Summary

大鼠背部机械敏感性评估用于慢性背痛的机制研究

Published: August 30, 2022
doi:

Summary

为了开发预防和管理背痛的新型治疗干预措施,需要动物模型从转化角度检查这些疗法的机制和有效性。本协议描述了BMS测试,这是一种评估大鼠背部机械敏感性的标准化方法。

Abstract

腰痛是全世界残疾的主要原因,具有严重的个人、经济和社会后果。为了开发新的治疗方法,需要动物模型从转化角度检查新疗法的机制和有效性。在当前的研究中使用了几种啮齿动物的背痛模型。然而,令人惊讶的是,没有标准化的行为测试被验证来评估背痛模型中的机械敏感性。这对于确认推定背痛的动物对伤害性刺激存在局部超敏反应以及在旨在缓解背痛的干预措施期间监测敏感性至关重要。这项研究的目的是制定一个简单易用的测试来评估大鼠背部的机械敏感性。专门为这种方法制造了一个测试笼;长 x 宽 x 高:50 x 20 x 7 厘米,顶部有不锈钢网。该测试笼允许在背部施加机械刺激。为了进行测试,在感兴趣的区域剃掉动物的背部,并根据需要标记测试区域以在不同日期重复测试。机械阈值是通过将冯弗雷细丝应用于椎旁肌肉来确定的,使用前面描述的上下方法。积极的反应包括(1)肌肉抽搐,(2)拱起(背部伸展),(3)颈部旋转(4)抓挠或舔背部,以及(5)逃跑。该行为测试(背部机械敏感性(BMS)测试)可用于啮齿动物背痛模型的机制研究,以开发预防和管理背痛的治疗干预措施。

Introduction

腰痛(LBP)是全球残疾的主要原因,具有严重的个人,经济和社会后果1234每年,约有37%的人口受到LBP5的影响。腰痛通常在几周内消退,但在 24%-33% 的个体中复发,在 5%-10% 的病例中变为慢性2.为了了解LBP的机制和影响以及不同治疗干预的效果,已经使用了几种LBP动物模型,模拟临床条件或LBP6的某些成分。这些小鼠和大鼠模型可分为以下一个或多个类别:(1)椎间盘性LBP7,8,,(2)根性LBP 8910,11,(3)小关节骨关节炎12和(4)肌肉诱导的LBP1314.由于疼痛不能直接在非人类物种中测量,因此已经开发了许多测试来量化这些模型中的类似疼痛的行为8。这些测试评估由有害刺激(机械力15,16,17,热刺激18192021,2223,24,25)或自发产生的行为26272829

使用机械刺激的方法包括冯弗雷检验1516和兰德尔-塞利托检验17。使用热刺激的方法包括尾部轻弹测试18、热板测试19、哈格里夫斯测试20和热探针测试21。使用冷刺激的方法包括冷板试验22、丙酮蒸发试验23和冷足底试验24自发行为的方法包括鬼脸秤26,挖洞27,负重和步态分析28,以及自动行为分析29尽管有这么多可用的测试,但没有一个是专门为背痛模型设计的。

这项研究的目的是制定一个简单易用的测试来评估大鼠背部的机械敏感性。该技术主要基于应用于后爪足底表面的冯弗雷测试1516。冯弗雷测试的基本原理是使用一系列单丝到感兴趣的区域,提供恒定的预定力。如果大鼠表现出伤害性行为,则反应被认为是积极的。然后可以根据引起响应的细丝计算机械阈值。在本研究中,提供了一种基于冯弗雷测试的简单易行的方法来确定大鼠背部的机械敏感性。

Protocol

该实验方案得到了魁北克三河大学动物护理委员会的批准,并符合加拿大动物护理理事会的指导方针和国际疼痛研究协会(IASP)的研究和伦理问题委员会的指导方针。本研究使用了六只雄性Wistar大鼠(体重:320-450克;年龄:18-22周)。这些动物是从商业来源获得的(见 材料表)。这些大鼠的数据来自先前研究30的较大样本。 1. 实验准备</stron…

Representative Results

该方法用于先前的研究,其中提供了完整的数据和统计数据,以比较CFA和对照大鼠之间的机械敏感性30。来自先前研究中包含的六只大鼠的代表性个体数据(左右阈值的平均值)如图 3 和 表1所示。在基线时,两组之间的机械敏感性相似。在腰肌肌内注射CFA导致CFA注射后机械敏感性从7天显着增加到28天。相比之下,对照(CTL)大鼠没有表现出这?…

Discussion

关键步骤
BMS测试是一种评估大鼠背部机械敏感性的简单方法,无论是在一个时间点还是在几天或几周内重复,当预期会发生变化(疼痛模型)或在药物或非药物干预后。该方法的关键问题包括测试笼,其尺寸必须确保大鼠舒适但不会移动太多。动物的背部必须通过网状天花板保持可接近,以实现可重复的机械刺激。为了限制阈值评估的变异性,必须剃掉被研究的背部区域,以便将?…

Divulgations

The authors have nothing to disclose.

Acknowledgements

这项工作得到了魁北克脊椎基金会和加拿大自然科学与工程研究理事会(MP:grant #06659)的资助。香港的贡献得到了魁北克三河大学(PAIR计划)的支持。BP的贡献得到了魁北克健康研究基金会(FRQS)和魁北克脊椎基金会的支持。TP的贡献得到了加拿大自然科学和工程研究理事会的支持。NE和EK的贡献得到了魁北克脊椎基金会的支持。MP 的贡献得到了 FRQS 的支持。

Materials

Aerrane (isoflurane, USP) – Veterinary Use Only Baxter NDC 10019-773-60 Inhalation Anaesthetic ; DIN 02225875, for inducing anasthesia
Complete Freund Adjuvant (CFA) Fisher Scientific #77140 Water-in-oil emulsion of Complete Freund Adjuvant (CFA) with killed cells of Mycobacterium butyricum.
Male Wistar Rats Charles River Laboratories body weight: 320–450 g; age: 18-22 weeks.
Penlon Sigma Delta Vaporizer Penlon 990-VI5K-SVEEK Penlon Sigma Delta Vaporizer used for anasthesia
Sharpie Permanent Marker Sharpie BC23636 Permanent Marker, Fine Point, Black
Test cage Custom-made Width: 20 cm;  Length: 50 cm; Height from the bottom to the top: 40 cm; Height from the bottom mesh to the top of the cage: 7 cm; Wall thickness: 5 mm; Mesh: 1 mm wire with an 8 mm inter-wire distance   
Von Frey Filaments Aesthesio, Precise Tactile Sensory Evaluator 514000-20C Filaments from 0.07 g to 26 g
Wahl Professional Animal, ARCO Cordless Pet Clipper, Trimmer Grooming  Wahl Kit #8786-1201 Animal hair trimmer, for shaving purposes, zero blade 

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Khosravi, H., Eskandari, N., Provencher, B., Paquette, T., Leblond, H., Khalilzadeh, E., Piché, M. Back Mechanical Sensitivity Assessment in the Rat for Mechanistic Investigation of Chronic Back Pain. J. Vis. Exp. (186), e63667, doi:10.3791/63667 (2022).

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