我们感谢JL博士。 Galzi（UMR7242法国国家科学研究中心;伊尔基希，法国）的支持。 这项工作是由国家科学研究中心，INSERM，大学德斯特拉斯堡，阿尔萨斯生物谷和由Conectus，法新社法国国家（ANR 08 EBIO 014.02）的资金支持行政法院区域阿尔萨斯（Pharmadol），城市社区斯特拉斯堡（Pharmadol） ICFRC（Pharmadol），OSEO（Pharmadol），向兴德企业经营者（Pharmadol）。

所有实验均严格按照实验动物（欧共体理事会指令86/609/ECC）以及对实验性疼痛的动物意识调查13道德准则的照顾欧洲指南。雄性C57BL6 / N西塔小鼠（10周，25 - 30克）被安置在一个机构动物设施与保健人员负责符合环保标准经营设施的。动物饲养在组（每笼最高五只小鼠）下12小时/ 12小时亮/暗周期在一个恒定的温度（21±1℃）免费获得食物和水。所有实验均在每天的同一时段（上午10:00至下午4:00）使用16个小鼠的队列进行。具体的材料和设备都显示在材料表。 五步程序监督吗啡诱导痛觉过敏和耐受性该协议是分为五个连续的步骤（AE）超过15天图1的持续时间。 1小鼠处理（步骤A，D-7 D-5） <ol><li>处理小鼠和习惯于他们自由进入限制器。这第一步减轻压力 - 从而减少应激诱导的镇痛任何混乱 - 和使动物的习惯的调查，处理和操作到鼠标限制器。每个鼠标轻轻5分钟每天要处理的。 </li></ol> 2，基础伤害性反应（步骤B，D-4 D-1） <ol><li>使用尾部浸渍试验（TIT）测量尾退缩潜伏期： <ol><li>温控器设置为48°C。 </li><li>轻轻地引入鼠标放到限制器。沾其尾部的突起2/3的一端插入水浴启动计时器。 </li><li>只要鼠标撤回它的尾巴从热水停止计时器并记录等待时间（以秒为单位）。在日ë没有任何伤害性反应的，一个25秒的截止是用来防止组织损伤。 </li><li>代替鼠标在笼子里，并做下一个，直到系列的结尾。 </li><li>重复的伤害性反应的测量两次，取测量从在同一顺序的动物。伤害性反应潜伏期（秒）为每只小鼠被确定为三个连续测定的平均值。 </li></ol></li><li>测量力学响应使用尾部压力试验（TPT） <ol><li>轻轻地引入鼠标放到限制器和analgesimeter的锥顶下放置它的尾巴。 </li><li>踩下脚踏开关适用均匀增加压力到尾部的近端部分，直到第一个伤害性反应（挣扎，吱吱）发生。在动物反应的时刻，记录当前的力（单位为克），该引出的疼痛反应。在没有任何反应时，600克截止值用于避免吨发出的损害。 </li><li>重复此测量在相同的小鼠的尾部的中间和远侧部分。至少30秒的时间间隔在给定的鼠标措施，以避免适应或应力偏压之间观察到。代替鼠标在笼子里，并测试下一个动物直到系列（ 即所有小鼠进行测试）结束。感受伤害值（克）对每只小鼠取为三个测量（ 即近端，中位数，每个动物的尾巴的顶端部分）的平均值。 </li></ol></li><li>重复伤害性测试（根据第2步中列出的所有程序），在随后的日子里，D-3 D-1。 </li></ol> 3，测量吗啡镇痛的（步骤C; D0） <ol><li>确定动物的最佳组合，使小鼠两次与稳定和可比的平均伤害值组（n = 8每组），取其伤害性方式（TIT或TPT）的选择考虑。这个值将被当作基底对未来“盐”和“吗啡”参照群体伤害性反应。 </li><li>测量体重每一个动物。 </li><li>在无菌的生理盐水（0.9％生理盐水），用于皮下施用（每千克动物体重5毫克吗啡）准备一个吗啡溶液（每毫升0.5毫克吗啡）。 </li><li>测量疼痛反应潜伏期（取时间点0）为“盐碱地”，并在TIT&#39;吗啡&#39;组（2.1根据以上所有步骤）的每只小鼠。然后测量痛觉的TPT（在2.2以上的所有步骤）。 </li><li>皮下注入吗啡（通常为0.25毫升每25克鼠标重量的0.5毫克/毫升吗啡溶液）和生理盐水（每25克鼠标重量0.25毫升）的&#39;吗啡&#39;和&#39;盐水&#39;组，分别为。 </li><li>测量在TIT和TPT伤害值（根据以上列出的所有程序步骤2.1和2.2，分别）在一个时间过程（在30分钟INTERV人），以评估吗啡（5毫克/公斤）的镇痛作用： <ol><li> 30分钟后喷射之后，测量疼痛反应（单测定）为“盐水”和“吗啡&#39;基团的各小鼠，使用TIT那么TPT。 </li><li> 1-1.5-2-2.5-3和3.5注射后：然后，在时间点（以小时为单位）衡量在所有小鼠疼痛反应值（TIT和TPT）。 </li></ol></li></ol> 4慢性吗啡处理 - 吗啡诱发的痛觉过敏。（步骤D，D1至D6） <ol><li>在第一天：D1 <ol><li>在如上所述的TIT和TPT测量疼痛反应值（步骤2.1和2.2）。仔细标注退缩潜伏期，并为每个动物的压力限制。 </li><li>准备一个新的吗啡溶液如在步骤3.2详述。 </li><li>皮下注入吗啡（5 mg / kg体重），以整个&#39;吗啡&#39;组和生理盐水（每25克鼠标重量0.25毫升）的&#39;盐&#39;组。让日ë动物休息，直到第二天。 </li></ol></li><li>关于天：D2，D3，D4，D5和D6重复下4.1节所述之业务</li></ol> 5，证据镇痛耐受性（步骤E; D7） <ol><li>根据第3节中已经详述的时间过程范式评估吗啡的镇痛作​​用。 </li></ol> 6，数据采集和统计分析<ol><li>的基础伤害性反应的评估值（步骤B） <ol><li>计算出的每一天（在D-4 D-1期）的平均值±标准误差值（N = 8），用于从TIT和TPT的“盐”和“吗啡”组内给予基础伤害性反应。 </li><li>情节意味着基础伤害值的时间（天）的函数两组图2。 </li></ol></li><li>吗啡镇痛时间，当然在天D0分析（步骤C）和D7（步骤E） <ol><li>计算，在吗啡后各时间点注射，平均值±标准误差值（N = 8），用于从TIT各组给予（以秒为单位）和TPT（克）伤害性反应。 </li><li>积平均伤害性反应的值作为时间的“盐”和“吗啡&#39;基团在天的函数0 图3和7天图5。 </li></ol></li><li>吗啡诱导的痛觉过敏（步骤D）的发展<ol><li>计算出的每一天（在D0-D7治疗期）的平均值±标准误差值（N = 8），用于从TIT和TPT中的“盐水处理”和“吗啡治疗团体给予基础伤害性反应值。 </li><li>情节意味着基础伤害性反应值随时间（天）为&#39;盐水处理“和”吗啡治疗团体图4的功能。 </li></ol></li><li>为镇痛耐受性的证据（步骤C和E） <ol><li>从吗啡时期间E确定xperiment在D0 图3诱导的最大止痛效果所需的吗啡的时间价值（或时间范围）进行。 </li><li>取这个值（通常为30分钟）作为基准时间来估计在D7 图5中的伤害感受基线值（生理盐水处理组）和实际止痛反应（吗啡处理组），以急性吗啡。 </li><li>在时间点来自于D0和D7为生理盐水治疗组和吗啡处理组进行吗啡时程实验30分钟采取伤害性的值，被作为直方图图6。 </li><li>统计：使用单向重复测量方差分析分析数据。变化的因素是治疗（受试者之间）和时间（内主体）。要单独检查各组中的差异，重复测量的方差分析。采用非配对t检验或配对t检验在适当的时候进行了两组之间的比较。 </li></ol></li><li>显着性水平设为P &lt;0.05。所有统计分析使用的是STATview软件进行。 </li></ol>

作者宣称，他们有没有竞争的财务权益。

<html> 关键步骤 动物模型的痛觉测量的选择 其中小鼠品系变异性疼痛和镇痛的敏感性已被审查（评论14-16）评论使用各种疼痛模型在不同的病因（疼痛，炎症，神经性），情态（热学，化学，机械），持续时间（急性，补虚，慢性）和站点管理（皮肤，皮下，内脏）。相较于其他菌株，C57BL/6J（“J”的杰克逊实验室）小鼠成为一个受欢迎的动物模型，为疼痛的研究，因为他们表现出高的基础痛觉敏感性17,18和稳健的镇痛反应阿片类药物14,19。下面慢性吗啡处理，他们也制定显著镇痛耐受20,21，痛觉过敏21,22和20,23的依赖。 NT“&gt;在这里，实验在C57BL/6N小鼠交咨会进行（”N“为美国国立卫生研究院和”西塔“的Taconics农场），其中属于B6血统的一个独立分支。虽然C57BL / 6小鼠已久认为是可以互换的，最近的研究指出，C57BL/6J和C57BL/6N菌株24之间显著行为上的差异，特别是，这三个C57BL/6N substrains（包括西塔1）急性热疼痛的较低灵敏度可被视为一个优势，为测试这种表型。 雄性小鼠被选为疼痛的研究绝大多数，利用老鼠作为动物模型，对未成年男性25执行。在我们的手中，他们提供了强大的和可重复的数据来看镇痛或痛觉检查点时。偶尔，我们注意到一个倾向C57BL/6N女性提供更多的变量的反应，无论是在TIT和TPT测试。虽然这可能会观察反映挂女性的荷尔蒙状态的自然变化，相关的疼痛和镇痛的性别差异整体机制目前仍争议的问题。这个热议的某些方面进行简要介绍一节中的下一个&#39;技术的局限性“。 动物习惯 小鼠首次获准以熟悉的动物设施内一个星期。类似于任何其他行为的研究中，试验进行以下3天的驯化期（ 图1，步骤A）。由于伤害性测试是压力敏感的，第一措施可能获得更长的等待时间比后续的，特别是在非习惯于小鼠26,27。习惯化步骤也允许的范围内的同一天和天之间更稳定的伤害感受响应值的获得方法图2和图4。为了减少疼痛和镇痛作用的昼夜灵敏度28,29，所有的性化验是上午10:00和下午4:00进行。 选择伤害性测试 感受伤害测试使用任一热，机械，化学或电刺激（综述26,27,30，如伤害性不同的方式可以通过不同的感受器和纤维18,31,32被处理他们的选择是至关重要的。 我们选择了尾部浸渍试验（TIT）33，经典的甩尾测试由D&#39;AMOUR和史密斯34研制的修改版本，以及尾部压力试验（TPT），改编自兰德尔和Selitto 35，作为热例子，机械方式来研究吗啡的镇痛作​​用，痛觉过敏和耐受小鼠。这两个测试已经被广泛地应用于大鼠。一个截止时间进行了系统的定义，以避免或限制组织损伤的风险。 吗啡电感编辑镇痛，痛觉过敏和耐受性吗啡，原型μ-阿片受体激动剂，在这里选择的，因为它是一种强效镇痛药和OIH诱导物，无论是在人类和小鼠1,2,36。吗啡的镇痛效力是已知的随小鼠品系，给药途径和伤害感受的方式。在C57BL / 6小鼠，可靠的镇痛效果通常可以得到以下的吗啡在1-20毫克/公斤的剂量范围14,21皮下注射。因此，我们选择了研究急性镇痛下单次给药吗啡（SC）在5毫克/公斤，接近其ED 50值（7-20毫克/千克），从热痛觉19,21评估。 吗啡反复给药常伴有镇痛耐受性和痛觉过敏（对疼痛stimul的灵敏度恶化（或者从剂量 - 反应曲线的向右移位或从镇痛响应振幅或持续时间的减少证明）我从基础伤害值减少为凭证）。这两种不良现象依赖于啮齿动物品系，其上选择了鸦片化合物及其剂量，治疗持续时间和伤害性方式21的性质。例如，实验范式来研究宽容和痛觉过敏家道或升级（高达50甚至200毫克/千克）20,21吗啡剂量高而稳定的（20〜40毫克/千克每天）22日常管理。超过8天期间，因此，我们通过每天的吗啡给药（SC 5毫克/公斤）促进痛觉过敏和耐受性的发展C57BL / 6小鼠。这种温和的吗啡剂量优于以更好地模拟临床用量则较高。 设立TIT操作窗口 在TIT一个可能的缺陷可能与尾巴的老鼠26,37的体温调节中的作用。由于环境温度是在nocicept的关键因素香港专业教育学院响应变化，它应保持不变（这里是21°C时）在整个实验38。热强度通常设置为检测在5至10秒27伤害性反应。实际上，更大的延迟可能增加风险监测动物的动作无关的伤害性刺激，而较短的可能降低了测试的差分功耗。我们在48℃的固定温度下进行测量TIT尾退缩潜伏期接近9秒（基础伤害值）和25秒从不同的4秒（痛觉过敏）（最大镇痛;切断）。除了现实的原因，在一个固定的温度疼痛反应值测量可先验涉及伤害感受器和电路的同一剧目，从而便于数据的判读。 可能的修改 镇痛和OIH measureme的TIT操作窗口的优化NTS 当专注于镇痛响应，低基准值（较高的热强度）可能有利于在响应延迟的检测。反过来，以解决疼痛刺激的后果或OIH的发展，较高的基准值（较低的热强度;这里48°C）可能有助于更快地响应检测图4。 虽然我们发现吗啡在5毫克/公斤的剂量方便诱发强大的止痛效果图3和推广（后重复给药）显著痛觉过敏图4，如前（关键一步中提到其用量可适应：吗啡的镇痛作用，痛觉过敏和耐受性）。例如，较低的剂量可用于减少镇痛振幅（从而避免截断的限制），而较高剂量可被选择以加速痛觉过敏发病和增加它的幅度。 奥雅纳拉尔中，“痛觉窗口&#39;的优化应适应所研究的小鼠的遗传背景，并考虑到的可能性对伤害感受器和电路的不同的阵列的参与。 另一种阿片受体激动剂（芬太尼，瑞芬太尼） 虽然大多数临床使用的阿片类药物靶向的μ-阿片受体的激动剂，它们明显不同相对于在体外和体内的药理性质。例如，瑞芬太尼和芬太尼，与吗啡反差明显，表现为完全激动剂，促进μ-阿片受体39的内化。阿片类镇痛药如吗啡和芬太尼的半衰期为40小时的范围内，而瑞芬太尼具有超短半衰期数分钟41。在人类中，OIH最好的证据是从谁在手术过程中接受阿片类药物，包括短效肝炎患者ompounds如瑞芬太尼2,42。因此，芬太尼和瑞芬太尼可能是有价值的工具太来研究痛觉过敏和耐受小鼠的发展，在TIT和TPT范式。 感应OIH的替代模式（慢性与急性给药） OIH被认为是在人类和动物模型作为阿片类施用的结果，无论是在非常低或非常高的剂量1,2。在这里，我们下面的慢性治疗的小鼠与中等剂量吗啡的报告OIH发展。有必要的治疗C57BL/6N小鼠的几天，以证明一个明确的和可重复的痛觉过敏状态图4每日吗啡注射，能充分替换植入吗啡颗粒：在他们搬迁，既热痛觉过敏和机械性异常疼痛已经已经报道在小鼠43。通过微渗透泵阿片类药物的输注是另一种可能性<html> 44，在啮齿动物中，持久的痛觉过敏也可以实现使用一个协议模仿人类手术36,45,46使用这种μ-阿片受体激动剂的下列芬太尼急性给药。 该技术的局限性 动物种类和型号的疼痛 众多小鼠品系的比较研究提供了证据之后4天的吗啡治疗22伤害性反应对疼痛刺激17,31,47和OIH水平都有很大的差异。无论是相关的疼痛处理和调制在动物模型（小鼠和大鼠）机制相关的慢性疼痛患者仍是一个根本性的，打开的问题。因此，更应谨慎支付给动物数据的解释和他们的预测效度为16人。 在疼痛和镇痛的性别差异 ntent“&gt;动物模型的疼痛大部分临床前研究已经对雄性啮齿动物16,25,48进行，尽管这样的选择偏差，新兴的观点是，考虑男性更好的反应，以鸦片止痛剂49,50，不易发生发展阿片诱发痛觉过敏51,52和更宽容吗啡镇痛53比女性（综述54），但是，对于伤害性镇痛药物疗效的性别差异并不在这种恢复&#39;一个一刀切&#39;的模式。事实上，大量数据，现在指示众多变量可能影响的性别差异，如阿片类药物的功效和选择性，伤害性测定法，遗传背景，年龄，性腺激素状况或社会互动48,54的大小和方向。在人类中，临床疼痛更普遍的妇女，但这个事实是否反映实际的性别差异仍然辩论48,55,56的问题。佛r例，五十临床试验的全球分析 ​​显示性别之间的镇痛作用无显著差异，而荟萃分析对患者控制受试者进行指出，在女性57显著更大的阿片类药物的疗效，后者的观察，这明显与什么已经对比在啮齿类动物中发现，再次提出了关于原点这种分歧16,48,55,57几个问题。总之，在镇痛的性别差异确实存在，并值得进一步专注于底层机制和临床意义。 关于伤害性测试 尾巴 ​​撤退试验是一种脊髓反射，但它可能会受到棘上的影响58。 TIT是相对容易的对大鼠进行，但需要在小鼠更多的专业知识。一个潜在的困难是保持鼠标在一个正确的姿势不引起不必要的压力。提出的协议可以根据需要调整队列的大小。 16动物（8控制和处理8）很容易，只要自己的基础伤害性反应值的测量（先用TIT，然后TPT为全系列的小鼠）正在关注管理。监测镇痛时间课程要求所传递的时间间隔（这里30分钟）内建立一个精确的时间安排和动物，可测试的最大数目的评价（TIT第一，然后TPT）。动物的整个队列可能因此被分成子组，以允许实验者尊重动力学限制。 相对于现有的/替代方法的技术意义 OIH在大鼠与小鼠 鼠已被广泛用于研究阿片类药物镇痛，痛觉过敏和耐受性，急性或慢性阿片管理46,59-61。事实上，对于一些实际原因，它们可以被认为优于小鼠作为动物模型实验的疼痛16,61。然而，直到最近，转基因鼠的产生不是一个简单的过程。众多的转基因小鼠品系已经上市，我们的模型提供了研究的OIH和宽容的发展在小鼠众多的单个基因的贡献的机会。 TIT和TPT与其他伤害性测试 TIT是在甩尾试验中，最明显的区别是刺激的区域的变体。与辐射热相比，尾部成热水浸泡导致其温度迅速而均匀增加。相对于其他形式的热痛觉测试（热板或哈格里夫斯测试）中，TIT提供了这两个问题之间和内部公平地重复的结果。 TPT是一个非常受欢迎的机械痛觉26,27,35这可能涉及到的试验研究不同的痛觉纤维和分子传感器然后TIT 32。它提供了快速，可靠的测量59，不过，需要实验者和大型动物同伙一定的专业知识。作为替代，在本研究中，其它程序或装置使用的analgesimeter依靠应变仪确实存在（评论27）。 TPT是最适合用于研究机械性痛觉过敏，而的von Frey细丝，通常采取评价机械性异常性疼痛（评论27）。 掌握这一技术后，未来的应用或路线 我们在这里介绍的实验OIH /公差模型可以类似地适用于转基因小鼠，以评估各个基因的疼痛的调制作用。它还提供了一个模型系统来调查潜在的治疗药物的有效性，以缓解慢性疼痛。

阿片类药物引起的痛觉过敏（OIH），镇痛耐受性限制了动物和人类的1-3阿片 ​​类药物的临床疗效。亲伤害性（抗阿片）6,7系统的促炎性4,5或参与正在探讨的假设。相关OIH和宽容机制的阐明就必须的组合在体内和体外的方法，使用合适的动物模型，实验方案和分子的工具。 行为药理学的主导范式，以监测和量化实验动物的镇痛和痛觉过敏状态（大鼠，小鼠）。有害刺激（热，机械或化学）的动物的一个方便的身体部位（后爪，尾）的应用导致nocifensive停药，可以轻松得分。 在这里我们提出一个方法途径诱导，记录和量化OIH和公差在野生型小鼠中，使用尾浸没和尾部压力测试。该程序允许一种简单，灵敏，重现性测定小鼠热和机械伤害的响应值。这表现在视频协议，C57BL / 6小鼠体验显著以下痛觉过敏慢性吗啡管理和维护这几天。热和机械痛觉值显著减少，相比于幼稚动物的基线测量。此外，我们的实验装置允许监视，除了OIH，镇痛回应吗啡（公差）的衰落的发展。给出的数据支持这样的观点痛觉过敏和耐受性可能涉及共同的细胞和分子机制8,9，尽管这是有争议的文献1,10〜12。最后，这个协议可以同样适应于转基因小鼠，以评估各个基因在调制中的作用化的痛苦。它还提供了一个模型系统来评估潜在的治疗药物的有效性，以提高阿片类药物的镇痛效果。

<table border="0" cellpadding="0" cellspacing="0" width="879">
	<tbody>
		<tr height="17">
			<td height="34" rowspan="2" style="height:34px;width:220px;">Name</td>
			<td rowspan="2" style="width:231px;">Company</td>
			<td rowspan="2" style="width:139px;">Catalog Number</td>
			<td rowspan="2" style="width:291px;">Comments/description</td>
		</tr>
		<tr height="17">
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:220px;">C57BL/6N Tac mice</td>
			<td style="width:231px;">Taconic, Ry, Denmark</td>
			<td style="width:139px;">C57BL/6N Tac B6-M</td>
			<td style="width:291px;">Male mice (25-30g)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Morphine hydrochloride</td>
			<td style="width:231px;">Francopia, Paris, France</td>
			<td style="width:139px;">CAS n&#176; 52-26-6</td>
			<td style="width:291px;">Delivered with special authorization</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Syringes (Terumo)</td>
			<td style="width:231px;">Dutscher, Brumath, France</td>
			<td style="width:139px;">050000</td>
			<td style="width:291px;">Polypropylene; Sterile; Volume : 1 ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Needles (Terumo)</td>
			<td style="width:231px;">Dutscher, Brumath, France</td>
			<td style="width:139px;">050101</td>
			<td style="width:291px;">G26 &#189; (Terumo reference : NN2613RO1)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:220px;">Mouse restrainer</td>
			<td style="width:231px;">Home-made</td>
			<td style="width:139px;">-</td>
			<td style="width:291px;">Two metallic grids (5x11cm) assembled with adhesive tape and staples.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Thermostated water bath GR150</td>
			<td style="width:231px;">Grant Instruments, Cambridge, UK</td>
			<td style="width:139px;">GP 0540003</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Analgesimeter</td>
			<td style="width:231px;">Panlab, Barcelona, Spain</td>
			<td style="width:139px;">LE 7306</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:220px;">Kaleidagraph software</td>
			<td style="width:231px;">Synergy software, Reading, PA, USA</td>
			<td style="width:139px;">Kaleidagraph 4.03&#160;</td>
			<td style="width:291px;">Scientific graphing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">STATview software</td>
			<td style="width:231px;"></td>
			<td style="width:139px;"></td>
			<td style="width:291px;">Free download; Statistics</td>
		</tr>
	</tbody>
</table>

assessment of morphine-induced hyperalgesia and analgesic tolerance in mice using thermal and mechanical nociceptive modalities

阿片类药物引起的痛觉过敏和耐受严重影响阿片类药物的临床疗效在动物和人类的止痛药。这两种现象背后的分子机制尚不十分清楚，并阐明他们应该从动物模型的研究，并从相应的实验方案的设计中获益。 我们在这里描述用于诱导，记录和量化吗啡诱导的痛觉过敏以及用于证明镇痛耐受性，使用尾浸没和尾部压力测试在野生型小鼠中的方法途径。如图所示，在视频中，协议分为五个连续的步骤。处理和习惯的阶段允许进行安全测定动物的基础伤害性反应。慢性吗啡诱导显著痛觉过敏如通过增加热和机械感度，而镇痛时间课程后，比较急性或代表eated吗啡治疗清楚地表明宽容在镇痛响应幅度的下降体现的发展。这个协议可以同样适应于转基因小鼠，以评估各个基因在伤害感受和吗啡镇痛的调节作用。它还提供了一个模型系统来调查潜在的治疗药物的有效性，以提高阿片类药物镇痛效果。

天真的小鼠基础伤害性价值评估（步骤B） TIT和TPT依次应用到小鼠的整个队列（N = 16），提供的平均疼痛反应值。动物的最佳组合，使2组（n = 8）小鼠的称为盐水和吗啡，其显示类似的，稳定的基础伤害值图2。两组的等价是有效的任何伤害性试验（的后验概率的定义多： 图2A; TPT： 图2B）被选中。 时间课程吗啡镇痛在第0天（步骤C） 以下吗啡单次注射（SC）（5毫克/千克）在幼稚小鼠同时使用TIT 图3A和TPT 图3B吗啡镇痛进行评价。在两种测试中，有一种方式重复测量ANOVA revea统计分析升，有用于治疗TIT和时间之间的显著交互作用（F（7，98）= 72，P &lt;0.001）和TPT（F（7，98）= 31，P &lt;0.001）。 TIT和TPT数据分析采用重复测量方差分析表明，有盐水注射没有影响（F（7，49）= 0.49，P&gt; 0.05）和F（7，49）= 1.85，P&gt; 0.05分别为TIT和TPT试验），而吗啡注射诱导小鼠较强的镇痛（F（7，49）= 92.46，P &lt;0.001）和F（7，49）= 34.37，P &lt;0.001分别为TIT和TPT测试）。后在TIT 30分钟，并在TPT 60分钟相比，注射了生理盐水的对照组（P &lt;0.001，配对t-检验）之后达到吗啡的最大止痛作用。 反复主管吗啡诱导痛觉过敏小鼠（步骤D） 基础伤害值的测定每一天生理盐水或吗啡给药前（见协议）。如示于图4中 ，每日一次给药吗啡在6天的治疗期间引起一个显著和逐步降低的热（F（7，56）= 11.6，P &lt;0.001，重复测量ANOVA， 图4A）和机械式（F（ 7,56）= 15,55，P &lt;0.001，重复测量的方差分析; 图4B）基础伤害值。痛觉过敏的发展很快，因为它开始是显著在第1天在TIT（P &lt;0.01，配对t-检验，相比于盐水注射的对照组）和在TPT 2天（P &lt;0.05，配对t-检验，相比于盐水喷射控制）。 时间过程的吗啡镇痛的7天，慢性吗啡处理（步骤E）后第7天，超过7天期间（D0到D6）每天接受吗啡或生理盐水注射的小鼠进行了检查T中IT 图5A和TPT 图5B第一为他们的基础伤害值，然后他们对急性吗啡镇痛的响应（5毫克/公斤，SC）。在与痛觉过敏的图4中所示的开发协议，即分别慢性吗啡处理后的小鼠的基础伤害值（时间为0）显着高于盐水注射的对照小鼠（P &lt;0.001，配对t-检验）的显著更低。以下急性吗啡，慢性吗啡处理组的疼痛反应显著增加，但仅稍微超过注射盐水的对照小鼠在TIT 30分钟和TPT（P &lt;0.01 和 p &lt;0.05， 配对 t测得的基底伤害性值测，分别）和在TIT 60分钟（P &lt;0.05，配对t-检验）。从2小时吗啡处理后直至实验结束，伤害性反应回到较低的值比对照组小鼠的（对&lt;0.001，配对t-检验）。 慢性吗啡处理（7天）的小鼠的最大镇痛回应吗啡比较前（0天）后。 如图6所示，痛觉阈值是从TIT（A）和示于图3（第7天）（第0天）和5 TPT（B）中进行30分钟的盐水或吗啡注射后。在吗啡镇痛一个强大的下降，观察小鼠后，在两个伤害性测试在第0天他们最初的镇痛反应（P &lt;0.001配对t检验）比较，慢性吗啡治疗7天。这些数据表明，宽容并发展过敏性疼痛的动物。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51264/51264fig1highres.jpg" width="500" /> 图1：五步亲cedure监控吗啡诱导的痛觉过敏和耐受性。协议分为五个连续的步骤（AE）超过15天的总工期。 <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/51264/51264fig2highres.jpg" width="500" /> 图2定义的基感受伤害响应值（步骤B，D-4 D-1）的尾部浸入（TIT）（A）和尾部压力（TPT）（B）的测试也适用于整个系列的动物为了评估其基础伤害值。此后，两组小鼠（ 每组 8 只 ），被称为“盐”和“吗啡&#39;基团，被定义为使得它们显示出稳定的和可比较的平均疼痛值，无论伤害性方式被考虑。 <img alt="图3" fo:content-width="5in" srC =“/ files/ftp_upload/51264/51264fig3highres.jpg”宽度=“500”/&gt; 图3。时间课程在第0天（步骤三）在TIT（A）和TPT（B）吗啡镇痛。小鼠的基础伤害性反应值确定单吗啡（5毫克/千克，皮下注射后每隔30分钟。 ）或生理盐水注射。数据表示为平均值±SEM，每组n = 8只小鼠。* P &lt;0.05，** P &lt;0.01，*** P &lt;0.001，配对t-检验比较对照组。 <img alt="图4" fo:content-width="5in" src="/files/ftp_upload/51264/51264fig4highres.jpg" width="500" /> 。以下吗啡反复给药（步骤D，D1至D6）图4发展痛觉过敏。小鼠的基础伤害值由TIT确定 （A）和TPT（B）每日一次前吗啡（5毫克/公斤，SC）或生理盐水给药。数据表示为平均值±SEM，每组n = 8只小鼠。 * P &lt;0.05，** P &lt;0.01，*** P &lt;0.001通过非配对t检验，与盐水处理的对照组进行比较。 <img alt="图5" fo:content-width="5in" src="/files/ftp_upload/51264/51264fig5highres.jpg" width="500" /> 图5。时间课程吗啡镇痛慢性吗啡处理的小鼠在第7天（步骤E）在TIT（A）和TPT（B）的小鼠进行了长期使用吗啡（黑点）或生理盐水（白色三角形）处理从第0天至第6天，第7天接受单次注射吗啡（5毫克/千克，皮下注射）或生理盐水，分别。小鼠的疼痛反应测定吗啡或生理盐水注射后每隔30分钟。数据表示为平均值±标准差，正每组= 8只小鼠。 * P &lt;0.05，** P＆＃60; 0.01通过非配对t检验，与盐水处理的对照组进行比较。误差线不超过符号的大小是隐藏的。 <img alt="图6" fo:content-width="5in" src="/files/ftp_upload/51264/51264fig6highres.jpg" width="500" /> 图6的小鼠的前（第0天）和后这里报道慢性吗啡治疗（7天）。的值是从图3和图5所示的实验最大的镇痛反应，吗啡（5毫克/千克，皮下注射）的比较 。伤害性值使用TIT（A）和TPT（B）吗啡或生理盐水注射后30分钟测量。数据表示为平均值±标准差，正每组= 8只小鼠。 *** P &lt;0.001通过配对t检验。

Watch this Scientific Journal Video about Assessment of Morphine-induced Hyperalgesia and Analgesic Tolerance in Mice Using Thermal and Mechanical Nociceptive Modalities at JoVE.com

Assessment of Morphine-induced Hyperalgesia and Analgesic Tolerance in Mice Using Thermal and Mechanical Nociceptive Modalities

我们描述了一个协议，研究阿片类药物引起的痛觉过敏和耐受小鼠的发展。基于对幼稚和吗啡处理的动物的热和机械伤害性反应的测量，它允许以量化的增加疼痛的敏感性（痛觉过敏）和减少在慢性阿片类施用相关镇痛（公差）。

吗啡诱发的痛觉过敏和镇痛耐受小鼠中利用热和机械伤害性的评估方式

Opioid-induced hyperalgesia and tolerance severely impact the clinical efficacy of opiates as pain relievers in animals and humans. The molecular ...

assessment-morphine-induced-hyperalgesia-analgesic-tolerance-mice

Research

Education

JoVE Core

JoVE Journal

Pharmacology

Neuroscience

Unit 1

Drugs for Pain Management: Opioid Analgesics and General Anesthetics

SCIENTISTS IN ACTION

33.0K 次观看.  Université de Strasbourg. 我们描述了一个协议，研究阿片类药物引起的痛觉过敏和耐受小鼠的发展。基于对幼稚和吗啡处理的动物的热和机械伤害性反应的测量，它允许以量化的增加疼痛的敏感性（痛觉过敏）和减少在慢性阿片类施用相关镇痛（公差）。 

视频: Assessment of Morphine-induced Hyperalgesia and Analgesic Tolerance in Mice Using Thermal and Mechanical Nociceptive Modalities

Assessment of Motor Balance and Coordination in Mice using the Balance Beam

Brain injury, genetic manipulations, and pharmacological treatments can result in alterations of motor skills in mice. Fine motor coordination and balance can be assessed by the beam walking assay. The goal of this test is for the mouse to stay upright and walk across an elevated narrow beam to a safe platform. This test takes place over 3 consecutive days: 2 days of training and 1 day of testing. Performance on the beam is quantified by measuring the time it takes for the mouse to traverse the beam and the number of paw slips that occur in the process. Here we report the protocol used in our laboratory, and representative results from a cohort of C57BL/6 mice. This task is particularly useful for detecting subtle deficits in motor skills and balance that may not be detected by other motor tests, such as the Rotarod.

Deficits in fine motor coordination can be assessed with the balance beam test. Performance on the beam is quantified by the speed at which the beam is traversed and the number of times the mouse slips on the beam.

在小鼠体内的汽车平衡和协调的评估使用的平衡木

Brain injury, genetic manipulations, and pharmacological treatments can result in alterations of motor skills in mice.  Fine motor coordination and ...

科研

神经科学

Rapid Determination of the Thermal Nociceptive Threshold in Diabetic Rats

Painful diabetic neuropathy (PDN) is characterized by hyperalgesia i.e., increased sensitivity to noxious stimulus, and allodynia i.e., hypersensitivity to normally innocuous stimuli1. Hyperalgesia and allodynia have been studied in many different rodent models of diabetes mellitus2. However, as stated by B&ouml;lcskei et al, determination of "pain" in animal models is challenging due to its subjective nature3. Moreover, the traditional methods used to determine behavioral responses to noxious thermal stimuli usually lack reproducibility and pharmacological sensitivity3. For instance, by using the hot-plate method of Ankier4, flinch, withdrawal and/or licking of either hind- and/or fore-paws is quantified as reflex latencies at constant high thermal stimuli (52-55 &deg;C). However, animals that are hyperalgesic to thermal stimulus do not reproducibly show differences in reflex latencies using those supra-threshold temperatures3,5. As the recently described method of B&ouml;lcskei et al.6, the procedures described here allows for the rapid, sensitive and reproducible determination of thermal nociceptive thresholds (TNTs) in mice and rats. The method uses slowly increasing thermal stimulus applied mostly to the skin of mouse/rat plantar surface. The method is particularly sensitive to study anti-nociception during hyperalgesic states such as PDN. The procedures described bellow are based on the ones published in detail by Alm&aacute;si et al 5 and B&ouml;lcskei et al 3. The procedures described here have been approved the Laboratory Animal Care and Use Committee (LACUC), Wright State University.

Here, we describe a rapid reliable and simple procedure to determine the lowest temperature at which rats or mice show nocifensive behavior, i.e. the thermal nociceptive threshold (TNT). This method applies a slowly increasing thermal stimulus allowing precise and reproducible estimation of TNTs with minimum, if any, stress to the animals.

糖尿病大鼠的热痛觉阈值的快速测定

Painful diabetic neuropathy (PDN) is characterized by hyperalgesia i.e., increased sensitivity to noxious stimulus, and allodynia i.e., hypersensitivity ...

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.
Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 &#176;C to 5 &#176;C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.
This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

The Cold Plantar Assay (CPA) measures cold responsiveness between 30 &#176;C and 5 &#176;C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

测定冷灵敏度和适应小鼠一种简单廉价的方法

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

Assessment of Hippocampal Dendritic Complexity in Aged Mice Using the Golgi-Cox Method

Dendritic spines are the protuberances from the neuronal dendritic shafts that contain&#160; excitatory synapses. The morphological and branching variations of the neuronal dendrites within the hippocampus are implicated in cognition and memory formation. There are several approaches to Golgi staining, all of which have been useful for determining the morphological characteristics of dendritic arbors and produce a clear background. The present Golgi-Cox method, (a slight variation of the protocol that is provided with a commercial Golgi staining kit), was designed to assess how a relatively low dose of the chemotherapeutic drug 5-flurouracil (5-Fu) would affect dendritic morphology, the number of spines, and the complexity of arborization within the hippocampus. The 5-Fu significantly modulated the dendritic complexity and decreased the spine density throughout the hippocampus in a region-specific manner. The data presented show that the Golgi staining method effectively stained the mature neurons in the CA1, the CA3, and the dentate gyrus (DG) of the hippocampus. This protocol reports the details for each step so that other researchers can reliably stain tissue throughout the brain with high quality results and minimal troubleshooting.

Here we present a Golgi-Cox protocol in extensive detail. This reliable tissue stain method allows for a high-quality assessment of the cytoarchitecture in the hippocampus, and throughout the entire brain, with minimal troubleshooting.

使用高尔基Cox方法评估老年小鼠海马树突状复杂性

Dendritic spines are the protuberances from the neuronal dendritic shafts that contain&#160; excitatory synapses. The morphological and branching ...

Assessment of Dopaminergic Homeostasis in Mice by Use of High-performance Liquid Chromatography Analysis and Synaptosomal Dopamine Uptake

Dopamine (DA) is a modulatory neurotransmitter controlling motor activity, reward processes and cognitive function. Impairment of dopaminergic (DAergic) neurotransmission is strongly associated with several central nervous system-associated diseases such as Parkinson&#39;s disease, attention-deficit-hyperactivity disorder and drug addiction1,2,3,4. Delineating disease mechanisms involving DA imbalance is critically dependent on animal models to mimic aspects of the diseases, and thus protocols that assess specific parts of the DA homeostasis are important to provide novel insights and possible therapeutic targets for these diseases.
Here, we present two useful experimental protocols that when combined provide a functional read-out of the DAergic system in mice. Biochemical and functional parameters on DA homeostasis are obtained through assessment of DA levels and dopamine transporter (DAT) functionality5. When investigating the DA system, the ability to reliably measure endogenous levels of DA from adult brain is essential. Therefore, we present how to perform high-performance liquid chromatography (HPLC) on brain tissue from mice to determine levels of DA. We perform the experiment on tissue from dorsal striatum (dStr) and nucleus accumbens (NAc), but the method is also suitable for other DA-innervated brain areas.
DAT is essential for reuptake of DA into the presynaptic terminal, thereby controlling the temporal and spatial activity of released DA. Knowing the levels and functionality of DAT in the striatum is of major importance when assessing DA homeostasis. Here, we provide a protocol that allows to simultaneously deduce information on surface levels and function using a synaptosomal6 DA uptake assay.
Current methods combined with standard immunoblotting protocols provide the researcher with relevant tools to characterize the DAergic system.

Synaptosomal dopamine uptake and high-performance liquid chromatography analysis represent experimental tools to investigate dopamine homeostasis in mice by assessing the function of the dopamine transporter and levels of dopamine in striatal tissue, respectively. Here we present protocols to measure dopamine tissue content and assess the functionality of the dopamine transporter.

高效液相色谱分析和触多巴胺摄取对小鼠多巴胺能稳态的评价

Dopamine (DA) is a modulatory neurotransmitter controlling motor activity, reward processes and cognitive function. Impairment of dopaminergic (DAergic) ...

Visual Evoked Potential Recordings in Mice Using a Dry Non-invasive Multi-channel Scalp EEG Sensor

For scalp EEG research environments with laboratory mice, we designed a dry-type 16 channel EEG sensor which is non-invasive, deformable, and re-usable because of the plunger-spring-barrel structural facet and mechanical strengths resulting from metal materials. The whole process for acquiring the VEP responses in vivo from a mouse consists of four steps: (1) sensor assembly, (2) animal preparation, (3) VEP measurement, and (4) signal processing. This paper presents representative measurements of VEP responses from multiple mice with a submicro-voltage signal resolution and sub-hundred millisecond temporal resolution. Although the proposed method is safer and more convenient compared to other previously reported animal EEG acquiring methods, there are remaining issues including how to enhance the signal-to-noise ratio and how to apply this technique with freely moving animals. The proposed method utilizes easily available resources and shows a repetitive VEP response with a satisfactory signal quality. Therefore, this method could be utilized for longitudinal experimental studies and reliable translational research exploiting non-invasive paradigms.

We designed a dry-type 16 channel EEG sensor which is non-invasive, deformable, and re-usable. This paper describes the whole process from manufacturing the proposed EEG electrode to signal processing of visual evoked potential (VEP) signals measured on a mouse scalp using a dry non-invasive multi-channel EEG sensor.

干无创多通道头皮脑电传感器在小鼠视觉诱发电位记录中的应用

For scalp EEG research environments with laboratory mice, we designed a dry-type 16 channel EEG sensor which is non-invasive, deformable, and re-usable ...

Assessment of Sensory Thresholds in Dogs Using Mechanical and Hot Thermal Quantitative Sensory Testing

Quantitative sensory testing (QST) is used to evaluate the function of the somatosensory system in dogs by assessing the response to applied mechanical and thermal stimuli. QST is used to determine normal dogs&#39; sensory thresholds and evaluate alterations in peripheral and central sensory pathways caused by various disease states, including osteoarthritis, spinal cord injury, and cranial cruciate ligament rupture. Mechanical sensory thresholds are measured by electronic von Frey anesthesiometers and pressure algometers. They are determined as the force at which the dog exhibits a response indicating conscious stimulus perception. Hot thermal sensory thresholds are the latency to respond to a fixed or ramped temperature stimulus applied by a contact thermode.
Following a consistent protocol for performing QST and paying attention to details of the testing environment, procedure, and individual study subjects are critical for obtaining accurate QST results for dogs. Protocols for the standardized collection of QST data in dogs have not been described in detail. QST should be performed in a quiet, distraction-free environment that is comfortable for the dog, the QST operator, and the handler. Ensuring that the dog is calm, relaxed, and properly positioned for each measurement helps produce reliable, consistent responses to the stimuli and makes the testing process more manageable. The QST operator and handler should be familiar and comfortable with handling dogs and interpreting dogs&#39; behavioral responses to potentially painful stimuli to determine the endpoint of testing, reduce stress, and maintain safety during the testing process.

This work describes a standard protocol for mechanical and hot thermal quantitative sensory testing to evaluate the somatosensory system in dogs. Sensory thresholds are measured using an electronic von Frey anesthesiometer, pressure algometer, and hot contact thermode.

使用机械和热热定量感官测试评估狗的感官阈值

Quantitative sensory testing (QST) is used to evaluate the function of the somatosensory system in dogs by assessing the response to applied mechanical ...

Recording Network Activity in Spinal Nociceptive Circuits Using Microelectrode Arrays

The roles and connectivity of specific types of neurons within the spinal cord dorsal horn (DH) are being delineated at a rapid rate to provide an increasingly detailed view of the circuits underpinning spinal pain processing. However, the effects of these connections for broader network activity in the DH remain less well understood because most studies focus on the activity of single neurons and small microcircuits. Alternatively, the use of microelectrode arrays (MEAs), which can monitor electrical activity across many cells, provides high spatial and temporal resolution of neural activity. Here, the use of MEAs with mouse spinal cord slices to study DH activity induced by chemically stimulating DH circuits with 4-aminopyridine (4-AP) is described. The resulting rhythmic activity is restricted to the superficial DH, stable over time, blocked by tetrodotoxin, and can be investigated in different slice orientations. Together, this preparation provides a platform to investigate DH circuit activity in tissue from na&#239;ve animals, animal models of chronic pain, and mice with genetically altered nociceptive function. Furthermore, MEA recordings in 4-AP-stimulated spinal cord slices can be used as a rapid screening tool to assess the capacity of novel antinociceptive compounds to disrupt activity in the spinal cord DH.

The combined use of microelectrode array technology and 4-aminopyridine-induced chemical stimulation for investigating network-level nociceptive activity in the spinal cord dorsal horn is outlined.

使用微电极阵列记录脊髓伤害感受回路中的网络活动

The roles and connectivity of specific types of neurons within the spinal cord dorsal horn (DH) are being delineated at a rapid rate to provide an ...

Mechanical Conflict-Avoidance Assay to Measure Pain Behavior in Mice

Pain comprises of both sensory (nociceptive) and affective (unpleasant) dimensions. In preclinical models, pain has traditionally been assessed using reflexive tests that allow inferences regarding pain's nociceptive component but provide little information about the affective or motivational component of pain. Developing tests that capture these components of pain are therefore translationally important. Hence, researchers need to use non-reflexive behavioral assays to study pain perception at that level. Mechanical conflict-avoidance (MCA) is an established voluntary non-reflexive behavior assay, for studying motivational responses to a noxious mechanical stimulus in a 3 chamber paradigm. A change in a mouse's location preference, when faced with competing noxious stimuli, is used to infer the perceived unpleasantness of bright light versus tactile stimulation of the paws. This protocol outlines a modified version of the MCA assay which pain researchers can use to understand affective-motivational responses in a variety of mouse pain models. Though not specifically described here, our example MCA data use the intraplantar complete Freund's adjuvant (CFA), spared nerve injury (SNI), and a fracture/casting model as pain models to illustrate the MCA procedure.

The mechanical conflict-avoidance assay is used as a non-reflexive readout of pain sensitivity in mice which can be used to better understand affective-motivational responses in a variety of mouse pain models.

机械冲突避免测定测量小鼠疼痛行为

Pain comprises of both sensory (nociceptive) and affective (unpleasant) dimensions. In preclinical models, pain has traditionally been assessed using ...

Assessment of Thermal Damage from Robot-Drilled Craniotomy for Cranial Window Surgery in Mice

Cranial window surgery allows for the imaging of brain tissue in live mice with the use of multiphoton or other intravital imaging techniques. However, when performing any craniotomy by hand, there is often thermal damage to brain tissue, which is inherently variable surgery-to-surgery and may be dependent on individual surgeon technique. Implementing a surgical robot can standardize surgery and lead to a decrease in thermal damage associated with surgery. In this study, three methods of robotic drilling were tested to evaluate thermal damage: horizontal, point-by-point, and pulsed point-by-point. Horizontal drilling utilizes a continuous drilling schematic, while point-by-point drills several holes encompassing the cranial window. Pulsed point-by-point adds a &#34;2 s on, 2 s off&#34; drilling scheme to allow for cooling in between drilling. Fluorescent imaging of Evans Blue (EB) dye injected intravenously measures damage to brain tissue, while a thermocouple placed under the drilling site measures thermal damage. Thermocouple results indicate a significant decrease in temperature change in the pulsed point-by-point (6.90 &#176;C &#177; 1.35 &#176;C) group compared to the horizontal (16.66 &#176;C &#177; 2.08 &#176;C) and point-by-point (18.69 &#176;C &#177; 1.75 &#176;C) groups. Similarly, the pulsed point-by-point group also showed significantly less EB presence after cranial window drilling compared to the horizontal method, indicating less damage to blood vessels in the brain. Thus, a pulsed point-by-point drilling method appears to be the optimal scheme for reducing thermal damage. A robotic drill is a useful tool to help minimize training, variability, and reduce thermal damage. With the expanding use of multiphoton imaging across research labs, it is important to improve the rigor and reproducibility of results. The methods addressed here will help inform others of how to better use these surgical robots to further advance the field.

Cranial windows have become a ubiquitously implemented surgical technique to allow for intravital imaging in transgenic mice. This protocol describes the use of a surgical robot that performs semi-automated bone drilling of cranial windows and can help reduce surgeon-to-surgeon variability and partially mitigate thermal blood-brain barrier damage.

机器人钻孔开颅术对小鼠颅窗手术热损伤的评估

Cranial window surgery allows for the imaging of brain tissue in live mice with the use of multiphoton or other intravital imaging techniques. However, ...