大鼠复杂区域疼痛综合征I型慢性缺血后疼痛模型
Summary
此处提供了一个协议，详细说明了建立慢性缺血后疼痛（CPIP）动物模型的步骤。这是一个公认的模型，模仿人类复杂的区域疼痛综合征I型。进一步评价机械和热超敏感性，以及在CPIP大鼠模型中观察到的辣椒素引起的角感行为。
Abstract
复杂的区域疼痛综合征I型（CRPS-I）是一种神经系统疾病，在患者中引起剧烈疼痛，并且仍然是一个未解决的医疗状况。然而，CRPS-I的基本机制尚未透露。众所周知，缺血/再灌注是导致CRPS-I的主要因素之一。通过长期缺血和后肢再灌注，建立了大鼠慢性缺血后疼痛（CPIP）模型，以模仿CRPS-I。CPIP模型已成为研究CRPS-I机制的公认动物模型。该协议描述了建立CPIP大鼠模型所涉及的详细程序，包括麻醉，然后是后肢缺血/再灌注。通过测量后肢的机械和热超敏感性以及急性辣椒素注射的角质反应，进一步评价大鼠CPIP模型的特征。大鼠CPIP模型表现出几种CRPS-I样的表现，包括建立后早期后肢水肿和高血症，持续热和机械过敏，以及急性辣椒素注射增加的角质反应。这些特性使其成为进一步研究CRPS-I中涉及的机制的合适动物模型。
Introduction
复杂的区域疼痛综合征 （CRPS） 引起骨折、创伤、手术、缺血或神经损伤1、2、3引起的复杂和慢性疼痛症状。CRPS分为2个子类别：CRPS类型I和II型（CRPS-I和CRPS-II）4。流行病学研究表明，CRPS的流行率约为1：20005。CRPS-I，显示没有明显的神经损伤，可能导致慢性疼痛，并极大地影响患者的生活质量。目前可用的治疗方法显示治疗效果不足。因此，CRPS-I 仍然是一个需要解决的重要且具有挑战性的临床问题。
建立模仿CRPS-I的临床前动物模型对于探索CRPS-I背后的机制至关重要。为了解决这个问题，Coderre等人设计了一个大鼠模型，将长期缺血和再灌注到后肢，以重述CRPS-I6。据了解，缺血/再灌注损伤是CRPS-I7的主要原因之一。大鼠CPIP模型表现出许多CRPS-I样的症状，包括后肢水肿和高血症在模型建立后的早期阶段，其次是持续热和机械高灵敏度6。在此模型的帮助下，建议中央疼痛敏化、外周TRPA1通道激活和活性氧物种生成等对CRPS-I8、9、10作出贡献。我们最近成功地建立了CPIP大鼠模型，并进行了背根结核（DRGs）的RNA测序，内骨对受影响的后爪11进行内脏测序。我们发现了一些潜在的机制，可能参与调解CRPS-I11的疼痛过度敏感。我们进一步确定DRG神经元中的瞬态受体电位万能1（TRPV1）通道是CRPS-I12的机械和热超灵敏度的重要原因。
在这项研究中，我们描述了建立CPIP的老鼠模型所涉及的详细程序。通过测量机械和热超敏感性及其对急性辣椒素挑战的反应能力，我们进一步评估了大鼠CPIP模型。提出大鼠CPIP模型是进一步研究CRPS-I中相关机制的可靠动物模型。
Protocol
动物规程经浙江医科大学动物伦理委员会批准。 1. 动物 从上海实验室动物中心获得雄性斯普拉格-道利（SD）大鼠（280~320克，8-10周龄）。在浙江中医药大学动物实验室动物中心饲养动物。请注意，繁殖条件应包括 12 h/ 2h 的光/暗周期，并保持温度稳定在 24°C。提供水和食物。请注意，本研究共使用了48只大鼠。有一点，在这个模型中，我们需要观察整个过程中的…
Representative Results
在脚踝上放置O环后，叶侧后爪皮肤出现青紫，指示组织缺氧（图1A）。切割O环后，叶侧后爪开始充满血液，并表现出强健的肿胀，这显示了高血症的强烈迹象（图1A）。爪子肿胀逐渐减少，并在缺血/再灌注程序后恢复正常的48小时（与Sidak的双向ANOVA，即位测试，图1B）。所有这些迹象都符合?…
Discussion
该协议描述了通过在大鼠后肢应用缺血/再灌注来建立大鼠CPIP模型的详细方法。它涉及评估后肢外观，水肿，机械/热过敏性，和急性角质行为，以回应辣椒素注射。
肢体缺血/再灌注是导致CRPS-I在人类患者12的常见因素。该协议描述了如何建立大鼠CPIP模型，这是一个常用的动物模型来概括人类CRPS-I6。在麻醉下，在大鼠的后肢诱导了缺血，在…
Declarações
The authors have nothing to disclose.
Acknowledgements
该项目由中国国家自然科学基金（81873365和81603676）、浙江省杰出青年自然科学基金（LR17H270001）和浙江中医药大学（）资助。Q2019J01， 2018ZY37， 2018ZY19）.
Materials
| 1.5 ml Eppendorf tube | Eppendorf | 22431021 | |
| DMSO | Sigma-Aldrich | D1435 | |
| Capsaicin | APEXBIO | A3278 | |
| Digital caliper | Meinaite | NA | |
| O-ring | O-Rings West | Nitrile 70 Durometer | 7/32 in. internal diameter |
| Plantar Test Apparatus | UGO Basile, Italy | 37370 | |
| von Frey filaments | UGO Basile, Italy | NC12775 |
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Citar este artigo
Hu, Q., Zheng, X., Chen, R., Liu, B., Tai, Y., Shao, X., Fang, J., Liu, B. Chronic Post-Ischemia Pain Model for Complex Regional Pain Syndrome Type-I in Rats. J. Vis. Exp. (155), e60562, doi:10.3791/60562 (2020).