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Médecine

局部或全身药物干预后评估的变化小鼠中挥发性全身麻醉药敏感性

Published: October 16, 2013
doi:
10.3791/51079
, ,
1Department of Anesthesiology and Critical Care,Perelman School of Medicine, University of Pennsylvania, 2Department of Pharmacology,Perelman School of Medicine, University of Pennsylvania, 3Department of Neuroscience,Perelman School of Medicine, University of Pennsylvania, 4Center for Sleep and Circadian Neurobiology,Perelman School of Medicine, University of Pennsylvania

Summary

翻正反射消失长期以来一直作为一个标准的代孕行为的无意识，也叫催眠，在实验室动物。改变中的挥发性麻醉剂的敏感性引起的药理学干预可以仔细控制的高通量的评估系统，该系统可适合用于递送任何吸入治疗进行检测。

Abstract

全身麻醉的一个理想的端点是无意识的状态，也称为催眠。限定在动物催眠状态是较复杂的比它是在人类患者。在啮齿类动物中广泛使用的代孕行为对催眠是翻正反射（翻正反射消失），或在哪个动物不再响应自己与生俱来的本能，以避免背部斜卧的脆弱点的损失。我们已经开发了一个系统，在24只小鼠同时评估翻正反射消失，同时小心地控制潜在的困惑，包括温度波动和变化的气流。这些商会允许麻药敏感性可靠的评估，通过延迟测量以下一个固定的麻醉曝光返回翻正反射（RORR）的。可替代地，在麻醉浓度采用逐步增加（或减少）时，腔室还能够确定一个群体的灵敏度感应（或出现），作为由测量EC 50和希尔斜率。最后，这里所描述的控制的环境腔室可适于用于各种其他用途，包括吸入输送的其他药物，毒物学研究及生命体征的同时实时监控。

Introduction

全身麻醉是由他们造成的各种各样的品种，但在解释如何毒品这样一个多样化的类都可以引出一个单一的端点仍然遥遥无期催眠的可逆状态的能力来定义。一些理论已经假定多年来，从麻醉效能和脂溶性的迈耶-奥弗顿的相关性，这表明一般膜破坏为基础进行催眠1,2。最近的证据表明，影响神经信号蛋白指标来麻醉作用作出贡献。小鼠已被证明是探索这些理论，因为小鼠和人的麻醉剂响应之间的同源性的一个不可缺少的模型。虽然鼠标不能询问其在全身麻醉下的主观意识，某些原始反射服务啮齿动物催眠作为有用的替代措施。在出生后的头几天，老鼠制定一个反身扶正RESPONSE，防止他们从被动地被放置在仰卧位3。麻醉的剂量在哪个鼠标失去其翻正反射以及相关人类催眠剂量4。

翻正反射（翻正反射消失）的损失评估，已经成为一种广泛使用的实验室标准在小鼠试验麻醉药的敏感性，以及其他多种物种，包括大鼠，豚鼠，兔，雪貂，羊，狗5-8。一个给定的麻醉剂在其翻正反射消失将发生一个物种的成员的剂量是非常一致的，但它可以显著由环境因素进行移位。例如，睡眠剥夺大鼠是易失性和静脉麻醉药9和大鼠高的有氧代谢能力更敏感是异氟醚10不太敏感。低温也已经显示减少的物种11-14的一个大范围所需的催眠许多麻醉药的剂量。为了可靠地识别麻醉剂的剂量在该LORR发生在一组实验动物，这是至关重要的评估环境被严格控制，以最大限度地减少应力，保持euthermia，并提供药物等量的所有受试者。这并不奇怪，遗传因素也被称为改变麻醉剂灵敏度15-18。因此，应认真考虑并给予控制的遗传背景19。

我们已经制定，以确保相同的麻醉气体输送到每个24小鼠的同时保持恒定37℃环境下的设备。我们的曝光室的透明圆柱形设计允许快速翻正反射消失评估和易于集成的遥测生理测量。此系统已被证明是准确测量的异氟醚，氟烷，七氟醚诱导EC 50和时间出现在野生型小鼠20。我们也使用这个系统观察小鼠的变化麻醉敏感性与基因突变和有针对性的下丘脑病变21-23。这里我们描述了两种方法，其中麻醉剂的敏感性可能以后使用我们的控制环境装置的药物干预进行评估。挥发性麻醉诱导和苏醒灵敏度稳态表型，需要8-10个小时，并因此最好专门针对研究中，实验条件不改变，如慢性或长效药物干预。然而，对于短效的治疗，其效果显著随时间消散，我们也提出了一个简单的程序，以评估下列立体定位，有针对性的显微注射或静脉注射药物治疗的显著影响麻醉中出现翻正反射的变化。这些测试代表一个小的子集的此受控环境系统中的潜在应用，这可以适用于任何数量的主旨的各种品种的学分收取任何类型的吸入治疗的。

Protocol

所有涉及此概括动物的程序已获宾夕法尼亚州的机构动物护理和使用委员会的大学。 1。该测试装置的概述该试验装置由24个透明的丙烯酸圆柱形腔室10厘米的长度和直径5厘米（2​​00毫升总体积）。这个尺寸适用于典型的25克成年小鼠。钱伯斯有端口每端的气体入口和出口。出口端是可移动的，这样的动物可以被很容易地装入腔室中。气口开口仔细密封带聚四氟乙烯?…

Representative Results

图1显示了逐步LORR测定用于确定药物干预的长期效果的工具。鹅膏蕈氨酸（IBA）是谷氨酸的N-甲基-D-天冬氨酸（NMDA）受体是经常被用来作为兴奋性毒素引起永久性神经病变的激动剂。在这里，我们注入的10 NL 1％IBA的双边入C57BL/6J小鼠的腹外侧视前区（VLPO）前一周的测试。在这个核心的大多数神经元具有发射率低清醒时和在非快速眼动睡眠，快速眼动睡眠，并与接触催眠剂量的全身麻?…

Discussion

虽然在一个单一的鼠标评估LORR的是一个看似简单的任务，但它仍然是必不可少的，以保持学科之间是相同的生理条件，以便收集从一组动物的可靠数据。这里提出的严格调控，高容量翻正反射消失设备提供了一种标准化的实验和最大限度地提高效率。按照体温调节和平等的流量分配的基本原则，这个系统可以很容易地重新创建和定制，以适应个人的实验者的需求。室尺寸可以按比例用于其它物种…

Divulgations

The authors have nothing to disclose.

Acknowledgements

这项工作是由R01 GM088156和T32 HL007713-18的支持。我们要感谢比尔彭涅和迈克尔·卡曼来自宾夕法尼亚州研究仪器中心的大学为他们在我们的组装翻正反射装置的帮助。

Materials

Name of the Reagent Company Catalogue Number Comments
Oxygen Airgas OX300
Isoflurane Butler Schein Any volatile anesthetic of interest may be substituted
Name of Material Company Catalogue Number Comments
Mass flow meter- 10 SLPM Omega Engineering FMA-A2309
Mass flow meter- 500 SCCM Omega Engineering FMA-A2305
Anesthetic agent analyzer/gas indicator AM Bickford FI-21 Riken
Heating water pump Fisher Scientific 13-874-175
Temperature transponders BMDS IPTT-300
RF temperature reader BMDS DAS-6007

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References

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Tags

Keywords: General AnesthesiaHypnosisLoss Of Righting Reflex (LORR)Return Of Righting Reflex (RORR)Anesthetic SensitivityEC50Hill SlopeControlled Environmental ChambersInhaled Drug DeliveryToxicology StudiesVital Signs Monitoring
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McCarren, H. S., Moore, J. T., Kelz, M. B. Assessing Changes in Volatile General Anesthetic Sensitivity of Mice after Local or Systemic Pharmacological Intervention. J. Vis. Exp. (80), e51079, doi:10.3791/51079 (2013).
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