利用瞬态受体电位辣椒1型 Ultrapotent 激动剂建立单纯小纤维神经病变的小鼠模型
Summary
本研究建立了树酯毒素 (RTX) 纯小纤维神经病变的实验模型。独特剂量的 RTX (50 µg/千克) 是最佳的发展一个小纤维神经病变模型, 模仿病人的特点, 并可以帮助调查的伤害性分子意义下的神经病理性疼痛。
Abstract
糖尿病患者 (DM) 或那些经历了化疗药物的神经毒性作用的人可能会产生感觉障碍, 由于退化和损伤小直径感觉神经元, 称为小纤维神经病变。目前小纤维神经病变的动物模型对大直径和小口径感觉纤维都有影响, 从而造成神经病理学太复杂, 无法正确评估小直径感觉纤维受损的影响。因此, 有必要建立一个纯小纤维神经病变实验模型, 以充分研究这些问题。该协议描述了小纤维神经病变的实验模型, 具体影响小直径感觉神经与树酯毒素 (RTX), ultrapotent 激动剂的瞬态受体电位辣椒类型 1 (TRPV1), 通过单一剂量的腹腔注射, 称为 RTX 神经病。这种 RTX 神经病变的病理表现和行为异常, 模仿小纤维神经病变患者的临床特点, 包括 intraepidermal 神经纤维 (IENF) 变性, 特别是损伤小直径神经元, 并诱导热 hypoalgesia 和机械痛觉。该协议分别测试了三剂量的 RTX (200, 50 和10µg/千克), 并得出结论认为, 关键剂量的 RTX (50 µg/千克) 是必要的发展典型的小纤维神经病变的表现, 并准备了一个改进的染色程序, 以研究 IENF 变性和神经元躯体损伤。修改后的程序快速、系统、经济。神经性疼痛的行为评价对于揭示小直径感觉神经的功能至关重要。对实验性啮齿动物的机械阈值的评估特别具有挑战性, 该协议描述了一种适合于啮齿类动物评估的定制金属网格。总之, RTX 神经病变是一种新的、容易建立的实验模型, 用于评价神经性疼痛的分子意义和干预作用, 为治疗药物的发展提供依据。
Introduction
小纤维神经病变涉及神经病理性疼痛, 这是明显的退化 IENFs, 是常见的各种类型的条件, 如 DM, 并由于化疗药物的神经毒性作用1,2, 3,4,5。IENFs 是位于背根神经节的小直径神经元的外围终端, 并在 IENF 变性6的情况下平行地受到影响。例如, 上调激活转录 factor-3 (ATF3)6,7, 证明了改变的神经元胞体的上游基因转录。此外, 评估 IENFs 神经支配与皮肤活检有助于诊断小纤维神经病变5,8,9。传统上, 皮肤活检 IENFs 的概况依赖于蛋白质基因产品 9.5 (PGP 9.5)1,10,11的免疫组化演示。结合, 神经节和 IENFs 的病理剖面反映小纤维神经病变的功能状况, 可能是这类神经病变对小直径神经元功能性后果的指示。
以前, 有几个实验模型讨论了化疗诱发神经病变的 IENF 变性问题12,13和由压缩或横断导致的神经损伤14,15,16. 这些实验模型也影响大口径神经;因此, 不可能排除受影响的大口径神经在观察到的小纤维神经病变中的贡献;例如, 通过有害的撤退检查 thermosensation 障碍取决于功能性马达神经纤维17,18,19。因此, 建立单纯的小纤维神经病变模型, 系统地研究小直径神经元中神经元胞体及其周围皮神经纤维的病理状态是必要的和必要的。
RTX 是一种辣椒素模拟剂和一个强有力的激动剂对瞬态受体电位辣椒受体 1 (TRPV1), 它介导伤害处理20,21,22。最近, 外围 RTX 治疗缓解神经源性疼痛23,24,25和 intraganglionic 注射 RTX 引起的背根神经节神经元的不可逆转的损失22。外围 RTX 管理的效果是剂量依赖性20,26,27, 这导致 IENFs 的瞬变脱敏或变性。有趣的是, 系统性的大剂量 RTX 治疗导致神经病理性疼痛28, 小纤维神经病变的症状。这些发现表明, RTX 的治疗方式和剂量会产生明显的病理效应和神经元反应;对机智, 外围管理防止痛苦传输由地方作用29并且影响了神经胞体发展神经病变行为 6.总的来说, 这些发现表明, RTX 有一个多向分化潜能的影响, 并提出了问题是否有一个特定剂量的 RTX, 可能有系统地影响周围神经, 如外围 IENFs 和中枢神经元胞体。如果是这样, RTX 可能是一个潜在的代理人, 专门影响小直径神经元和模拟小纤维神经病变在诊所。例如, DM 在临床上是一个复杂的问题, 包括代谢紊乱和神经病理学的周围神经, 这是主要特点的小纤维神经病变。糖尿病相关的小纤维神经病变的机制不能排除可能不是影响周围神经的主要药物代谢紊乱的贡献。因此, DM 相关的小纤维神经病变需要一个纯动物模型, 可以排除系统性代谢紊乱的影响。该协议描述了 RTX 的工作剂量, 以发展一个典型的小纤维神经病变模型, 包括 IENF 变性和小直径神经元损伤, 通过改进的染色分析表明。
Protocol
所描述的所有程序均符合实验室动物的道德准则30, 该议定书已获台湾高雄医科大学动物委员会批准。 1. RTX 神经病变的建立 警告:RTX 是神经毒素和危险的。在接触时, 它会对眼睛、粘膜和上呼吸道起到刺激性作用。在 RTX 准备期间, 避免吸入和佩戴实验室眼镜和大衣。用大量的水冲洗, 以防皮肤接触或处理后。 添加1毫?…
Representative Results
该协议描述了一种新的 RTX 神经病变的小鼠模型, 它特别影响到与感官紊乱相关的微小的神经元, 包括 IENF 变性 (图 2)。根据本文所述的协议, 动物在 RTX 注射 D7 后表现出热 hypoalgesia 和机械痛觉。为了建立这种小纤维神经病变模型, 三剂量的 RTX: 200, 50, 10 µg/公斤由 ip 路线管理。RTX 剂量 (50 µg/千克) 被认为是至关重要的, 初步研究表明, 高剂量 RTX (200 µg/?…
Discussion
为提高患者的功能恢复和生活质量, 需要在临床上有效地治疗小纤维神经病变。目前, 由于缺乏对小直径神经元损伤的分子机制的全面了解, 缺乏针对小纤维神经病变的感觉障碍的治疗指南。以往的神经病变模型通常会影响大直径和小口径的感觉神经;例如, 化疗诱发神经病变的模型12,32,33和机械诱发神经病变34<s…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了科学和技术部 (106-2320-037-024)、高雄医科大学 (KMU-M106028、KMU-S105034) 的赠款和台湾高雄医科大学顶尖大学补助金 (TP105PR15) 的资助。
Materials
| Chemical reagent | |||
| Resiniferatoxin | Sigma | R8756 | |
| Tween 80 | Sigma | P1754 | |
| 3,3’-diaminobenzidine | Sigma | D8001 | |
| avidin-biotin complex | Vector | PK-6100 | |
| Name | Company | Catalog Number | Comments |
| Primary Antisera | |||
| Peripherin | Chemicon | MAB-1527 | |
| ATF3 | Santa Cruz | SC-188 | |
| PGP9.5 | UltraClone | RA95101 | |
| Name | Company | Catalog Number | Comments |
| Secondary Antisera | |||
| Biotinylated goat anti-rabbit IgG | Vector | BA-1000 | |
| Texas Red-conjugated goat anti-mouse | Jackson ImmunoResearch | 115-075-146 | |
| Isothiocyanate (FITC)-conjugated donkey anti-rabbit | Jackson ImmunoResearch | 711-095-152 | |
| Name | Company | Catalog Number | Comments |
| Equipment | |||
| Hot plate | IITC | Model 39 | |
| von Frey filament | Somedic Sales AB | 10-600-0001 | |
| Name | Company | Catalog Number | Comments |
| Material | |||
| Shandon coverplate | Thermo scientific | 72110017 | |
| Slide rack | Thermo scientific | 73310017 |
References
- Shun, C. T., et al. Skin denervation in type 2 diabetes: correlations with diabetic duration and functional impairments. Brain. 127 (Pt 7), 1593-1605 (2004).
- Polydefkis, M., et al. The time course of epidermal nerve fibre regeneration: studies in normal controls and in people with diabetes, with and without neuropathy. Brain. 127 (Pt 7), 1606-1615 (2004).
- Holland, N. R., et al. Small-fiber sensory neuropathies: clinical course and neuropathology of idiopathic cases. Ann Neurol. 44 (1), 47-59 (1998).
- Chaudhry, V., Rowinsky, E. K., Sartorius, S. E., Donehower, R. C., Cornblath, D. R. Peripheral neuropathy from taxol and cisplatin combination chemotherapy: clinical and electrophysiological studies. Ann Neurol. 35 (3), 304-311 (1994).
- Mellgren, S. I., Nolano, M., Sommer, C. The cutaneous nerve biopsy: technical aspects, indications, and contribution. Handb Clin Neurol. 115, 171-188 (2013).
- Hsieh, Y. L., Chiang, H., Lue, J. H., Hsieh, S. T. P2X3-mediated peripheral sensitization of neuropathic pain in resiniferatoxin-induced neuropathy. Exp Neurol. 235 (1), 316-325 (2012).
- Fukuoka, T., et al. Re-evaluation of the phenotypic changes in L4 dorsal root ganglion neurons after L5 spinal nerve ligation. Pain. 153 (1), 68-79 (2012).
- Joint Task Force of the, E., et al. European Federation of Neurological Societies/Peripheral Nerve Society Guideline on the use of skin biopsy in the diagnosis of small fiber neuropathy. Report of a joint task force of the European Federation of Neurological Societies and the Peripheral Nerve Society. J Peripher Nerv Syst. 15 (2), 79-92 (2010).
- Hsieh, S. T. Pathology and functional diagnosis of small-fiber painful neuropathy. Acta Neurol Taiwan. 19 (2), 82-89 (2010).
- Kennedy, W. R., Wendelschafer-Crabb, G. Utility of the skin biopsy method in studies of diabetic neuropathy. Electroencephalogr Clin Neurophysiol Suppl. 50, 553-559 (1999).
- Kennedy, W. R. Opportunities afforded by the study of unmyelinated nerves in skin and other organs. Muscle Nerve. 29 (6), 756-767 (2004).
- Verdu, E., et al. Physiological and immunohistochemical characterization of cisplatin-induced neuropathy in mice. Muscle Nerve. 22 (3), 329-340 (1999).
- Ko, M. H., Hu, M. E., Hsieh, Y. L., Lan, C. T., Tseng, T. J. Peptidergic intraepidermal nerve fibers in the skin contribute to the neuropathic pain in paclitaxel-induced peripheral neuropathy. Neuropeptides. 48 (3), 109-117 (2014).
- Hsieh, S. T., Chiang, H. Y., Lin, W. M. Pathology of nerve terminal degeneration in the skin. J Neuropathol Exp Neurol. 59 (4), 297-307 (2000).
- Tseng, T. J., Hsieh, Y. L., Ko, M. H., Hsieh, S. T. Redistribution of voltage-gated sodium channels after nerve decompression contributes to relieve neuropathic pain in chronic constriction injury. Brain Res. 1589, 15-25 (2014).
- Hsieh, Y. L., Lin, W. M., Lue, J. H., Chang, M. F., Hsieh, S. T. Effects of 4-methylcatechol on skin reinnervation: promotion of cutaneous nerve regeneration after crush injury. J Neuropathol Exp Neurol. 68 (12), 1269-1281 (2009).
- Tseng, T. J., Chen, C. C., Hsieh, Y. L., Hsieh, S. T. Effects of decompression on neuropathic pain behaviors and skin reinnervation in chronic constriction injury. Exp Neurol. 204 (2), 574-582 (2007).
- Hsieh, Y. L., Chiang, H., Tseng, T. J., Hsieh, S. T. Enhancement of cutaneous nerve regeneration by 4-methylcatechol in resiniferatoxin-induced neuropathy. J Neuropathol Exp Neurol. 67 (2), 93-104 (2008).
- Hsieh, Y. L., et al. Role of Peptidergic Nerve Terminals in the Skin: Reversal of Thermal Sensation by Calcitonin Gene-Related Peptide in TRPV1-Depleted Neuropathy. PLoS One. 7 (11), e50805 (2012).
- Neubert, J. K., et al. Peripherally induced resiniferatoxin analgesia. Pain. 104 (1-2), 219-228 (2003).
- Almasi, R., Petho, G., Bolcskei, K., Szolcsanyi, J. Effect of resiniferatoxin on the noxious heat threshold temperature in the rat: a novel heat allodynia model sensitive to analgesics. Br J Pharmacol. 139 (1), 49-58 (2003).
- Karai, L., et al. Deletion of vanilloid receptor 1-expressing primary afferent neurons for pain control. J Clin Invest. 113 (9), 1344-1352 (2004).
- Apostolidis, A., et al. Capsaicin receptor TRPV1 in urothelium of neurogenic human bladders and effect of intravesical resiniferatoxin. Urology. 65 (2), 400-405 (2005).
- Helyes, Z., et al. Antiinflammatory and analgesic effects of somatostatin released from capsaicin-sensitive sensory nerve terminals in a Freund’s adjuvant-induced chronic arthritis model in the rat. Arthritis Rheum. 50 (5), 1677-1685 (2004).
- Kissin, I., Bright, C. A., Bradley, E. L. Selective and long-lasting neural blockade with resiniferatoxin prevents inflammatory pain hypersensitivity. Anesth Analg. 94 (5), 1253-1258 (2002).
- Helyes, Z., et al. Inhibitory effect of anandamide on resiniferatoxin-induced sensory neuropeptide release in vivo and neuropathic hyperalgesia in the rat. Life Sci. 73 (18), 2345-2353 (2003).
- Kissin, I. Vanilloid-induced conduction analgesia: selective, dose-dependent, long-lasting, with a low level of potential neurotoxicity. Anesthesia and analgesia. 107 (1), 271-281 (2008).
- Pan, H. L., Khan, G. M., Alloway, K. D., Chen, S. R. Resiniferatoxin induces paradoxical changes in thermal and mechanical sensitivities in rats: mechanism of action. J Neurosci. 23 (7), 2911-2919 (2003).
- Iadarola, M. J., Mannes, A. J. The vanilloid agonist resiniferatoxin for interventional-based pain control. Current topics in medicinal chemistry. 11 (17), 2171-2179 (2011).
- Zimmermann, M. Ethical guidelines for investigations of experimental pain in conscious animals. Pain. 16 (2), 109-110 (1983).
- Chaplan, S. R., Bach, F. W., Pogrel, J. W., Chung, J. M., Yaksh, T. L. Quantitative assessment of tactile allodynia in the rat paw. J Neurosci Methods. 53 (1), 55-63 (1994).
- Cliffer, K. D., et al. Physiological characterization of Taxol-induced large-fiber sensory neuropathy in the rat. Ann Neurol. 43 (1), 46-55 (1998).
- Lipton, R. B., et al. Taxol produces a predominantly sensory neuropathy. Neurology. 39 (3), 368-373 (1989).
- Bennett, G. J., Xie, Y. K. A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man. Pain. 33 (1), 87-107 (1988).
- Ko, M. H., Hsieh, Y. L., Hsieh, S. T., Tseng, T. J. Nerve demyelination increases metabotropic glutamate receptor subtype 5 expression in peripheral painful mononeuropathy. Int J Mol Sci. 16 (3), 4642-4665 (2015).
- Jeftinija, S., Liu, F., Jeftinija, K., Urban, L. Effect of capsaicin and resiniferatoxin on peptidergic neurons in cultured dorsal root ganglion. Regul Pept. 39 (2-3), 123-135 (1992).
- Caudle, R. M., et al. Resiniferatoxin-induced loss of plasma membrane in vanilloid receptor expressing cells. Neurotoxicology. 24 (6), 895-908 (2003).
- Acs, G., Biro, T., Acs, P., Modarres, S., Blumberg, P. M. Differential activation and desensitization of sensory neurons by resiniferatoxin. J Neurosci. 17 (14), 5622-5628 (1997).
- Athanasiou, A., et al. Vanilloid receptor agonists and antagonists are mitochondrial inhibitors: how vanilloids cause non-vanilloid receptor mediated cell death. Biochem Biophys Res Commun. 354 (1), 50-55 (2007).
- Wu, C. H., Ho, W. Y., Lee, Y. C., Lin, C. L., Hsieh, Y. L. EXPRESS: NGF-trkA signaling modulates the analgesic effects of prostatic acid phosphatase in resiniferatoxin-induced neuropathy. Mol Pain. 12, (2016).
- Hsiao, T. H., Fu, Y. S., Ho, W. Y., Chen, T. H., Hsieh, Y. L. Promotion of thermal analgesia and neuropeptidergic skin reinnervation by 4-methylcatechol in resiniferatoxin-induced neuropathy. Kaohsiung J Med Sci. 29 (8), 405-411 (2013).
- Chao, C. C., et al. Pathophysiology of neuropathic pain in type 2 diabetes: skin denervation and contact heat-evoked potentials. Diabetes Care. 33 (12), 2654-2659 (2010).
- Kim, S. H., Chung, J. M. An experimental model for peripheral neuropathy produced by segmental spinal nerve ligation in the rat. Pain. 50 (3), 355-363 (1992).
Cite This Article
Lee, Y., Lu, S., Hsieh, Y. Establishing a Mouse Model of a Pure Small Fiber Neuropathy with the Ultrapotent Agonist of Transient Receptor Potential Vanilloid Type 1. J. Vis. Exp. (132), e56651, doi:10.3791/56651 (2018).