耐药性颞叶癫痫大鼠海马组织γ-氨基丁酸A、B受体表达观察
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摘要
目的观察耐药性颞叶癫痫大鼠海马组织γ-氨基丁酸A(GABAA)受体、γ-氨基丁酸B(GABAB)受体表达的变化,以探讨二者在颞叶癫痫大鼠耐药机制中的作用。方法随机选取雄性SD大鼠60只,制作氯化锂-匹罗卡品大鼠癫痫模型,根据癫痫大鼠对抗癫痫药物苯妥英钠及苯巴比妥的反应分为耐药组、药物敏感组,选择8只正常SD大鼠作为对照组。处死各组动物,取出大脑海马组织,分别采用免疫组织化学法、蛋白质免疫印迹法检测海马组织GABAA、GABAB受体。结果免疫组织化学染色结果显示,耐药组大鼠海马组织GABAA、GABAB受体平均积分光密度(MOD)值分别为0.157±0.016、0.158±0.026,药物敏感组分别为0.262±0.016、0.263±0.027,对照组分别为0.294±0.040、0.307±0.032;耐药组、药物敏感组与对照组相比,P<0.05;耐药组、药物敏感组相比,P<0.05。蛋白质免疫印迹法检测结果显示,耐药组GABAA、GABAB受体累积积分光密度(IOD)值(代表GABAA、GABAB蛋白相对表达量)分别为0.121±0.029、0.071±0.016,药物敏感组分别为0.223±0.036、0.149±0.012,对照组分别为0.261±0.034、0.180±0.011,耐药组、药物敏感组与对照组相比,P<0.05;耐药组、药物敏感组相比,P<0.05。结论 GABAA、GABAB受体在耐药性颞叶癫痫大鼠海马组织中表达降低,二者可能与癫痫耐药发生有关。
Objective To observe the expression changes of γ-aminobutyric acid receptor A(GABAA) and γ-aminobutyric acid receptor B(GABAB) in the hippocampus of pharmacoresistant temporal lobe epilepsy rats, and to explore their role in the mechanism of resistance in rats with temporal lobe epilepsy.Methods Sixty adult male SD rats were selected to establish the epilepsy models by pilocarpine introperitonial injection. The models of pharmacoresistant epilepsy were selected by their response to antiepileptic drugs phenobabital and phynitoin. The epileptic rats were divided into the pharmacosensitive epileptic group(PSE group, 8 rats) and the pharmacoresistant epileptic group(PRE group, 8 rats). Another normal rats were used as the normal control group(NRC group, 8 rats). We executed the selected rats and removed the brain hippocampus. Immunohistochemistry and Western blotting were performed to assess GABAA and GABAB expression in the hippocampal tissues in the three groups.Results Immunohistochemistry showed that the mean optical density(MOD) of GABAA and GABAB in the hippocampi were 0.157±0.016 and 0.158±0.026 in the PRE group, 0.262±0.016 and 0.263±0.027 in the PSE group, and 0.294±0.040 and 0.307±0.032 in the NCR group; significant difference was found between the PRE and PSE groups and the NCR group(P<0.05), and between the PRE group and PSE group(P<0.05). Western blotting showed that the integrated optical density(IOD) of GABAA and GABAB were 0.121±0.029 and 0.071±0.016 in the PRE group, 0.223±0.036 and 0.149±0.012 in the PSE group, and 0.261±0.034 and 0.180±0.011 in the NCR group; significant difference was found between the PRE and PSE groups and the NCR group(P<0.05), and between the PRE group and PSE group(P<0.05). Conclusion The expression of GABAA and GABAB in the pharmacoresistant epileptic rats decreases, and both of them may be related to the epileptogenesis of pharmacoresistance.
Objective To observe the expression changes of γ-aminobutyric acid receptor A(GABAA) and γ-aminobutyric acid receptor B(GABAB) in the hippocampus of pharmacoresistant temporal lobe epilepsy rats, and to explore their role in the mechanism of resistance in rats with temporal lobe epilepsy.Methods Sixty adult male SD rats were selected to establish the epilepsy models by pilocarpine introperitonial injection. The models of pharmacoresistant epilepsy were selected by their response to antiepileptic drugs phenobabital and phynitoin. The epileptic rats were divided into the pharmacosensitive epileptic group(PSE group, 8 rats) and the pharmacoresistant epileptic group(PRE group, 8 rats). Another normal rats were used as the normal control group(NRC group, 8 rats). We executed the selected rats and removed the brain hippocampus. Immunohistochemistry and Western blotting were performed to assess GABAA and GABAB expression in the hippocampal tissues in the three groups.Results Immunohistochemistry showed that the mean optical density(MOD) of GABAA and GABAB in the hippocampi were 0.157±0.016 and 0.158±0.026 in the PRE group, 0.262±0.016 and 0.263±0.027 in the PSE group, and 0.294±0.040 and 0.307±0.032 in the NCR group; significant difference was found between the PRE and PSE groups and the NCR group(P<0.05), and between the PRE group and PSE group(P<0.05). Western blotting showed that the integrated optical density(IOD) of GABAA and GABAB were 0.121±0.029 and 0.071±0.016 in the PRE group, 0.223±0.036 and 0.149±0.012 in the PSE group, and 0.261±0.034 and 0.180±0.011 in the NCR group; significant difference was found between the PRE and PSE groups and the NCR group(P<0.05), and between the PRE group and PSE group(P<0.05). Conclusion The expression of GABAA and GABAB in the pharmacoresistant epileptic rats decreases, and both of them may be related to the epileptogenesis of pharmacoresistance.
引文
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[6] Racine RJ. Modification of seizure activity by electrical stimulation. Ⅱ. Motor seizure [J]. Electroencephalogr Clin Neurophysiol, 1972,32(3):281-294.
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[9] Pahuja M, Mehla J, Reeta KH, et al. Effect of Anacyclus pyrethrum on pentylenetetrazole-induced kindling, spatial memory, oxidative stress and rho-kinase Ⅱ expression in mice [J]. Neurochem Res, 2013,38(3):547-556.
[10] Bethmann K, Brandt C, Loscher W. Resistance to phenobarbital extends to phenytoin in a rat model of temporal lobe epilepsy [J]. Epilepsia, 2007,48(4):816-826.
[11] Xiang J, Jiang Y. Antiepileptic potential of matrine via regulation the levels of gamma-aminobutyric acid and glutamic acid in the brain [J]. Int J Mol Sci, 2013,14(12):23751-23761.
[12] Toth K, Eross L, Vajda J, et al. Loss and reorganization of calretinin-containing interneurons in the epileptic human hippocampus [J]. Brain, 2010,133(9):2763-2777.
[13] Joshi S, Rajasekaran K, Kapur J. GABAergic transmission in temporal lobe epilepsy: the role of neurosteroids [J]. Exp Neurol, 2013,244:36-42.
[14] Wang A, Chi Z, Wang S, et al. Calcineurin-mediated GABA(A) receptor dephosphorylation in rats after kainic acid-induced status epilepticus [J]. Seizure, 2009,18(7):519-523.
[15] Ghafouri S, Fathollahi Y, Semnanian S, et al. Effects of low frequency stimulation on spontaneous inhibitory and excitatory post-synaptic currents in hippocampal CA1 pyramidal cells of kindled rats [J]. Cell J, 2017,18(4):547-555.
[16] Nishiki T, Augustine GJ. Synaptotagmin I synchronizes transmitter release in mouse hippocampal neurons [J]. J Neurosci, 2004,24(27): 6127-6132.
[17] Teichgraber LA, Lehmann TN, Meencke HJ, et al. Impaired function of GABA(B) receptors in tissues from pharmacoresistant epilepsy patients [J]. Epilepsia, 2009,50(7):1697-1716.
[18] Soukupova M, Binaschi A, Falcicchia C, et al. Impairment of GABA release in the hippocampus at the time of the first spontaneous seizure in the pilocarpine model of temporal lobe epilepsy [J]. Exp Neurol, 2014,257:39-49.
[19] Bethmann K, Fritschy JM, Brandt C, et al. Antiepileptic drug resistant rats differ from drug responsive rats in GABA A receptor subunit expression in a model of temporal lobe epilepsy [J]. Neurobiol Dis, 2008,31(2):169-187.
[20] Marrosu F, Serra A, Maleci A, et al. Correlation between GABA(A) receptor density and vagus nerve stimulation in individuals with drug-resistant partial epilepsy [J]. Epilepsy Res, 2003,55(1-2):59-70.
[21] Mohler H, Fritschy JM, Rudolph U. A new benzodiazepine pharmacology [J]. J Pharmacol Exp Ther, 2002,300(1):2-8.
[22] Volk HA, Arabadzisz D, Fritschy JM, et al. Antiepileptic drug-resistant rats differ from drug-responsive rats in hippocampal neurodegeneration and GABA(A) receptor ligand binding in a model of temporal lobe epilepsy [J]. Neurobiol Dis, 2006,21(3):633-646.
[2] Skjei KL, Dlugos DJ. The evaluation of treatment-resistant epilepsy [J]. Semin Pediatr Neurol, 2011,18(3):150-170.
[3] Macdonald RL, Olsen RW. GABAA receptor channels [J]. Annu Rev Neurosci, 1994,17:569-602.
[4] Hernandez CC, Klassen TL, Jackson LG, et al. Correction: deleterious rare variants reveal risk for loss of GABAA receptor function in patients with genetic epilepsy and in the general population [J]. PLoS One, 2016,11(11):e0167264.
[5] Billinton A, Baird VH, Thom M, et al. GABA(B) receptor autoradiography in hippocampal sclerosis associated with human temporal lobe epilepsy [J]. Br J Pharmacol, 2001,132(2):475-480.
[6] Racine RJ. Modification of seizure activity by electrical stimulation. Ⅱ. Motor seizure [J]. Electroencephalogr Clin Neurophysiol, 1972,32(3):281-294.
[7] Korff CM, Scheffer IE. Epilepsy classification: a cycle of evolution and revolution [J]. Curr Opin Neurol, 2013,26(2):163-167.
[8] Pohlmann-Eden B, Weaver DF. The puzzle(s) of pharmacoresistant epilepsy[J]. Epilepsia, 2013,54 (Suppl 2):1-4.
[9] Pahuja M, Mehla J, Reeta KH, et al. Effect of Anacyclus pyrethrum on pentylenetetrazole-induced kindling, spatial memory, oxidative stress and rho-kinase Ⅱ expression in mice [J]. Neurochem Res, 2013,38(3):547-556.
[10] Bethmann K, Brandt C, Loscher W. Resistance to phenobarbital extends to phenytoin in a rat model of temporal lobe epilepsy [J]. Epilepsia, 2007,48(4):816-826.
[11] Xiang J, Jiang Y. Antiepileptic potential of matrine via regulation the levels of gamma-aminobutyric acid and glutamic acid in the brain [J]. Int J Mol Sci, 2013,14(12):23751-23761.
[12] Toth K, Eross L, Vajda J, et al. Loss and reorganization of calretinin-containing interneurons in the epileptic human hippocampus [J]. Brain, 2010,133(9):2763-2777.
[13] Joshi S, Rajasekaran K, Kapur J. GABAergic transmission in temporal lobe epilepsy: the role of neurosteroids [J]. Exp Neurol, 2013,244:36-42.
[14] Wang A, Chi Z, Wang S, et al. Calcineurin-mediated GABA(A) receptor dephosphorylation in rats after kainic acid-induced status epilepticus [J]. Seizure, 2009,18(7):519-523.
[15] Ghafouri S, Fathollahi Y, Semnanian S, et al. Effects of low frequency stimulation on spontaneous inhibitory and excitatory post-synaptic currents in hippocampal CA1 pyramidal cells of kindled rats [J]. Cell J, 2017,18(4):547-555.
[16] Nishiki T, Augustine GJ. Synaptotagmin I synchronizes transmitter release in mouse hippocampal neurons [J]. J Neurosci, 2004,24(27): 6127-6132.
[17] Teichgraber LA, Lehmann TN, Meencke HJ, et al. Impaired function of GABA(B) receptors in tissues from pharmacoresistant epilepsy patients [J]. Epilepsia, 2009,50(7):1697-1716.
[18] Soukupova M, Binaschi A, Falcicchia C, et al. Impairment of GABA release in the hippocampus at the time of the first spontaneous seizure in the pilocarpine model of temporal lobe epilepsy [J]. Exp Neurol, 2014,257:39-49.
[19] Bethmann K, Fritschy JM, Brandt C, et al. Antiepileptic drug resistant rats differ from drug responsive rats in GABA A receptor subunit expression in a model of temporal lobe epilepsy [J]. Neurobiol Dis, 2008,31(2):169-187.
[20] Marrosu F, Serra A, Maleci A, et al. Correlation between GABA(A) receptor density and vagus nerve stimulation in individuals with drug-resistant partial epilepsy [J]. Epilepsy Res, 2003,55(1-2):59-70.
[21] Mohler H, Fritschy JM, Rudolph U. A new benzodiazepine pharmacology [J]. J Pharmacol Exp Ther, 2002,300(1):2-8.
[22] Volk HA, Arabadzisz D, Fritschy JM, et al. Antiepileptic drug-resistant rats differ from drug-responsive rats in hippocampal neurodegeneration and GABA(A) receptor ligand binding in a model of temporal lobe epilepsy [J]. Neurobiol Dis, 2006,21(3):633-646.