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神经科学

戊四氮诱导点燃小鼠模型

Published: June 12, 2018
doi:
10.3791/56573
,
1Synaptic Plasticity Project,Tokyo Metropolitan Institute of Medical Science

Summary

本协议描述了一种戊四氮化学点燃方法, 并提供了一种小鼠癫痫模型。该协议还可用于调查小鼠癫痫发作后癫痫诱导和发病机制的脆弱性。

Abstract

戊四氮 (PTZ) 是 GABA-受体拮抗剂。腹腔内注射 PTZ 进入动物诱导急性, 严重癫痫的高剂量, 而顺序注射 subconvulsive 剂量已用于发展化学点燃, 癫痫模型。单次低剂量的 PTZ 诱导轻度发作, 不抽搐。然而, 反复低剂量的 PTZ 注射降低了诱发痉挛性癫痫发作的门槛。最后, 云台连续低剂量管理诱发严重的挛癫痫发作。这种方法简单, 广泛适用于研究癫痫的病理生理学, 它被定义为一种涉及重复性癫痫的慢性疾病。这种化学点燃协议导致动物反复发作。利用这种方法, 估计易受云台介导的癫痫发作或癫痫发作加重的程度。这些优势导致了使用这种方法筛选抗癫痫药物和癫痫相关的基因。此外, 该方法已被用来调查癫痫发作后的神经细胞损伤, 因为在癫痫患者的大脑中观察到的组织学改变也出现在化学点燃动物的大脑中。因此, 该协议对于方便地制作癫痫动物模型是有益的。

Introduction

癫痫是一种慢性神经系统疾病, 其特点是反复发作, 影响大约1% 的人。在临床研究中不能充分阐明癫痫患者负反馈和癫痫产生的机制。因此, 对癫痫1的研究需要一个适宜的动物模型。

各种动物模型的癫痫已被用来调查癫痫的生理和识别抗癫痫药物2,3。在这些模型中, 药物癫痫诱导是一种常见的方法, 用于生成动物模型, 以调查4的癫痫病理。这种方法既便宜又简单。电极介导的点燃也是一种常用的方法, 但这一过程的成本较高, 该方法需要手术和电气技能, 以诱发重复性发作5。

药理诱导也有利, 因为发作的时间和数量容易控制。在癫痫的研究中也使用了表现为自发癫痫的基因小鼠模型。然而, 预测在这些基因模型中癫痫发作的时间和频率可能是不可能的6。为了观察基因修饰小鼠6的癫痫行为, 需要一个监测系统。

海仁酸、匹戊四氮 (PTZ) 被广泛用作癫痫诱导药物7。海仁酸是谷氨酸受体的促激动剂, 而匹属激活胆碱受体。PTZ 是一种伽玛丁酸 (GABA)-受体拮抗剂8。PTZ 抑制抑制性突触的功能, 导致神经元活动增加。这个章程导致普遍的缉获在动物9。单一注射海仁酸和匹特能诱发急性癫痫发作, 特别是癫痫持续状态 (SE)10,11和海仁酸-或匹特式介导硒促进慢性自发性和复发性癫痫12,13. 脑电图 (EEG) 录音和行为分析表明, 在一次注射12、13后一个月内, 自然复发性癫痫发作。单一注射的抽搐剂量的 PTZ 也诱发急性发作。然而, 单一注射 PTZ 后的慢性自发性癫痫很难推广。需要对 PTZ 进行慢性管理, 以诱发重复性癫痫14。在这两种方法中, 重复性癫痫的产生都能诱发一种与人类癫痫相似的病理, 而非急性癫痫的发生。在 PTZ 的情况下, 每次注射都能引起癫痫发作, 每次注射时, 检出的严重性都会逐步加重。最后, 单低剂量 PTZ 注射诱发严重的挛癫痫发作。在这个阶段, 每次注射都能唤起严重的癫痫发作。此外, 在注射过程中, 癫痫潜伏期和持续时间也会发生变化。在点燃15的后阶段, 补剂发作的潜伏期往往会缩短。此外, 癫痫发作加重还伴有长期发作期16。研究控制癫痫发作严重性、潜伏期和持续时间的分子机制对于筛查抗癫痫药物17、18、19有很重要的意义。

癫痫发作通常是由一个单一的系统管理的 PTZ, 恢复速度非常快, 在30分钟4,5。因此, 在云台点火模型中, 癫痫发作的次数更能控制。然而, 脑电图监测表明, 在云台介导的癫痫发作20后, 一般峰值可达12小时。因此, 动物最好保持观察24小时后, 挛或补药发作21 , 以更精确的分析点燃机制。

抗癫痫药物, 如 ethosuximide, 丙戊酸, 苯巴比妥, 氨乙烯酸, 和 retigabine3, 在 PTZ 注射之前或之后, 降低发作严重性3,22的恶化, 23。同样, 没有基因发作加重的击倒小鼠, 如基质 metalloproteinase-924, FGF-2225和 neuritin26, 已被证明在多台 PTZ 注射后, 检出严重程度降低。此外, 通过这种方法可以观察癫痫发作后的组织病理改变。颞叶癫痫患者有典型的组织学改变, 如苔藓纤维发芽27,28, 异常颗粒神经元迁移29, astrogliosis30, 神经细胞死亡在海马31,32和海马硬化症33。癫痫模型动物也观察到类似的变化。在现有的方法中, 云台介导的化学点火是一种很好的, 重现性和廉价的方法来产生一种动物模型的癫痫。在一个由匹凡达介导的 se 模型中, 癫痫控制是困难的, 许多小鼠死亡或无法发展 se34。相比之下, 在 PTZ 模型中, 死亡率和癫痫严重程度更可控制。此外, PTZ 比海仁酸便宜, 而且在小鼠脑外科的技能是不需要药物管理。

Protocol

所有实验程序均经东京都医学科学院动物护理和使用委员会批准。建议产后 8-16 周大鼠。任何近交菌株都是可以接受的实验。C57BL/6 小鼠对 ptz 更有抵抗力, 而 BALB/c 和瑞士白化小鼠对 ptz 更敏感。C57BL/6 在这项研究中被使用。对 PTZ 的脆弱性也取决于鼠标的年龄。与小鼠相比, 大鼠更难在 PTZ35。这种方法使用的动物数量可能会有所不同, 但每种情况下至少需要 6-10 只动物。 <p class…

Representative Results

反复注射 PTZ 诱发癫痫发作的严重性增加。对六 C57BL/6 小鼠进行了 PTZ 治疗, 另有6只小鼠以生理盐水作为对照组治疗。PTZ 剂量为35毫克/千克, 10 次注射。随着 PTZ 注射, 癫痫评分逐渐增加, 而生理盐水注射没有诱发癫痫发作或异常行为 (图 2)。方差分析后的 Bonferroni 试验显示, 云台治疗组与生理盐水治疗组有显著性差异。 <p class="jove_content" fo:keep-togethe…

Discussion

在这里, 我们提出了一个广泛的可访问的协议, 建立一个药理动物模型的癫痫。云台介导的化学点火具有悠久的历史, 是一种普遍接受的模型, 研究的病理学和细胞病理学的癫痫41。Suzdak 和詹森对癫痫的化学点燃模型进行了回顾, 199542。药物癫痫诱导, 特别是与 PTZ, 是一种简单和简单的方法来唤起严重癫痫发作。注射剂量的变化与癫痫严重程度相关。因此, 通过改?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

这项工作得到部分支持的 jsp KAKENHI 赠款号 24700349, 24659093, 25293239, JP18H02536 和 17K07086, 下个 KAKENHI 赠款号码25110737和 23110525, 阿曼德赠款号码 JP18ek0109311, SENSHIN 医学研究基金会和日本癫痫研究基金会。

Materials

Pentylenetetrazole Sigma-Aldrich P6500
Sodium chloride MANAC 7647-14-5
Mouse CLEA Japan C57Bl/6NJcl, postnatal 8 week, male
Syringe (1mL) Terumo SS-01T
Needle(27G x 3/4") (0.40 x 19 mm) Terumo NN-2719S
Weighing scale Mettler PE2000 This item is a discontinued product. Almost equivalent to FX-2000i with FXi-12-JA from A&D company.
Paraformaldehyde Sigma-Aldrich P6148
Sodium hydroxide nacalai tesque 31511-05
Peristatic pump ATTO SJ1211
Sucrose nacalai tesque 30404-45
Microtome Yamato REM-700 This item is a discontinued product. Almost equivalent to REM-710
Microtome blade Feather S35
Triton X-100 Sigma-Aldrich X-100
anti-synaptoporin antibody Synaptic systems 102 002
anti-ZnT3 antibody Synaptic systems 197 002
anti-doublecortin Santa Cruz sc-8066 This item is a discontinued product. We did not test equivalent product (sc-271390).
Contextual fear discrimination test apparatus O'hara
Three chamber test apparatus Muromachi
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References

  1. Löscher, W., Brandt, C. Prevention or Modification of Epileptogenesis after Brain Insults: Experimental Approaches and Translational Research. Pharmacological Reviews. 62 (4), 668-700 (2010).
  2. Loscher, W. Animal Models of Seizures and Epilepsy: Past, Present, and Future Role for the Discovery of Antiseizure Drugs. Neurochem Res. , (2017).
  3. Löscher, W. Critical review of current animal models of seizures and epilepsy used in the discovery and development of new antiepileptic drugs. Seizure. 20 (5), 359-368 (2011).
  4. Kandratavicius, L., et al. Animal models of epilepsy: use and limitations. Neuropsychiatr Dis Treat. 10, 1693-1705 (2014).
  5. Pitkänen, A., Schwartzkroin, P. A., Moshé, S. L. . Models of Seizures and Epilepsy. , xvii (2006).
  6. Yang, Y., Frankel, W. N. Genetic approaches to studying mouse models of human seizure disorders. Adv Exp Med Biol. 548, 1-11 (2004).
  7. Leite, J. P., Garcia-Cairasco, N., Cavalheiro, E. A. New insights from the use of pilocarpine and kainate models. Epilepsy Res. 50 (1-2), 93-103 (2002).
  8. Squires, R. F., Saederup, E., Crawley, J. N., Skolnick, P., Paul, S. M. Convulsant potencies of tetrazoles are highly correlated with actions on GABA/benzodiazepine/picrotoxin receptor complexes in brain. Life Sci. 35 (14), 1439-1444 (1984).
  9. Tourov, A., et al. Spike morphology in PTZ-induced generalized and cobalt-induced partial experimental epilepsy. Funct Neurol. 11 (5), 237-245 (1996).
  10. Furtado Mde, A., Braga, G. K., Oliveira, J. A., Del Vecchio, F., Garcia-Cairasco, N. Behavioral, morphologic, and electroencephalographic evaluation of seizures induced by intrahippocampal microinjection of pilocarpine. Epilepsia. 43, 37-39 (2002).
  11. Hosford, D. A. Animal models of nonconvulsive status epilepticus. J Clin Neurophysiol. 16 (4), 306-313 (1999).
  12. Medina-Ceja, L., Pardo-Pena, K., Ventura-Mejia, C. Evaluation of behavioral parameters and mortality in a model of temporal lobe epilepsy induced by intracerebroventricular pilocarpine administration. Neuroreport. , (2014).
  13. Bragin, A., Azizyan, A., Almajano, J., Wilson, C. L., Engel, J. Analysis of chronic seizure onsets after intrahippocampal kainic acid injection in freely moving rats. Epilepsia. 46 (10), 1592-1598 (2005).
  14. Schmidt, J. Changes in seizure susceptibility in rats following chronic administration of pentylenetetrazol. Biomed Biochim Acta. 46 (4), 267-270 (1987).
  15. Angelatou, F., Pagonopoulou, O., Kostopoulos, G. Changes in seizure latency correlate with alterations in A1 adenosine receptor binding during daily repeated pentylentetrazol-induced convulsions in different mouse brain areas. Neuroscience Letters. 132 (2), 203-206 (1991).
  16. Löscher, W. Animal models of epilepsy for the development of antiepileptogenic and disease-modifying drugs. A comparison of the pharmacology of kindling and post-status epilepticus models of temporal lobe epilepsy. Epilepsy Research. 50 (1), 105-123 (2002).
  17. Ilhan, A., Iraz, M., Kamisli, S., Yigitoglu, R. Pentylenetetrazol-induced kindling seizure attenuated by Ginkgo biloba extract (EGb 761) in mice. Progress in Neuro-Psychopharmacology and Biological Psychiatry. 30 (8), 1504-1510 (2006).
  18. Emami, S., Kebriaeezadeh, A., Ahangar, N., Khorasani, R. Imidazolylchromanone oxime ethers as potential anticonvulsant agents: Anticonvulsive evaluation in PTZ-kindling model of epilepsy and SAR study. Bioorganic & Medicinal Chemistry Letters. 21 (2), 655-659 (2011).
  19. Klitgaard, H. Levetiracetam: The Preclinical Profile of a New Class of Antiepileptic Drugs?. Epilepsia. 42, 13-18 (2001).
  20. Kellinghaus, C., et al. Dissociation between in vitro and in vivo epileptogenicity in a rat model of cortical dysplasia. Epileptic Disord. 9 (1), 11-19 (2007).
  21. Koutroumanidou, E., et al. Increased seizure latency and decreased severity of pentylenetetrazol-induced seizures in mice after essential oil administration. Epilepsy Res Treat. 2013, 532657 (2013).
  22. Krall, R. L., Penry, J. K., White, B. G., Kupferberg, H. J., Swinyard, E. A. Antiepileptic drug development: II. Anticonvulsant drug screening. Epilepsia. 19 (4), 409-428 (1978).
  23. White, H. S. Preclinical development of antiepileptic drugs: past, present, and future directions. Epilepsia. 44, 2-8 (2003).
  24. Mizoguchi, H., et al. Matrix metalloproteinase-9 contributes to kindled seizure development in pentylenetetrazole-treated mice by converting pro-BDNF to mature BDNF in the hippocampus. J Neurosci. 31 (36), 12963-12971 (2011).
  25. Lee, C. H., Umemori, H. Suppression of epileptogenesis-associated changes in response to seizures in FGF22-deficient mice. Front Cell Neurosci. 7, 43 (2013).
  26. Shimada, T., Yoshida, T., Yamagata, K. Neuritin Mediates Activity-Dependent Axonal Branch Formation in Part via FGF Signaling. J Neurosci. 36 (16), 4534-4548 (2016).
  27. Parent, J. M., et al. Dentate granule cell neurogenesis is increased by seizures and contributes to aberrant network reorganization in the adult rat hippocampus. J Neurosci. 17 (10), 3727-3738 (1997).
  28. Sutula, T., He, X. X., Cavazos, J., Scott, G. Synaptic reorganization in the hippocampus induced by abnormal functional activity. Science. 239 (4844), 1147-1150 (1988).
  29. Parent, J. M., Elliott, R. C., Pleasure, S. J., Barbaro, N. M., Lowenstein, D. H. Aberrant seizure-induced neurogenesis in experimental temporal lobe epilepsy. Ann Neurol. 59 (1), 81-91 (2006).
  30. Sofroniew, M. V. Molecular dissection of reactive astrogliosis and glial scar formation. Trends Neurosci. 32 (12), 638-647 (2009).
  31. Arzimanoglou, A., et al. Epilepsy and neuroprotection: an illustrated review. Epileptic Disord. 4 (3), 173-182 (2002).
  32. Represa, A., Niquet, J., Pollard, H., Ben-Ari, Y. Cell death, gliosis, and synaptic remodeling in the hippocampus of epileptic rats. Journal of Neurobiology. 26 (3), 413-425 (1995).
  33. Blumcke, I. Neuropathology of focal epilepsies: a critical review. Epilepsy Behav. 15 (1), 34-39 (2009).
  34. Buckmaster, P. S., Haney, M. M. Factors affecting outcomes of pilocarpine treatment in a mouse model of temporal lobe epilepsy. Epilepsy Res. 102 (3), 153-159 (2012).
  35. Nokubo, M., et al. Age-dependent increase in the threshold for pentylenetetrazole induced maximal seizure in mice. Life Sci. 38 (22), 1999-2007 (1986).
  36. Ferraro, T. N., et al. Mapping Loci for Pentylenetetrazol-Induced Seizure Susceptibility in Mice. The Journal of Neuroscience. 19 (16), 6733-6739 (1999).
  37. Kosobud, A. E., Cross, S. J., Crabbe, J. C. Neural sensitivity to pentylenetetrazol convulsions in inbred and selectively bred mice. Brain Res. 592 (1-2), 122-128 (1992).
  38. Shimada, T., Takemiya, T., Sugiura, H., Yamagata, K. Role of Inflammatory Mediators in the Pathogenesis of Epilepsy. Mediators of Inflammation. 2014, 1-8 (2014).
  39. Itoh, K., et al. Magnetic resonance and biochemical studies during pentylenetetrazole-kindling development: the relationship between nitric oxide, neuronal nitric oxide synthase and seizures. 神经科学. 129 (3), 757-766 (2004).
  40. Racine, R. J. Modification of seizure activity by electrical stimulation. II. Motor seizure. Electroencephalogr Clin Neurophysiol. 32 (3), 281-294 (1972).
  41. Bialer, M., White, H. S. Key factors in the discovery and development of new antiepileptic drugs. Nat Rev Drug Discov. 9 (1), 68-82 (2010).
  42. Suzdak, P. D., Jansen, J. A. A review of the preclinical pharmacology of tiagabine: a potent and selective anticonvulsant GABA uptake inhibitor. Epilepsia. 36 (6), 612-626 (1995).
  43. Seyfried, T. N., Glaser, G. H. A review of mouse mutants as genetic models of epilepsy. Epilepsia. 26 (2), 143-150 (1985).
  44. Upton, N., Stratton, S. Recent developments from genetic mouse models of seizures. Current Opinion in Pharmacology. 3 (1), 19-26 (2003).
  45. Rao, M. S., Hattiangady, B., Reddy, D. S., Shetty, A. K. Hippocampal neurodegeneration, spontaneous seizures, and mossy fiber sprouting in the F344 rat model of temporal lobe epilepsy. J Neurosci Res. 83 (6), 1088-1105 (2006).
  46. Hellier, J. L., Patrylo, P. R., Buckmaster, P. S., Dudek, F. E. Recurrent spontaneous motor seizures after repeated low-dose systemic treatment with kainate: assessment of a rat model of temporal lobe epilepsy. Epilepsy Res. 31 (1), 73-84 (1998).
  47. Turski, L., et al. Seizures produced by pilocarpine: neuropathological sequelae and activity of glutamate decarboxylase in the rat forebrain. Brain Res. 398 (1), 37-48 (1986).
  48. Itoh, K., Watanabe, M. Paradoxical facilitation of pentylenetetrazole-induced convulsion susceptibility in mice lacking neuronal nitric oxide synthase. 神经科学. 159 (2), 735-743 (2009).
  49. Deng, Y., Wang, M., Wang, W., Ma, C., He, N. Comparison and Effects of Acute Lamotrigine Treatment on Extracellular Excitatory Amino Acids in the Hippocampus of PTZ-Kindled Epileptic and PTZ-Induced Status Epilepticus Rats. Neurochemical Research. 38 (3), 504-511 (2013).
  50. Kupferberg, H. Animal models used in the screening of antiepileptic drugs. Epilepsia. 42, 7-12 (2001).
  51. Berg, A. T., Plioplys, S. Epilepsy and autism: is there a special relationship?. Epilepsy Behav. 23 (3), 193-198 (2012).
  52. Robinson, S. J. Childhood epilepsy and autism spectrum disorders: psychiatric problems, phenotypic expression, and anticonvulsants. Neuropsychol Rev. 22 (3), 271-279 (2012).
  53. Tuchman, R. Autism and Cognition Within Epilepsy: Social Matters. Epilepsy Curr. 15 (4), 202-205 (2015).
  54. Takechi, K., Suemaru, K., Kiyoi, T., Tanaka, A., Araki, H. The alpha4beta2 nicotinic acetylcholine receptor modulates autism-like behavioral and motor abnormalities in pentylenetetrazol-kindled mice. Eur J Pharmacol. 775, 57-66 (2016).
  55. Abdel-Zaher, A. O., Farghaly, H. S. M., Farrag, M. M. Y., Abdel-Rahman, M. S., Abdel-Wahab, B. A. A potential mechanism for the ameliorative effect of thymoquinone on pentylenetetrazole-induced kindling and cognitive impairments in mice. Biomed Pharmacother. 88, 553-561 (2017).
  56. Jia, F., et al. Effects of histamine H(3) antagonists and donepezil on learning and mnemonic deficits induced by pentylenetetrazol kindling in weanling mice. Neuropharmacology. 50 (3), 404-411 (2006).
  57. Pahuja, M., Mehla, J., Reeta, K. H., Tripathi, M., Gupta, Y. K. Effect of Anacyclus pyrethrum on pentylenetetrazole-induced kindling, spatial memory, oxidative stress and rho-kinase II expression in mice. Neurochem Res. 38 (3), 547-556 (2013).

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Pentylenetetrazole-Induced KindlingMouse ModelEpilepsySeizureHistological ChangesNeuropsychiatric DisordersPTZSodium ChlorideIntraperitoneal Injection129 SV MiceThree-Chamber ApparatusHabituationSocial InteractionBehavioral Parameters

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Shimada, T., Yamagata, K. Pentylenetetrazole-Induced Kindling Mouse Model. J. Vis. Exp. (136), e56573, doi:10.3791/56573 (2018).
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