiment were used to detect the spatial recognition and memory ability, and Morris water maze experiment was used to detect the spatial memory ability. Results:(1) Twenty-four rats (48.00%) survived in the lithium chloride-pilocarpine group, 25 (78.00%) in the pilocarpine-pilocarpine group, and 21 (65.62%) in the pilocarpine-carbamylcholine group; and ultimately 7, 9, and 8 drug-resistant epileptic rat models were identified, respectively; frequency and duration of SRSs in the pilocarpine-pilocarpine group and pilocarpine-carbamylcholine group were significantly higher/longer than those in the lithium chloride-pilocarpine group ( P<0.05). (2) The pilocarpine-pilocarpine group and pilocarpine-carbamylcholine group had significantly higher amplitude of the epileptic waves in EEG compared with the lithium chloride-pilocarpine group ( P<0.05); the frequency of the epileptic waves in EEG increased gradually in the lithium chloride-pilocarpine group, pilocarpine-pilocarpine group, and pilocarpine-carbamylcholine group ( P<0.05). (3) Discrimination index, accuracy, ratio of distance traveled in novel arm to total distance, and time of novel arm entries gradually decreased in the normal control group, lithium chloride-pilocarpine group, pilocarpine-pilocarpine group, and pilocarpine-carbamylcholine group, with significant differences ( P<0.05). (4) Compared with the normal control group, the pilocarpine-pilocarpine group and pilocarpine-carbamylcholine group had significantly decreased frequency in crossing the original platform ( P<0.05); compared with the normal control group, lithium-pilocarpine chloride group and pilocarpine-pilocarpine group, the pilocarpine-carbamylcholine group had statistically shorter distance of target quadrant activity ( P<0.05); number of entries in the target quadrant gradually decreased in the normal control group, lithium chloride-pilocarpine group, pilocarpine-pilocarpine group, and pilocarpine-carbamylcholine group, with significant differences ( P<0.05). Conclusion:Drug-resistant epileptic rat models established by intracranial injection of carbamylcholine after intraperitoneal injection of lithium chloride-pilocarpine have high survival rate, high SRSs rate, and severe cognitive impairment, which is suitable for studying drug-resistant epilepsy combined with cognitive impairment.