低频重复经颅磁刺激对癫痫大鼠行为及GAD65、NMDAR1表达的影响
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摘要
目的观察低频重复经颅磁刺激(rTMS)对癫痫大鼠模型痫性发作行为及海马区谷氨酸脱羟酶65(GAD65)、N-甲基-D-天门冬氨酸受体1(NMDAR1)表达的影响,探讨rTMS的抗痫作用及可能机制。
方法选择健康雄性wistar大鼠50只,随机分为磁刺激组(包括0.3Hz刺激组、0.5Hz刺激组、0.8Hz刺激组、1.0Hz刺激组)及假刺激组,共5组,每组10只,各组进行相应预处理后,制作氯化锂-匹罗卡品癫痫急性模型,腹腔注射氯化锂3 mmol/kg,注射20h后腹腔注射东莨菪碱1mg/kg,30min后腹腔注射匹鲁卡品30 mg/kg,观察大鼠行为学变化。Ⅳ级以上痫性发作1h后腹腔注射10%水合氯醛(300mg/kg)终止发作,观察一周后,断头取脑切片,行HE染色及免疫组织化学染色观察海马区GAD65和NMDAR1的表达。
结果rTMS刺激组大鼠刺激期间未见痫性发作及颅内出血征象,各组大鼠注射匹鲁卡品后均出现痫性发作,表现为点头、甩尾、须动、头面抽动、前肢、后肢抽动、全身抽动、窜动、直立等,49只达到Ⅳ级以上发作,其中有2只终止后死亡。rTMS刺激组大鼠(0.3Hz刺激组、0.5Hz刺激组、0.8Hz刺激组、1.0Hz刺激组)痫性发作潜伏期均比rTMS假刺激组延长,差异均有统计学意义(P＜0.05或P＜0.01),其中0.5Hz刺激组最长;0.5Hz刺激组大鼠出现的痫性发作行为次数均比0.3Hz刺激组、0.8Hz刺激组、1.0Hz刺激组少,且整体发作程度轻,差异有统计学意义(P＜0.05)。rTMS刺激各组大鼠海马神经元GAD65的表达量明显高于rTMS假刺激组(均P＜0.05或P＜0.01),NMDAR1则明显低于rTMS假刺激组,差异有统计学意义(P＜0.05或P＜0.01),以0.5Hz刺激组最为明显。
结论适量的低频rTMS是比较安全的,可延缓大鼠痫性发作潜伏期、减少痫性发作次数、减轻痫性发作程度;能够降低癫痫大鼠海马区NMDAR1的表达及增强GAD65的表达,可能与其抗痫作用有关。
Objective Observing the changes of attack behavior and contents of glutamicaciddecarboxylase(GAD)、N-methyl-D-aspartateR1(NMDAR1)in kindled rat models of epilepsy after Low-frequency transcranial magnetic stimulation, to study Low-frequency transcranial magnetic stimulation have anti-epilepsy effect and perhapers how running.
Method Fifty healthy male Wistar rats were randomly divided into five groups with 10 rats in each group: 0.3Hz repetive transcranial magnetic stimulation epilepsy model group、0.5Hz epilepsy model group、0.8Hz epilepsy model group、1.0Hz epilepsy model group and the false repetive transcranial magnetic stimulation group. Making the acute epilepsy model by Lithium-Pilocarpine after pretreatment, all groups use the sub-eclampsia dosage of 3 mol/kg Lithium to abdominal cavity injection, 20 hour late the sub-eclampsia dosage of 1mg/kg methylscopolamine were injected to abdominal, after thirty minutes the sub-eclampsia dosage of 30mg/kg Pilocarpine were injected, observed the performance of epilepsy seizures after intraperitoneal injection of Pilocarpine, and then over the status epileptieus with the sub-eclampsia dosage (300mg/kg) of 10% chloral hydrate, after one week, HE stain and imnmnohistoehemistry stain were applied, study the efects afte repetive transcranial magnetic stimulation on GAD65、NMDAR1 expression in hippocampus of rats.
Results 0.3Hz、0.5Hz、0.8Hz、1.0Hz repetive transcranial magnetic stimulation epilepsy model groups rats are not looking out epilepsy、cerebral hemorrhage evidence, et al. All groups were taking epileptic attack behavior sincluding nodding, tail-flick , beard shaking , anterior, hind limbtic, systemic tic and jump, et al. 49 rats were induced successfully status epileptieus in all, 2 rats were dead. The latency period each of 0.3Hz、0.5Hz、0.8Hz and 1.0Hz groups were prolonger than the false repetive transcranial magnetic stimulation group (P<0. 05 or P<0. 01). The times of epileptic attack behavior in 0. 5Hz group was less than the 0.3Hz、0.5Hz、0.8Hz、1.0Hz repetive transcranial magnetic stimulation epilepsy model groups respectively (P<0. 05 or P<0. 01), and the whole seizure level was less than 0.3Hz、0.5Hz、0.8Hz、1.0Hz repetive transcranial magnetic stimulation epilepsy model groups too(P<0.05). The number of GAD65 expression in hippocampus of all repetive transcranial magnetic stimulation groups rats were lower than the false repetive transcranial magnetic stimulation group Rats(P<0. 05 or P<0. 01), NMDAR1 expression was higher than that (P<0. 05 or P<0. 01), especially that of 0. 5Hz groups were evidence.
Conclusion Appropriate low-frequency transcranial magnetic stimulation is safety, it could prolong the latent period of epilepsy seizures, decreased the times of epilepsy attack and diminish the symptom level of epilepsy attack; It can efectively decreased the content of excitatory amino acid Glu's receptor-NMDAR1 in hippocampus, at the same time, it can also enhance the expression of in hippocampus acid's receptor- GAD, that perhapers the one role of anti-epilepsy.
方法选择健康雄性wistar大鼠50只,随机分为磁刺激组(包括0.3Hz刺激组、0.5Hz刺激组、0.8Hz刺激组、1.0Hz刺激组)及假刺激组,共5组,每组10只,各组进行相应预处理后,制作氯化锂-匹罗卡品癫痫急性模型,腹腔注射氯化锂3 mmol/kg,注射20h后腹腔注射东莨菪碱1mg/kg,30min后腹腔注射匹鲁卡品30 mg/kg,观察大鼠行为学变化。Ⅳ级以上痫性发作1h后腹腔注射10%水合氯醛(300mg/kg)终止发作,观察一周后,断头取脑切片,行HE染色及免疫组织化学染色观察海马区GAD65和NMDAR1的表达。
结果rTMS刺激组大鼠刺激期间未见痫性发作及颅内出血征象,各组大鼠注射匹鲁卡品后均出现痫性发作,表现为点头、甩尾、须动、头面抽动、前肢、后肢抽动、全身抽动、窜动、直立等,49只达到Ⅳ级以上发作,其中有2只终止后死亡。rTMS刺激组大鼠(0.3Hz刺激组、0.5Hz刺激组、0.8Hz刺激组、1.0Hz刺激组)痫性发作潜伏期均比rTMS假刺激组延长,差异均有统计学意义(P＜0.05或P＜0.01),其中0.5Hz刺激组最长;0.5Hz刺激组大鼠出现的痫性发作行为次数均比0.3Hz刺激组、0.8Hz刺激组、1.0Hz刺激组少,且整体发作程度轻,差异有统计学意义(P＜0.05)。rTMS刺激各组大鼠海马神经元GAD65的表达量明显高于rTMS假刺激组(均P＜0.05或P＜0.01),NMDAR1则明显低于rTMS假刺激组,差异有统计学意义(P＜0.05或P＜0.01),以0.5Hz刺激组最为明显。
结论适量的低频rTMS是比较安全的,可延缓大鼠痫性发作潜伏期、减少痫性发作次数、减轻痫性发作程度;能够降低癫痫大鼠海马区NMDAR1的表达及增强GAD65的表达,可能与其抗痫作用有关。
Objective Observing the changes of attack behavior and contents of glutamicaciddecarboxylase(GAD)、N-methyl-D-aspartateR1(NMDAR1)in kindled rat models of epilepsy after Low-frequency transcranial magnetic stimulation, to study Low-frequency transcranial magnetic stimulation have anti-epilepsy effect and perhapers how running.
Method Fifty healthy male Wistar rats were randomly divided into five groups with 10 rats in each group: 0.3Hz repetive transcranial magnetic stimulation epilepsy model group、0.5Hz epilepsy model group、0.8Hz epilepsy model group、1.0Hz epilepsy model group and the false repetive transcranial magnetic stimulation group. Making the acute epilepsy model by Lithium-Pilocarpine after pretreatment, all groups use the sub-eclampsia dosage of 3 mol/kg Lithium to abdominal cavity injection, 20 hour late the sub-eclampsia dosage of 1mg/kg methylscopolamine were injected to abdominal, after thirty minutes the sub-eclampsia dosage of 30mg/kg Pilocarpine were injected, observed the performance of epilepsy seizures after intraperitoneal injection of Pilocarpine, and then over the status epileptieus with the sub-eclampsia dosage (300mg/kg) of 10% chloral hydrate, after one week, HE stain and imnmnohistoehemistry stain were applied, study the efects afte repetive transcranial magnetic stimulation on GAD65、NMDAR1 expression in hippocampus of rats.
Results 0.3Hz、0.5Hz、0.8Hz、1.0Hz repetive transcranial magnetic stimulation epilepsy model groups rats are not looking out epilepsy、cerebral hemorrhage evidence, et al. All groups were taking epileptic attack behavior sincluding nodding, tail-flick , beard shaking , anterior, hind limbtic, systemic tic and jump, et al. 49 rats were induced successfully status epileptieus in all, 2 rats were dead. The latency period each of 0.3Hz、0.5Hz、0.8Hz and 1.0Hz groups were prolonger than the false repetive transcranial magnetic stimulation group (P<0. 05 or P<0. 01). The times of epileptic attack behavior in 0. 5Hz group was less than the 0.3Hz、0.5Hz、0.8Hz、1.0Hz repetive transcranial magnetic stimulation epilepsy model groups respectively (P<0. 05 or P<0. 01), and the whole seizure level was less than 0.3Hz、0.5Hz、0.8Hz、1.0Hz repetive transcranial magnetic stimulation epilepsy model groups too(P<0.05). The number of GAD65 expression in hippocampus of all repetive transcranial magnetic stimulation groups rats were lower than the false repetive transcranial magnetic stimulation group Rats(P<0. 05 or P<0. 01), NMDAR1 expression was higher than that (P<0. 05 or P<0. 01), especially that of 0. 5Hz groups were evidence.
Conclusion Appropriate low-frequency transcranial magnetic stimulation is safety, it could prolong the latent period of epilepsy seizures, decreased the times of epilepsy attack and diminish the symptom level of epilepsy attack; It can efectively decreased the content of excitatory amino acid Glu's receptor-NMDAR1 in hippocampus, at the same time, it can also enhance the expression of in hippocampus acid's receptor- GAD, that perhapers the one role of anti-epilepsy.
引文
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