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。

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<ol><li>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生料带，而橡胶O型圈垫片用来密封的圆柱形腔的可拆卸的。 </li><li>每个室被安装在一个机架上，它位于内部的水浴中。齿条配合，使得腔室（下面进气口），仅下部被淹没。为稳定，该腔室的后端靠在支承，这样整个CHAMB呃坐镇水平。这将确保同浴的整个腔甚至接触。 </li><li>聚乙烯管材氧气罐连接到麻醉剂蒸发器，然后通过一个10升/分钟流量仪表。管子分成长度相等的25小直径的电阻，以确保相等的流量输送到各腔室24中和至代理分析器。 </li><li>真空管线退出每个腔室在气体入口的另一端。这促进了单向流动，消除呼出的二氧化碳的再呼吸。真空线结合在一个歧管连接到一个内部的吸入管路。沿主真空线的弹出式截止阀，确保每个腔室中的大气压力条件。 </li><li>浴填充有足够的水充分接触的每个腔室的底部。的水通过浴用泵循环，并保持在一恒定37℃。 </li></ol> 2。检查系统之前曝光<ol><li> Ç赫克说水浴的温度在整个浴37℃。 </li><li>氧气流的速度为5升/分（200毫升/每室+代理分析器分钟）。淹没每个腔室在水中，寻找气泡或水进入室内，这两者是指示泄漏。在开始实验前封住泄漏。 </li><li>对于每个腔室，连接在线路500毫升/分钟的流量计腔后，以确保流在每个25个气体管道的平衡。这确保了输入5升/分钟流量将被均匀地分布，使得每个室接收200毫升/分钟流动。没有收到预期的任何流室应该有它的流入和流出管道检查障碍物。 </li><li>校准试剂分析仪，以确保当100％的氧气流过0.00％的异氟醚的读数。 </li></ol> 3。注入温度转发<ol><li>前一星期习惯，麻醉每只小鼠用2％异氟醚。 </li><li>消毒用优碘背颈部。 </li><li>注射用无菌的，预包装注射器针头肩胛骨之间的温度下转发皮下注射。 </li><li>监视注射部位每日进行感染和应答器的迁移。 </li></ol> 4。习惯于对动物测试钱伯斯<ol><li>第一次评估前四天，将所有小鼠成2小时100％的氧气流单独的腔室。 </li><li>四个日前评估，避免因新的环境压力的影响因子重复步骤4.1日。 </li></ol> 5。进行药物干预是您希望测试效果上麻醉灵敏度<ol><li>这种干预可能是一个立体定位注射入脑24，静脉内或腹膜内注射25或递送药物至特定脑区域的一个特定的部分通过套管26。 </li><li>因为相对于一个天真的动物这些程序本身可能会改变麻醉剂的敏感性，适当的对照组应该接受同样的程序与车辆注射。 </li><li>确保药物干预行动有一个适当的时间长，如果你打算做一个逐步提高和/或降低测定灵敏度麻醉剂如步骤6所示，否则，请跳到步骤7。 </li></ol> 6。采用逐步EC 50测定感应和出苗评估麻醉灵敏度<ol><li>将每个动物成单独的腔室与100％的氧气流动。 </li><li>设置在异氟醚浓度为0.4％* 15分钟。在此期间的最后2分钟，轻轻滚动腔直到鼠标被放置在它的后面评估每个动物的翻正反射。该翻正反射被认为是完整的，当且仅当鼠标能够恢复所有它的爪子在2分钟内室的地板上。 <ol><li> *请注意，0.4％异氟醚是一种subhypnotic剂量C57BL/6J小鼠。如果任何小鼠失去其翻正反射在第一步骤中，初始剂量为过大，应减少后的日子。 </li></ol></li><li>记录翻正反射为每个鼠标的状态，并扫描每个小鼠为温度数据。模板记录如表1所示。 </li><li>增加的异氟烷浓度〜0.05％，持续15分钟，并重复步骤7.2。继续这样做，直到所有的动物已经失去了它们的翻正反射。 </li><li>可选:重复相同的步骤，以减少分步进行异氟醚的剂量，直到所有的动物都恢复了其翻正反射（见步骤6.3）。 </li><li>要结束实验，关闭异氟醚和冲洗整个系统与100％氧气15分钟。这将有助于防止缺氧的小鼠被送回他们的家笼前恢复，并会保护experimeNTER从任何麻醉风险。 </li><li>可选:如果动物或麻醉剂浓度的数的个数是由于资源或时间约束的限制，曲线拟合参数估计值，尤其是山的斜坡，可能有低估的，虚假的低误差估计。在这种情况下，可能有必要重复在上最多两个额外的实验日步骤6.1-6.6充分获得真实Hill斜率的参数和其相应的误差估计中描述的麻醉剂敏感性测定。 </li></ol> 7。评估麻醉灵敏度的短期变化与时间的出现<ol><li>将每个动物成单独的腔室与100％的氧气流动。 </li><li>设置在异氟醚浓度为1.2％，其对应于感应ED 99为野生型C57BL/6J小鼠20。保持30-60分钟取决于急性干预行动的预期持续时间。 </li><li>在确认翻正反射消失所有的动物轻轻滚动每个腔室，直到老鼠被放置在他们的后面。 </li><li>关闭异氟醚和流量100％的氧气。测量时间，直到每个动物恢复其翻正反射。这是由所有4爪放置在腔室的地板上定义并通过三个连续的测试，一个完整的翻正反射的存在证实。 </li></ol>

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Neurol. , (2013).</li><li>Solt, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methylphenidate+Actively+Induces+Emergence+from+General+Anesthesia.">Methylphenidate Actively Induces Emergence from General Anesthesia.</a> Anesthesiology. 115 (4), 791-803 (2011).</li></ol>

作者什么都没有透露。

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，例如大鼠，和额外的腔室可以通过将更多的分支点，在流入和真空被容纳。所有受试者都容易看到透过透明的丙烯酸室中，这使得它可以将视频记录实验结果。二次事后确认。丙烯酸类也与射频遥测系统，该系统可以用来监视温度兼容TURE，血压，生物电势。 我们提出了两种不同的方法来评估麻醉敏感性继药物干预。两者的时间出现和逐步感应测试需要实验者入网，翻正反射的存在与否。即使翻正反射消失的明确定义，如"无法将所有四只爪子上腔地板两分钟被卷到它的后面内"，评估可能有点主观。最好是有相同的待遇盲个人得分每个动物在实验期间，以确保一致性。当选择使用麻醉剂的敏感性的评估，测试，从药物干预效果的预期长度应是决定性的因素。许多药物具有作用时间短，为此，急性的时间出现的范式可以提供麻醉剂的敏感性有用的信息，在有限的时间的时间。然而，药物可以优先影响动物的敏感性感应催眠而不是出现;变化时进行感应往往难以检测，因为感应迅速发生，因此需要连续的评估。较长的测试逐步诱导和苏醒的EC 50可以给两个入口，并从催眠退出信息。实验的总长度将取决于所采用的增量麻醉剂浓度的改变在每个步骤中，以持续约8小时，典型的感应+出苗试验的尺寸。减小围绕预期的EC 50的麻醉剂的步长，增加各组动物的数量将提供更好的拟合剂量-响应曲线，但也将延长，以完成检测所需的时间。 时相比，他们控制了一些药物干预可以改变差异实验动物的每分钟通气量。 Ŧ他可能会导致一组以比其他更迅速呼出的挥发性麻醉剂的时候出现测试，从而混淆的结果。 SOLT 等 。描述了在这种情况下35测试麻药敏感性一个很好的替代方法。在他们的实验中，全身哌甲酯是在恒定的异氟醚暴露在那些已经与平衡麻醉动物的交付。在连续麻醉风险，因此被排除在每分钟通气量潜在的影响因子为在稳定的状态下麻醉剂的吸收和分布正是代谢和消除平衡。我们所描述的腔室可以被容易地修改一个额外的气密端口，以允许管道的通道，用于静脉内或脑内给药。还应当指出的是平衡的描述15分钟至麻醉剂在分步进行测定各浓度可能不足以在某些情况下。 AnesthetICS比异氟醚，如氟烷具有较高的溶解度，将需要更长的时间才能充分发挥其浓度的组织。较大的动物和动物进行麻醉浓度较大的步骤也可能需要更多的时间来平衡。以确定是否15分钟是真正足够达到平衡，麻醉组织水平在麻醉的同时在上行和曝光的降肢体同一浓度应进行测量。 在移动动物的能力，在物理上或药理学受阻的情况下，翻正反射消失，可能无法作为催眠的良好替代指标。最可靠的和广泛使用的另一种方法是皮层脑电图（EEG）记录。尽管脑电图可能能够更好地拿起麻药敏感性更微妙的变化，这是显著较昂贵的设置比我们所描述的设备。植入EEG电极是一种侵入性和耗时的过程，并且要想获取的能力从多个鼠标数据同时往往是由设备的可用性的限制。此外，脑电图记录的分析是概念比较抽象和难以解释比LORR评估的简单的二进制输出。由于这些原因，行为测试，如上述介绍的往往是比较可行的方法来快速筛选麻醉剂的敏感性。需要注意的是脑电模式暗示的觉醒和催眠可能无法与行为良好的相关性。翻正反射消失及脑电图是截然不同的端点这两者可能提供有关麻醉剂的灵敏度有用的信息。 除了在每分钟通气量和流动性的潜在药物引起的变化外，还有其他一些限制于本文描述的方法。虽然翻正反射消失是一个标准的替代物穿过田野催眠，用于其测量标准和方法各实验室不同。一些主张，小鼠应该以恒定的速度旋转，以评估正位反射。持续评估逻辑上缩小了精确定时与翻正反射消失及/或回报，但是，被翻仰卧的行为可能比单纯其余仰卧更刺激。此外，阶梯式翻正反射消失的评估是，可进一步延长，如果平衡在每一步在15分钟被发现为不足一个耗时测定。 尽管有这些限制，此协议的潜在应用远远超出了我们所提出的具体实例。显然，药物干预是不是唯一的方法，通过它麻醉剂的敏感性可能会改变;可能都使用相同的逐步EC 50测定被测试有针对性的病变，解剖异常和基因突变。这里提出的受控环境中的系统可以用来提供任何种类的吸入药物，如皮质类固醇，抗生素，或实验治疗学。许多小鼠暴露于相同的古物古迹办事处的能力UNT药一次使这个设置非常适合毒理学研究。此外，商会作为与监管环境温度和新鲜氧气流量的理想手术后恢复环境。这个装置是用于任何实例基本动物的生命体征需要监测和控制的，其中有用的。

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。

<table border="0" cellpadding="0" cellspacing="0" width="461">
	<colgroup>
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	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:115px;">Name of the Reagent</td>
			<td style="width:88px;">Company</td>
			<td style="width:88px;">Catalogue Number</td>
			<td style="width:171px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:115px;">Oxygen</td>
			<td style="width:88px;">Airgas</td>
			<td style="width:88px;">OX300</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:115px;">Isoflurane</td>
			<td style="width:88px;">Butler Schein</td>
			<td style="width:88px;"></td>
			<td style="width:171px;">Any volatile anesthetic of interest may be substituted</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:115px;">Name of Material</td>
			<td style="width:88px;">Company</td>
			<td style="width:88px;">Catalogue Number</td>
			<td style="width:171px;">Comments</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:115px;">Mass flow meter- 10 SLPM</td>
			<td style="width:88px;">Omega Engineering</td>
			<td style="width:88px;">FMA-A2309</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:115px;">Mass flow meter- 500 SCCM</td>
			<td style="width:88px;">Omega Engineering</td>
			<td style="width:88px;">FMA-A2305</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:115px;">Anesthetic agent analyzer/gas indicator</td>
			<td style="width:88px;">AM Bickford</td>
			<td style="width:88px;">FI-21 Riken</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:115px;">Heating water pump</td>
			<td style="width:88px;">Fisher Scientific</td>
			<td style="width:88px;">13-874-175</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:115px;">Temperature transponders</td>
			<td style="width:88px;">BMDS</td>
			<td style="width:88px;">IPTT-300</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:115px;">RF temperature reader</td>
			<td style="width:88px;">BMDS</td>
			<td style="width:88px;">DAS-6007</td>
			<td style="width:171px;"></td>
		</tr>
	</tbody>
</table>

assessing changes in volatile general anesthetic sensitivity of mice after local or systemic pharmacological intervention

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。

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醉药23,27-29专门增加他们的活动。成功的病变在VLPO应引起抗异氟醚诱导催眠。在异氟醚的每增加一级，已失去翻正反射消失的小鼠的分数被绘制的对数刻度10麻醉药浓度。数据对各组小鼠（溶媒注入和IBA喷射）然后用合适的S形剂量响应曲线。因为该测定法总是与所有的动物开始直立并总是结束与失去了所有的动物翻正反射，底部和顶部常数限制在0和1，分别。曲线的剩余自由参数是选举委员会50，麻醉剂或在其中小鼠的50％已经失去了他们的翻正反射的浓度和山的斜坡，其中他们的催眠状态转换过程中体现的总体方差。 F检验是用来查询与共享的EC 50和希尔斜率参数的单一感应曲线是否最符合双方车辆和IBA组或不同的参数，更好地分离诱导曲线是否适合数据。从相关曲线拟合的原始数据点产生自由的在本次测试的程度，因而依赖于麻醉剂浓度进行测试的数量和参数被网络连接的数叔EC 50和在这种情况下，Hill斜率。逐步出现的数据进行了分析和建模相同为感应数据。需要注意的是欧盟50出现，几乎总是比诱导下，由于麻醉剂的滞后也被称为神经惯性30。相反，预期结果，接受律师协会在VLPO动物表现出的EC 50或山边坡感应或出现相对于车辆注入控制（F 2,80 = 1.73和P = 0.184诱导，F 2无显著差异， 88 = 2.89 和p = 0.061的出现）。这表明，小鼠VLPO神经元是耐损伤，用1％的IBA，这一事实与验尸组织学（未示出）证实。 Lu 等人 。已经证实，10％的IBA剂量须病变大鼠VLPO 31，但鼠标VLPO的组织学检查以下注射10％IBA还没有表现出显着细胞的损失（未显示）。大鼠VLPO是已知的表达NMDA受体32。由于10％的IBA能够被注入VLPO上施加麻醉剂敏感的急性效应（ 见图2，下面的讨论），这一点证明了鼠标VLPO还必须具备必要的IBA的行动NMDA受体。因此，对于物种之间的差异的原因仍不清楚。使用靶向甘丙肽-皂草素23成功鼠标VLPO病变已经实现。 虽然IBA当注入VLPO不会对异氟醚的敏感性长期效果，这种药物的急性兴奋性质，预计将刺激VLPO神经元和瞬时增加麻醉的敏感性。在图2中，我们使用的时候出现测试，以显示大急转变，异氟醚的敏感性立即双边IBA显微注射下面进入VLPO可见一斑明显延长停止麻醉分娩（P &lt;0.001）后催眠。相反，国际律师协会显微注射入附近的内侧隔导致时间出现没有变化相比，车辆注射对照组（P&gt; 0.05）。这一发现增加了一个有趣的方面到以前的工作表明，这种细胞核失活时间延长至出现33,34。对实验组和对照组中的时间出现的测试数据进行平均，并用单因素方差分析进行比较。 <table border="0" cellpadding="0" cellspacing="0" width="558"><colgroup><col /><col /><col span="4" /></colgroup><tbody><tr height="25"><td height="25" style="height:25px;width:69px;">时间</td><td style="width:137px;">异氟醚（％大气压） </td><td style="width:88px;">鼠标＃1 </td><td style="width:88px;">鼠标＃2 </td><td style="width:88px;">鼠标＃3 </td><td style="width:88px;"> ... </td></tr><tr height="21"><td he洞察力="21"的风格="高度:21px;宽度:69px;"&gt;下午12:00 </td><td style="width:137px;"> 0.4 </td><td style="width:88px;"> - </td><td style="width:88px;"> - </td><td style="width:88px;"> - </td><td style="width:88px;"> - </td></tr><tr height="21"><td height="21" style="height:21px;width:69px;"> 12:15 PM </td><td style="width:137px;"> 0.45 </td><td style="width:88px;"> - </td><td style="width:88px;"> X </td><td style="width:88px;"> - </td><td style="width:88px;"> - </td></tr><tr height="21"><td height="21" style="height:21px;width:69px;">下午12点30分</td><td style="width:137px;"> 0.5 </td><td style="width:88px;"> - </td><td style="width:88px;"> X </td><td style="width:88px;"> X </td><td style="width:88px;"> - </td></tr><tr height="21"><td height="21" style="height:21px;width:69px;"> 12:45 PM </td><td style="width:137px;"> 0.55 </td><td style="width:88px;"> - </td><td style="width:88px;"> X </td><td style="width:88px;"> X </td><td sTYLE ="宽度:88px;"&gt; - </td></tr><tr height="21"><td height="21" style="height:21px;width:69px;"> ... </td><td style="width:137px;"> 0.6 </td><td style="width:88px;"> - </td><td style="width:88px;"> X </td><td style="width:88px;"> X </td><td style="width:88px;"> X </td></tr></tbody></table> 表1示例日志表的长期麻醉敏感性评价的:每15分钟的麻醉剂量增加了0.05％和翻正反射进行了评估每个动物。 "X"表示该已失去正位反射一个给定的时间点和动物" - "表示那些保持其翻正反射。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51079/51079fig1.jpg" width="500" /> 图1。评估翻正反射后一周鹅膏蕈氨酸注射液在腹外侧视前国统会的leus:本公司长期麻药敏感性测定法与任何车辆或鹅膏蕈氨酸（IBA）进行了小鼠注射到腹外侧视前区（VLPO）前一周的测试。感应和出苗数据为每组适合与S形剂量 - 响应曲线（感应在实线所示，出现在虚线）随95％置信区间包围的最佳拟合曲线（阴影柱）。麻醉药浓度绘制在对数10的规模。重叠的95％置信区间都显示为紫色。的S形剂量repsonse适合为汽车和IBA组提出没有证据的基础上EC 50和希尔斜率不同的最适合的曲线。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51079/51079fig1highres.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/51079/51079fig2.jpg" width="500" /> 图2。时间到E合并鹅膏蕈氨酸本地显微注射后:紧接之前的评估，小鼠接受了N-甲基-D-天冬氨酸（NMDA）受体激动剂鹅膏蕈氨酸（IBA）的显微注射入腹外侧视前核（VLPO）。这个区域被称为在异氟醚诱导催眠被激活。 IBA注射导致了时间的急性增加，返回翻正反射相比，注射赋形剂的对照组（P &lt;0.001）。时间出现，为动物IBA注入内侧隔没有从控制差异（P&gt; 0.05）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51079/51079fig2highres.jpg" target="_blank">点击这里查看大图</a> 。

Watch this Scientific Journal Video about Assessing Changes in Volatile General Anesthetic Sensitivity of Mice after Local or Systemic Pharmacological Intervention at JoVE.com

Assessing Changes in Volatile General Anesthetic Sensitivity of Mice after Local or Systemic Pharmacological Intervention

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。

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

One desirable endpoint of general anesthesia is  the state of unconsciousness, also known as hypnosis. Defining the hypnotic state in animals is  less ...

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Nanyang Technological University

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Education

JoVE Journal

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Pharmacology

Medicine

Unit 1

Local Anesthetics

Drugs for Pain Management: Opioid Analgesics and General Anesthetics

SCIENTISTS IN ACTION

13.5K 次观看.  Perelman School of Medicine, University of Pennsylvania. 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。 

视频: Assessing Changes in Volatile General Anesthetic Sensitivity of Mice after Local or Systemic Pharmacological Intervention

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.

In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.

在斑马鱼模型的胎儿酒精暴露评估致畸的变化

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

科研

医学

Modeling Intracerebral Hemorrhage in Mice: Injection of Autologous Blood or Bacterial Collagenase

Spontaneous intracerebral hemorrhage (ICH) defines a potentially life-threatening neurological malady that accounts for 10-15% of all stroke-related hospitalizations and for which no effective treatments are available to date1,2. Because of the heterogeneity of ICH in humans, various preclinical models are needed to thoroughly explore prospective therapeutic strategies3. Experimental ICH is commonly induced in rodents by intraparenchymal injection of either autologous blood or bacterial collagenase4. The appropriate model is selected based on the pathophysiology of hemorrhage induction and injury progression. The blood injection model mimics a rapidly progressing hemorrhage. Alternatively, bacterial collagenase enzymatically disrupts the basal lamina of brain capillaries, causing an active bleed that generally evolves over several hours5. Resultant perihematomal edema and neurofunctional deficits can be quantified from both models. In this study, we described and evaluated a modified double injection model of autologous whole blood6 as well as an ICH injection model of bacterial collagenase7, both of which target the basal ganglia (corpus striatum) of male CD-1 mice. We assessed neurofunctional deficits and brain edema at 24 and 72 hr after ICH induction. Intrastriatal injection of autologous blood (30 &mu;l) or bacterial collagenase (0.075U) caused reproducible neurofunctional deficits in mice and significantly increased brain edema at 24 and 72 hr after surgery (p&lt;0.05). In conclusion, both models yield consistent hemorrhagic infarcts and represent basic methods for preclinical ICH research.

Clinically relevant animal models of intracerebral hemorrhage (ICH) are needed to extend our knowledge of hemorrhagic stroke and to examine novel therapeutic strategies. In this study, we describe and evaluate two ICH models that implement unilateral injections of either autologous whole blood or bacterial collagenase into the basal ganglia (corpus striatum) of mice.

在小鼠的脑出血模型:注射自体血液或细菌胶原酶

Spontaneous intracerebral hemorrhage (ICH) defines a potentially life-threatening neurological malady that accounts for 10-15% of all stroke-related ...

Generation of Subcutaneous and Intrahepatic Human Hepatocellular Carcinoma Xenografts in Immunodeficient Mice

In vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate the human disease provide a valuable platform for research into disease pathophysiology and for the preclinical evaluation of novel therapies. We present a variety of methods to generate subcutaneous or orthotopic human HCC xenografts in immunodeficient mice that could be utilized in a variety of research applications. With a focus on the use of primary tumor tissue from patients undergoing surgical resection as a starting point, we describe the preparation of cell suspensions or tumor fragments for xenografting. We describe specific techniques to xenograft these tissues i) subcutaneously; or ii) intrahepatically, either by direct implantation of tumor cells or fragments into the liver, or indirectly by injection of cells into the mouse spleen. We also describe the use of partial resection of the native mouse liver at the time of xenografting as a strategy to induce a state of active liver regeneration in the recipient mouse that may facilitate the intrahepatic engraftment of primary human tumor cells. The expected results of these techniques are illustrated. The protocols described have been validated using primary human HCC samples and xenografts, which typically perform less robustly than the well-established human HCC cell lines that are widely used and frequently cited in the literature. In comparison with cell lines, we discuss factors which may contribute to the relatively low chance of primary HCC engraftment in xenotransplantation models and comment on technical issues that may influence the kinetics of xenograft growth. We also suggest methods that should be applied to ensure that xenografts obtained accurately resemble parent HCC tissues.

Human tumor xenografts in immunodeficient mice are valuable tools to study cancer biology. Specific protocols to generate subcutaneous and intrahepatic xenografts from human hepatocellular carcinoma cells or tumor fragments are described. Liver regeneration induced by partial hepatectomy in recipient mice is presented as a strategy to facilitate intrahepatic engraftment.

皮下及肝内人肝癌裸鼠移植瘤的免疫缺陷小鼠的产生

In  vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate  the human disease provide a valuable platform for research into disease  ...

Monitoring of Systemic and Hepatic Hemodynamic Parameters in Mice

The use of mouse models in experimental research is of enormous importance for the study of hepatic physiology and pathophysiological disturbances. However, due to the small size of the mouse, technical details of the intraoperative monitoring procedure suitable for the mouse were rarely described. Previously we have reported a monitoring procedure to obtain hemodynamic parameters for rats. Now, we adapted the procedure to acquire systemic and hepatic hemodynamic parameters in mice, a species ten-fold smaller than rats. This film demonstrates the instrumentation of the animals as well as the data acquisition process needed to assess systemic and hepatic hemodynamics in mice. Vital parameters, including body temperature, respiratory&#160;rate&#160;and heart&#160;rate were recorded throughout the whole procedure. Systemic hemodynamic parameters consist of carotid artery pressure (CAP) and central venous pressure (CVP). Hepatic perfusion parameters include portal vein pressure (PVP), portal flow rate as well as the flow rate of the common hepatic artery (table 1). Instrumentation and data acquisition to record the normal values was completed within 1.5 h. Systemic and hepatic hemodynamic parameters remained within normal ranges during this procedure.
This procedure is challenging but feasible. We have already applied this procedure to assess hepatic hemodynamics in normal mice as well as during 70% partial hepatectomy and in liver lobe clamping experiments. Mean PVP after resection (n= 20), was 11.41&#177;2.94 cmH2O which was significantly higher (P&#60;0.05) than before resection (6.87&#177;2.39 cmH2O). The results of liver lobe clamping experiment indicated that this monitoring procedure is sensitive and suitable for detecting small changes in portal pressure and portal flow rate. In conclusion, this procedure is reliable in the hands of an experienced micro-surgeon but should be limited to experiments where mice are absolutely needed.

This film demonstrates how to acquire systemic and hepatic hemodynamics in mice. The whole monitoring includes acquisition of vital parameters, systemic blood pressure, central venous pressure, common hepatic artery flow rate, and portal vein pressure as well as the portal flow rate in mice.

全身及肝脏血流动力学参数的监测小鼠

The use of mouse models in experimental research is of enormous importance for the study of hepatic physiology and pathophysiological disturbances. ...

The bm12 Inducible Model of Systemic Lupus Erythematosus (SLE) in C57BL/6 Mice

Systemic lupus erythematosus (SLE) is an autoimmune disease with diverse clinical and immunological manifestations. Several spontaneous and inducible animal models mirror common components of human disease, including the bm12 transfer model. Upon transfer of bm12 splenocytes or purified CD4 T cells, C57BL/6 mice rapidly develop large frequencies of T follicular helper cells (Tfh), germinal center (GC) B cells, and plasma cells followed by high levels of circulating anti-nuclear antibodies. Since this model utilizes mice on a pure C57BL/6 background, researchers can quickly and easily study disease progression in transgenic or knockout mouse strains in a relatively short period of time. Here we describe protocols for the induction of the model and the quantitation Tfh, GC B cells, and plasma cells by multi-color flow cytometry. Importantly, these protocols can also be used to characterize disease in most mouse models of SLE and identify Tfh, GC B cells, and plasma cells in other disease models.

The bm12 Inducible Model of Systemic Lupus Erythematosus SLE in C57BL/6 Mice

The transfer of bm12 lymphocytes into a C57BL/6 recipient is an established model of systemic lupus erythematosus. Here we describe how to initiate disease using this model and how to characterize T follicular helper cells, germinal center B cells and plasma cells by flow cytometry.

系统性红斑狼疮的BM12诱导模型（SLE）的C57BL / 6小鼠

Systemic lupus erythematosus (SLE) is an autoimmune disease with diverse clinical and immunological manifestations. Several spontaneous and inducible ...

Visualization of Vascular and Parenchymal Regeneration after 70% Partial Hepatectomy in Normal Mice

A modified silicone injection procedure was used for visualization of the hepatic vascular tree. This procedure consisted of in-vivo injection of the silicone compound, via a 26 G catheter, into the portal or hepatic vein. After silicone injection, organs were explanted and prepared for ex-vivo micro-CT (&#181;CT) scanning. The silicone injection procedure is technically challenging. Achieving a successful outcome requires extensive microsurgical experience from the surgeon. One of the challenges of this procedure involves determining the adequate perfusion rate for the silicone compound. The perfusion rate for the silicone compound needs to be defined based on the hemodynamic of the vascular system of interest. Inappropriate perfusion rate can lead to an incomplete perfusion, artificial dilation and rupturing of vascular trees.
The 3D reconstruction of the vascular system was based on CT scans and was achieved using preclinical software such as HepaVision. The quality of the reconstructed vascular tree was directly related to the quality of silicone perfusion. Subsequently computed vascular parameters indicative of vascular growth, such as total vascular volume, were calculated based on the vascular reconstructions. Contrasting the vascular tree with silicone allowed for subsequent histological work-up of the specimen after &#181;CT scanning. The specimen can be subjected to serial sectioning, histological analysis and whole slide scanning, and thereafter to 3D reconstruction of the vascular trees based on histological images. This is the prerequisite for the detection of molecular events and their distribution with respect to the vascular tree. This modified silicone injection procedure can also be used to visualize and reconstruct the vascular systems of other organs. This technique has the potential to be extensively applied to studies concerning vascular anatomy and growth in various animal and disease models.

Tools used for visualizing vascular regeneration require methods for contrasting the vascular trees. This film demonstrated a delicate injection technique used to achieve optimal contrasting of the vascular trees and illustrate the potential benefits resulting from a detailed analysis of the resulting specimen using &#181;CT and histological serial sections.

之后，正常小鼠70％部分肝切除血管和实质再生的可视化

A modified silicone injection procedure was used for visualization of the hepatic vascular tree. This procedure consisted of in-vivo injection of the ...

Intrasplenic Transplantation of Hepatocytes After Partial Hepatectomy in NOD.SCID Mice

Partial hepatectomy is a versatile and reproducible method to study liver regeneration and the effect of cell based therapeutics in various pathological conditions. Partial hepatectomy also facilitates the increased engraftment and proliferation of transplanted cells by accelerating neovascularization and cell migration towards the liver. Here, we describe a simple protocol for performing 30% hepatectomy and transplantation of cells in the spleen of a non-obese diabetic/severe combined immunodeficient NOD.SCID (NOD.CB17-Prkdcscid/J) mouse. 
In this procedure, two small incisions are made. The first incision is to expose and resect the left lobe of the liver, and another small incision is made to expose the spleen for the intrasplenic transplantation of cells. This procedure does not require any specialized surgical skills, and it can be completed in 5-7 minutes with less stress and pain, faster recovery, and better survival. We have demonstrated the transplantation of hepatocytes isolated from a green fluorescent protein (GFP) expressing mouse (Transgenic C57BL/6-Tg (UBC-GFP) 30Scha/J), as well as hepatocyte like cells of human origin (NeoHep) in partially hepatectomized NOD.SCID mice.

We have described a protocol for performing partial hepatectomy (PHx) and cell transplantation via spleen in NOD.SCID (NOD.CB17-Prkdcscid/J) mice. In this protocol, an incision is made to expose and resect the left lobe of the liver followed by another incision for the intrasplenic transplantation of cells.

肝细胞部分肝切除术后脾移植。小鼠

Partial hepatectomy is a versatile and reproducible method to study liver regeneration and the effect of cell based therapeutics in various pathological ...

In Vivo Photolabeling of Cells in the Colon to Assess Migratory Potential of Hematopoietic Cells in Neonatal Mice

Enteric bacterial communities are established early in life and influence immune cell development and function. The neonatal microbiota is susceptible to numerous external influences including antibiotics use and diet, which impacts susceptibility to autoimmune and inflammatory diseases. Disorders such as Inflammatory Bowel Disease (IBD) are characterized by a massive influx of immune cells to the intestines. However, immune cells conditioned by the microbiota may additionally emigrate out of the intestines to influence immune responses at extra-intestinal sites. Thus, there is a need to identify and characterize cells that may carry microbial messages from the intestines to distal sites. Here, we describe a method to label cells in the colon of newborn mice in vivo that enables their identification at extra-intestinal sites after migration.

In Vivo Photolabeling of Cells in the Colon to Assess Migratory Potential of Hematopoietic Cells in Neonatal Mice

The protocol described here utilizes a photolabeling approach in newborn mice to specifically identify immune cells that emigrate from the colon to extra-intestinal sites. This strategy will be useful to study host-microbiome interactions in early life.

体内结肠细胞 Photolabeling 对新生小鼠造血细胞迁移潜能的评价

Enteric bacterial communities are established early in life and influence immune cell development and function. The neonatal microbiota is susceptible to ...

Sleeve Gastrectomy in Mice using Surgical Clips

The number of people who are overweight and obese is continually increasing both in the adult and adolescent populations. This coincides with the increased universal phenomenon of type 2 diabetes (T2D) and other metabolic problems. Bariatric surgery, such as SG, is currently one of the most effective and commonly used long-term treatment for obesity and T2D, but the association between them is not completely explored yet. The mechanisms underlying the outcomes seen after bariatric surgery in humans can be investigated based on preclinical animal studies. The SG reduces body weight, glucose levels and many metabolic parameters, and is easy to perform with a low incidence of complications. The goal of this work is to provide a simple method and an uncomplicated preclinical model of bariatric surgery in animals for researchers.

The prevalence of diabetes and obesity is continuously increasing worldwide. The mechanisms between diabetes, obesity and their associated mortality and co-morbidities need to be further investigated. Here, we present a protocol for sleeve gastrectomy (SG) in animals as an uncomplicated preclinical model of bariatric surgery.

使用手术夹的小鼠的套管胃切除术

The number of people who are overweight and obese is continually increasing both in the adult and adolescent populations. This coincides with the ...

Assessing Whole-Body Lipid-Handling Capacity in Mice

Assessing lipid metabolism is a cornerstone of evaluating metabolic function, and it is considered essential for in vivo metabolism studies. Lipids are a class of many different molecules with many pathways involved in their synthesis and metabolism. A starting point for evaluating lipid hemostasis for nutrition and obesity research is needed. This paper describes three easy and accessible methods that require little expertise or practice to master, and that can be adapted by most labs to screen for lipid-metabolism abnormalities in mice. These methods are (1) measuring several fasting serum lipid molecules using commercial kits (2) assaying for dietary lipid-handling capability through an oral intralipid tolerance test, and (3) evaluating the response to a pharmaceutical compound, CL 316,243, in mice. Together, these methods will provide a high-level overview of lipid handling capability in mice.

This paper provides three easy and accessible assays for assessing lipid metabolism in mice.

评估小鼠全身脂质处理能力

Assessing lipid metabolism is a cornerstone of evaluating metabolic function, and it is considered essential for in vivo metabolism studies. Lipids are a ...