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Biologia

鼻电位差量化小鼠跨上皮离子转运

Published: July 04, 2018
doi:
10.3791/57934
,
1Louvain Center for Toxicology and Applied Pharmacology (LTAP), Institut de Recherche Expérimentale et Clinique (IREC),Université Catholique de Louvain

Summary

在这里, 我们提出了一个测量小鼠鼻电位差异的协议。该试验量化了囊性纤维化跨膜电导调节剂和上皮钠通道等跨膜离子转运蛋白的功能。评价新疗法对囊性纤维化的疗效有重要意义。

Abstract

鼻电位差试验已用于近三年来协助诊断囊性纤维化 (CF)。它已证明是有益的, 在减毒, 寡聚或单症状形式的 cf 通常诊断的生活后期, 和 CF 相关的障碍, 如先天性双侧缺失输精管, 特发性慢性胰腺炎, 过敏支气管肺曲霉病和支气管扩张症。在临床和前期的设置, 该测试已被用作一个生物标志, 以量化反应的靶向治疗策略的 CF. 将测试调整为鼠标是有挑战性的, 可能会导致相关的死亡率。本文介绍了保持鼻腔导管原位连续灌注所需的足够的麻醉深度。它列出了避免支气管在鼻腔内灌注溶液的方法。它还描述了动物护理在测试结束时, 包括管理麻醉药物解毒剂的组合, 导致迅速逆转麻醉与完全恢复的动物。从 cf 和野生型鼠标获得的代表性数据表明, 该测试在 cf 和非 cf 之间有区别。总之, 这里描述的协议允许可靠地测量在自发性呼吸小鼠中的反式上皮氯和钠转运体的功能状态, 以及在同一动物中进行多次试验, 同时减少与试验有关的死亡率。

Introduction

近三年来, 电电位差 (PD) 测量已被用来评估跨膜离子转运蛋白在鼻腔粘膜表达的功能状态, 作为远端气管1的代表。作为一个多步法的动态测试2,3, 鼻腔 PD 允许功能解剖的囊性纤维化跨膜电导调节剂 (CFTR) 和上皮钠通道 (ENaC) 活动, 均在顶端膜局部上皮细胞及在气道表面水化中发挥关键作用。鼻腔 PD 试验的主要临床应用是协助诊断 CF, 这是高加索人群中最常见的致命遗传紊乱, 平均发病率为 1, 在欧洲国家2500活产。这项试验长期证明有助于诊断的减毒, 寡聚或单症状的 cf 通常诊断的形式, 在以后的生活, 和 cf 相关的障碍, 如先天性双侧缺输精管, 特发性慢性胰腺炎, 过敏支气管肺曲霉病, 支气管扩张症4。最近, clinometric 评估的治疗调制基本 CFTR 缺损5,6,7,8,9,10,11 ,12,13,14,15,16已利用鼻腔 PD 在临床试验的新 CF 疗法。在临床前的设置, 测试已经适应了鼠标17 , 以允许调查的生物活性的新 CF 靶治疗18,19,20,21。在小鼠, 该技术是微妙的, 基于物种相关的解剖差异的鼻子区域之间的啮齿动物和人类, 主要是对感官输入的重要作用, 从 nasofacial 地区的啮齿动物。它需要训练有素和熟练的操作员、专用设备和用品。

CF 是一种多系统的外分泌腺体紊乱, 其中慢性呼吸道疾病占主导地位的临床图片。该病是由基因编码的突变所造成的循环腺苷磷酸 (阵营) 调节 CFTR 氯通道22。到目前为止, 已经发现了2000多 CFTR 突变23。最常见的突变24,25, 发现在约90% 的 CF 等位基因, 对应于删除的苯丙氨酸在位置508的多肽链的蛋白质 (F508del-CFTR)。CFTR 蛋白是纯欧姆的小电导氯通道。还有大量证据表明, CFTR 管制其他运输机制, 特别是 ENaC26、27。有缺陷的电解质输送, 包括降低 CFTR 依赖氯电导率和增加 ENaC 依赖性钠电导率, 是 CF 上皮的一个标志。前者的缺陷是由减少或废除的复极化反应的电化学梯度有利于氯外流和添加肾上腺素 (β肾上腺素激动剂, 增加细胞内的阵营) 或佛司可林 (腺苷酸酶激动剂, 不批准用于临床使用)。后者的缺陷是由鼻粘膜基底极化 (一个更负的 PD) 反映, 并增加对阿米洛利的反应, 一种利尿药物, 阻止 ENaC28。

cf 小鼠模型在 cf 研究中得到了广泛的应用, 对 cf 病理学的解剖具有重要的价值。目前, 至少有十五种模型被描述为29, 其中三是纯合的, 最临床相关的 F508del 变异30,31,32。其中一个三菌株30, 在鹿特丹的伊拉斯大学开发, 已经使用了近20年在大学天主教 de 鲁汶 (伦敦学院) 实验室。Cftrtm1Eur模型30已证明是非常有用的研究的多器官病理生理学的 CF 疾病和测试疗效的新的治疗策略18,19,20, 21。小鼠鼻腔 PD 试验 (< 24 小时) 后或术后早期可能出现许多问题。本文叙述了保持鼻腔导管原位连续灌注所需的足够的麻醉深度, 以及避免鼻内灌注的支气管的方法。在测试结束时的动物护理也被描述, 包括管理麻醉药物解毒剂的组合, 导致迅速逆转麻醉与完全恢复的动物。总之, 这些程序允许可靠的测量在自发呼吸的老鼠, 减少测试相关的死亡率和重复测试在同一动物。显示并讨论了在 CF 和野生型小鼠鼻腔 PD 试验中获得的代表性数据。

小鼠鼻腔 PD 测试协议报告在三次会议: 评估和管理之前, 期间和之后的测试。在试验前评估和管理中, 详细介绍了双腔鼻腔导管的制备和连续鼻腔灌注的解决方案。在测试的评估和管理部分, 对鼠标的实验设置和处理进行了详细的分析。最后, 对动物在试验结束时的管理进行描述, 以改善动物的完整恢复。

Protocol

研究和规程由学院的动物研究的道德委员会 (2017/伦敦大学/马里兰州/015) 批准, 并与欧洲共同体关于研究动物使用的条例 (中欧 n° 86/609) 达成一致。调查人员在欧洲议会 2010/63/欧盟和2010年9月22日理事会关于保护用于科学目的的动物的指导下, 有资格进行动物试验。 1. 试验前评估和管理 准备双腔鼻导管。注: 鼻导管为双腔毛细管, 一腔用于连续灌注溶液, 另一种为测量?…

Representative Results

为了说明 CF 中的特征离子传输异常, 在 F508del-CF 小鼠的上述协议和 FVB/129 遗传背景的野生型控制下, 进行了鼻 PD 测量, 从布鲁塞尔殖民地Cftrtm1Eur小鼠30。这个临床相关的模型, 窝藏最常见的和最严重的 F508del-CFTR 突变23,24,25, 是最好的目前可用 CF 鼠标模型30,…

Discussion

本文的目的是描述一个适当的协议, 以测量连续灌注的解决方案, 在自发呼吸小鼠的时间长度, 以测试的完整性, 离子转运体, 主要是 CFTR 和 ENaC。该议定书的所有步骤都经过仔细的优化, 以确保完整的动物恢复和良好的质量和可重现的数据。特别是, 关键步骤是麻醉评估和管理, 并在测试期间和之后适当的动物位置和护理。

先前的研究表明, 2 级的麻醉暴露, 可以通过应用这里使?…

Declarações

The authors have nothing to disclose.

Acknowledgements

作者感谢 j. Lebacq 教授对手稿的严谨编辑。Cftrtm1Eur (纯合 F508del-CFTR (FVB/129) 小鼠是由荷兰鹿特丹的伊拉斯 MC 开发的, 支持欧洲经济共同体欧洲协调行动, 为囊性纤维化的研究欧盟 FP6LHHM-CT-2005-018932。

Materials

Portex polyethylene tube  Smiths Medical, Hythe, Kent, England CT21 6JL Portex 800/100/500;2.0mm ID, 3.0 mmOD to prepare capillary tubes for nasal probe
Electrode cream Parker, Fairfield, NJ, USA Redux cream to build electrode bridges
Ag/AgCl electrodes Biomedical, Clinton Township, MI, USA JNS BNT131-1,0 measuring and reference electrodes
amiloride hydrochloride Sigma, St Louis, MI, USA A7410 to prepare perfusion solutions
forskolin Sigma, St Louis, MI, USA F6886 to prepare perfusion solutions
Knick Portamess voltmeter Elektronisch Meβgeräte, Berlin, Germany Portavo 904 pH to measure potential difference
Paraly SW 112 Software  Elektronisch Meβgeräte, Berlin, Germany Paraly SW112 software to capture potential difference data
midazolam  Mylan, Hoeilaart, Belgium Dormicum 15mg/3ml to serve as anaesthetic premedication
fentanyl Janssen Cilag, Berchem, Belgium Fentanyl-Janssen 0.05 mg/ml to serve as anaesthetic medication
medetomidine Orion Pharma, Espoo, Finland Domitor 1 mg/ml to serve as anaesthetic medication
droperidol  Janssen  Cilag, Berchem, Belgium Dehydrobenzperidol 2.5 mg/ml to serve as anaesthetic medication
clonidine  Boehringer Ingelheim Pharma KG, Ingelheim am Rhein, Germany Catapressan 0.15 mg/ml, to serve as anaesthetic medication
refernce IV catheter Becton Dickinson, Sandy, UT, USA 24 GA x 0.75 IN, BD Insyte-W to build electrode bridges
forceps  Fine science Tools, Heidelberg, Germany Dumont #5, Fine science Tools to place the nasal catheter
naloxone  Braun Medical, Brussels, Belgium Narcan, 0.4 mg/ml to serve as anaesthetic antagonist
atipamezole  Zoetis, Bloomberg, Belgium Antisedan, 5 mg/ml to serve as a medetomedine specific antidote 
Heating pads  Harvard Apparatus, Holliston, MA, USA 18,8×37,5 cm; 15,5×15,5 cm to avoid hypothermia during and after the test
Peristaltic pump P1 GE Life Sciences, Uppsala, Sweden 18111091 to perfuse solutions in the mouse nose
cyanoacrylate glue Loctite, Henkel, Düsseldorf, Germany  super glue 3 to glue together two capillary tubes  for nasal probe
NaCl Sigma, St Louis, MI, USA RES0926S-A7 Pharma-Grade, USP
CaCl2.2H2O Sigma, St Louis, MI, USA M7304 Pharma-Grade, USP
MgCl2.6H2O Sigma, St Louis, MI, USA 1551128 Pharma-Grade, USP
K2HPO4 Sigma, St Louis, MI, USA 1551139 Pharma-Grade, USP
Na gluconate Sigma, St Louis, MI, USA S2054 Pharma-Grade, USP
Ca gluconate Sigma, St Louis, MI, USA C8231 Pharma-Grade, USP
MgSO4.7H2O Sigma, St Louis, MI, USA RES0089M-A7 Pharma-Grade, USP
BD needle  Becton Dickinson, Franklin Lakes, USA BD 26G (0.45×10 mm) intraperitoneal injection
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Nasal Potential DifferenceCystic FibrosisTransgenic MiceIon TransportDouble Lumen Nasal CatheterElectrolyte CreamVoltmeterPeristaltic PumpData Capture Software
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Beka, M., Leal, T. Nasal Potential Difference to Quantify Trans-epithelial Ion Transport in Mice. J. Vis. Exp. (137), e57934, doi:10.3791/57934 (2018).
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