这项工作得到了科学和技术部 (106-2320-037-024)、高雄医科大学 (KMU-M106028、KMU-S105034) 的赠款和台湾高雄医科大学顶尖大学补助金 (TP105PR15) 的资助。

所描述的所有程序均符合实验室动物的道德准则30, 该议定书已获台湾高雄医科大学动物委员会批准。
1. RTX 神经病变的建立
警告:RTX 是神经毒素和危险的。在接触时, 它会对眼睛、粘膜和上呼吸道起到刺激性作用。在 RTX 准备期间, 避免吸入和佩戴实验室眼镜和大衣。用大量的水冲洗, 以防皮肤接触或处理后。
<ol><li>添加1毫克的 RTX 粉到200µL 的混合物相同体积的吐温80和绝对乙醇 (100 µL 为每个溶剂)。</li>
	<li>整除的 RTX 解决方案 (12 µL/瓶) 和存储在-20 °c, 长达3月。这构成了 RTX 的股票;在到期时丢弃剩余的 RTX 解决方案。</li>
	<li>将 RTX 的普通盐水稀释成600µL 的最终体积。RTX 溶液的最终浓度应为 0.01%, 在10µL 车溶液中等于1µg RTX。</li>
	<li>使用8周大的成年雄性小鼠 (35-40 克) 作为实验动物, 并管理一个单一剂量的 RTX 溶液 (剂量: 200, 50 和10µg/千克, 分别) 腹腔 (ip) 与注射注射器的小鼠。小鼠用吸入器麻醉, 用5% 异氟醚进行深度麻醉。如果小鼠在 RTX 注射过程中表现出肢体的退缩作用, 则小鼠应采取更长的麻醉吸入。 例如: 如果鼠标重40克, 那么它将收到 RTX 溶液的20µL, 表示剂量为50µg/千克。</li>
	<li>给一组小鼠同等数量的车辆 (10% 吐温80和10% 绝对乙醇在盐水溶液中), 作为一个控制。</li>
	<li>RTX 注射后, 将老鼠送回一个12小时 light/12-h 黑循环的塑料笼, 并提供食物和水的ad 随意。</li>
</ol>2. 神经病学行为的评价
注意: 在舒适的环境中保持动物 (步骤 1.6), 以便在注射后恢复。在7天后 RTX 注射液 (D7), 每一个动物执行热板和冯弗雷头发灯丝测试在同一天, 以减少时间偏差和提高行为测试的效率。把动物带到一个安静的房间, 保持在稳定的湿度 (40%) 和温度 (27 °c), 以优化动物驯化和减少环境影响, 在行为测试。在试验期间请勿打扰动物;行为测试是每周计划的。
<ol><li>热板试验测量热延迟<ol>
<li>将动物轻轻地放在金属热板上 (27 厘米 x 29 厘米), 透明有机玻璃笼 (长度 x 宽度 x 高度:22 厘米 x 22 厘米 x 25 厘米;图 1A)。将金属热板的温度设置为52摄氏度。</li>
		<li>开始测量在热板上的动物的热潜伏期的持续时间, 一旦动物的 hindpaws 触摸热板, 并观察动物的 hindpaws 反应, 热板的内置定时器。如果动物显示震动, 舔 hindpaws, 或跳跃, 而在热板上, 删除它, 并记录的持续时间, 动物留在热板。此时间长度定义了单个动物的热滞后时间。将热滞后时间记录到最近的0.1 秒。</li>
		<li>对于每个测试会话, 在最后一个热板测试之后, 执行三个具有30分钟间隔的试验, 以响应正常化。如果动物在热板上没有反应, 停止在二十五年代以后的会议, 以避免潜在的组织损伤。</li>
	</ol>
</li>
	<li>用弗雷毛细丝试验测量机械阈值<ol>
<li>将动物放在定制的金属网格上 (网格尺寸: 5 毫米 x 5 毫米), 带有半透明的圆筒形有机玻璃笼 (直径:13 厘米; 高度:12 厘米) (图 1B), 用于至少 2 h 的驯化。</li>
		<li>使用上下方法31, 将不同口径的 hindpaw 毛细丝应用到足底区域。从一组冯弗雷发丝的中间力开始初步应用 5-8 s 的长丝应用。</li>
		<li>在灯丝应用程序之间使用2分钟间隔来优化动物规范化。根据动物的最后反应改变应用的灯丝力。 注: 一组冯弗雷头发花丝包括 0.064, 0.085, 0.145, 0.32, 0.39, 1.1, 和 1.7 g 力量应用。例如, 如果 hindpaw 退出时发生的初始力为0.32 克, 则应用 0.145 g。在没有爪子撤退, 然后使用0.39 克力量。然后根据前面的响应应用四种不同力的附加丝, 并根据已发布的公式31计算机械阈值。</li>
		<li>对于每个测试会话, 包括双边 hindpaws。为每个 hindpaw 执行三项试验。将这六机械阈值的平均值表示为每种动物的平均机械阈值 (毫克)。</li>
	</ol>
</li>
</ol><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56651/56651fig1.jpg"> 图1。自制的有机玻璃笼和金属网, 用于评价树酯毒素 (RTX) 诱导的小纤维神经病变模型小鼠神经病理性疼痛。(A, B)这些图表显示用于测量 (A) 热延迟的设备, 金属热板 (27 厘米 x 29 厘米) 与透明有机玻璃笼 (长度 x 宽度 x 高度:22 厘米 x 22 厘米 x 25 厘米) 并通过自定义元计算 (B) 机械阈值l 网格 (网格尺寸: 5 毫米 x 5 毫米), 具有半透明的圆筒形有机玻璃笼 (直径:13 厘米; 高度:12 厘米), RTX 诱导的小纤维神经病变的小鼠。<a href="//cloudflare.jove.com/files/ftp_upload/56651/56651fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
3. 皮肤活检的制备和 IENFs 神经支配的评价
<ol><li>在行为测试以后, 麻醉动物与5% 异氟醚和牺牲动物通过心脏灌注与 0.1 m 磷酸盐缓冲 (PB) (pH 7.4) 跟随4% 多聚甲醛 (4P) 在 0.1 M PB。</li>
	<li>在灌注后切开两 hindpaws 的第一脚垫, 并在4P 后将其贴上6小时. 将都安组织转移到0.1 米 PB, 以4摄氏度为长期贮存。</li>
	<li>Cryoprotect 脚垫与30% 蔗糖在 PB 过夜, 并在30µm 厚切片垂直到足底表面的方式切割。按顺序对都安部分进行标签, 然后在-20 摄氏度的防冻剂中贮存。 注: 防冻剂成分如下: 蒸馏水, 乙烯甘油, 甘油, 和 2x PB 在 3:3: 3:1 的比例。</li>
	<li>为确保充分取样, 请选择都安的每第三个部分。<ol>
<li>将所选的都安部分放在涂覆的玻璃滑梯上, 晾干。</li>
		<li>在幻灯片上盖上塑料 coverplate, 并使用标准染色程序进行处理。<ol>
<li>在甲醇中用 1% h2O2的都安部分淬火30分钟, 并用0.5% 米三缓冲器 (三) 0.1% 个 X-100 的0.5 个脱脂干牛奶和个海卫一。</li>
			<li>孵化都安部分与血清对 pan 轴突标记, PGP 9.5 (上升在兔子; 1:1, 000), 隔夜在4°c。</li>
			<li>用生物素化山羊抗兔 IgG 二级抗体在室温 (RT) 上孵化都安段, 然后在 rt 中孵育亲和-生物素复合体, 45 分钟。</li>
			<li>四十五年代 0.05% 33 '-diaminobenzidine (民建联) 溶液可视化反应产物。然后用蒸馏水冲洗都安部分, 晾干后再进行安装。 注: 主要和次要血清稀释与0.5% 脱脂干牛奶在0.5 米三。</li>
		</ol>
</li>
	</ol>
</li>
</ol>4. 根背根节的制备及损伤小直径神经元的评价
<ol><li>解剖 4th和 5th腰椎节段和后修复为另2小时。</li>
	<li>Cryoprotect 背节组织与30% 蔗糖在 PB 过夜, 并削减到8µm 厚度依次, 在显微镜幻灯片和标签。在-80 摄氏度冷藏库中储存背根节段。</li>
	<li>Immunostain 节段在80µm 间隔, 以确保适当的取样。<ol>
<li>执行背节染色程序作为那些都安部分, 除了双重标记免疫荧光程序。另外, 包括 ATF3 (在兔子中上升, 1:100), 损伤标记, 和感光 (在小鼠; 1:800), 一个小直径神经元标记在主要血清。</li>
		<li>在4摄氏度的一夜之间, 用主要血清的混合物孵化根背节。</li>
	</ol>
</li>
	<li>用德州红或荧光素异硫氰酸酯 (FITC)-共轭次生血清 (1:200) 孵化根节, 对应于 RT 中适当的主要血清1小时, 然后装入定量。</li>
</ol>

作者没有什么可透露的

为提高患者的功能恢复和生活质量, 需要在临床上有效地治疗小纤维神经病变。目前, 由于缺乏对小直径神经元损伤的分子机制的全面了解, 缺乏针对小纤维神经病变的感觉障碍的治疗指南。以往的神经病变模型通常会影响大直径和小口径的感觉神经;例如, 化疗诱发神经病变的模型12,32,33和机械诱发神经病变34,35。因此, 在这些神经病变模型的行为测试中, 不能完全排除运动弱点和大直径感觉神经损伤的作用。本协议描述了小鼠纤维神经病变的新模型, 它只通过提供 IENFs 变性的病理和功能证据影响小直径感觉神经。
RTX 是 TRPV1 的 ultrapotent 激动剂和辣椒素模拟, 这可能会导致肽能背根神经节神经元的损失在文化36和体内系统 18, 19. 以往对 RTX 和辣椒素的研究主要集中在神经节神经元细胞体的形态学或功能丧失上, 揭示了 TRPV1 在热传导响应中的作用37,38,39。此外, 先前的一项研究表明, 有系统的高剂量 RTX 治疗 (200 µg/千克) 大鼠, 诱导机械痛觉和热 hypoalgesia, 可能是由于病理的大直径神经纤维28。然而, 剂量200µg/千克, 是一个致命剂量的小鼠, 这一目前的协议开发了纯小纤维神经病变模型通过减少 RTX 剂量 (50 µg/千克)。这种剂量的 RTX (50 µg/千克) 是建立一个纯小纤维神经病变模型的关键, 这是优于以前报告的28, 因为它的备件大纤维18。即它只影响小神经纤维;ATF3 上调6,40对小直径背节神经元和 IENFs 变性6,18,19 的归纳证实, 只有小直径神经元受伤. ,41, 与感官紊乱有关。这些病理表现综合模仿小纤维神经病变的临床症状。此外, 该模型诱发了小纤维神经病变的典型神经病理学和神经性疼痛, 其影响持续8周后 RTX 治疗 6, 18, 19.神经病理学和神经病理性疼痛的持续时间是等价的, 可以通过促进合成神经生长因子 (NGF) 18, 40, 41 来逆转.总的来说, 该协议建立了一个纯小纤维神经病变模型, 并强调了可能的治疗潜力的 NGF。
临床上, 调查神经病影响小直径伤害神经的金标准8,9是 biopsying 肢体皮肤评估皮肤神经支配。本研究报告将此技术应用于实验动物的都安皮肤, 评价小纤维神经病变模型的皮肤神经支配, 可模拟 IENFs 在临床上的病理, 并对根背根的形态特征进行了探讨。部分用损伤标记, ATF3, 揭示神经胞体的病理状态。显著的是, 表皮内 IENFs 的空间分布高度分支, 计数标准是导致群体间统计学差异的主要因素。例如, 我们的当前协议计算了每个 IENF, 只有在真皮和 IENFs 的分支点, 在表皮内的分支点作为一个单一的 IENF14,18,19。这一标准可能导致我们调查的 IENFs 密度低于其他组。我们用我们目前修改的协议, 以系统和批量评价的方式, 对实验动物的皮肤和根背节进行了处理。因此, 这些系统的研究 IENF 变性和神经元损伤可以避免小直径神经元在小纤维神经病变的功能和病理条件的视学偏差。
小直径神经的功能性评估, 特别是与无毒的头发灯丝应用, 传统上应用于患者皮肤诊断小纤维的机械敏感性神经病.对实验动物的机械痛觉的观察是很有挑战性的, 因为脚接地在金属网, 这被认为是外源机械刺激, 和动物在试验期间非常活跃。目前的协议优化了一个特定大小的网格线地板 (5 毫米 x 5 毫米) 在一个半透明的塑料笼中的环境适应实验动物的行为测试。这种大小的网状地板可以减少外源刺激脚接地, 避免徒步下降。
这种 RTX 小鼠神经病变模型可应用于不同类型的小纤维神经病变, 如糖尿病, 这与 IENF 变性1,42有关。然而, 这种模式仍然有限。例如, 脊髓神经结扎的动物模型43具有小纤维神经病变的特点, 称为临床神经根病, 也可能影响脊髓支根中的大纤维。

小纤维神经病变涉及神经病理性疼痛, 这是明显的退化 IENFs, 是常见的各种类型的条件, 如 DM, 并由于化疗药物的神经毒性作用1,2, 3,4,5。IENFs 是位于背根神经节的小直径神经元的外围终端, 并在 IENF 变性6的情况下平行地受到影响。例如, 上调激活转录 factor-3 (ATF3)6,7, 证明了改变的神经元胞体的上游基因转录。此外, 评估 IENFs 神经支配与皮肤活检有助于诊断小纤维神经病变5,8,9。传统上, 皮肤活检 IENFs 的概况依赖于蛋白质基因产品 9.5 (PGP 9.5)1,10,11的免疫组化演示。结合, 神经节和 IENFs 的病理剖面反映小纤维神经病变的功能状况, 可能是这类神经病变对小直径神经元功能性后果的指示。
以前, 有几个实验模型讨论了化疗诱发神经病变的 IENF 变性问题12,13和由压缩或横断导致的神经损伤14,15,16. 这些实验模型也影响大口径神经;因此, 不可能排除受影响的大口径神经在观察到的小纤维神经病变中的贡献;例如, 通过有害的撤退检查 thermosensation 障碍取决于功能性马达神经纤维17,18,19。因此, 建立单纯的小纤维神经病变模型, 系统地研究小直径神经元中神经元胞体及其周围皮神经纤维的病理状态是必要的和必要的。
RTX 是一种辣椒素模拟剂和一个强有力的激动剂对瞬态受体电位辣椒受体 1 (TRPV1), 它介导伤害处理20,21,22。最近, 外围 RTX 治疗缓解神经源性疼痛23,24,25和 intraganglionic 注射 RTX 引起的背根神经节神经元的不可逆转的损失22。外围 RTX 管理的效果是剂量依赖性20,26,27, 这导致 IENFs 的瞬变脱敏或变性。有趣的是, 系统性的大剂量 RTX 治疗导致神经病理性疼痛28, 小纤维神经病变的症状。这些发现表明, RTX 的治疗方式和剂量会产生明显的病理效应和神经元反应;对机智, 外围管理防止痛苦传输由地方作用29并且影响了神经胞体发展神经病变行为 6.总的来说, 这些发现表明, RTX 有一个多向分化潜能的影响, 并提出了问题是否有一个特定剂量的 RTX, 可能有系统地影响周围神经, 如外围 IENFs 和中枢神经元胞体。如果是这样, RTX 可能是一个潜在的代理人, 专门影响小直径神经元和模拟小纤维神经病变在诊所。例如, DM 在临床上是一个复杂的问题, 包括代谢紊乱和神经病理学的周围神经, 这是主要特点的小纤维神经病变。糖尿病相关的小纤维神经病变的机制不能排除可能不是影响周围神经的主要药物代谢紊乱的贡献。因此, DM 相关的小纤维神经病变需要一个纯动物模型, 可以排除系统性代谢紊乱的影响。该协议描述了 RTX 的工作剂量, 以发展一个典型的小纤维神经病变模型, 包括 IENF 变性和小直径神经元损伤, 通过改进的染色分析表明。

<table><tbody><tr><td>&lt;strong&gt;Chemical reagent&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Resiniferatoxin</td><td>Sigma</td><td>R8756</td><td></td></tr><tr><td>Tween 80</td><td>Sigma</td><td>P1754</td><td></td></tr><tr><td>3,3&amp;rsquo;-diaminobenzidine</td><td>Sigma</td><td>D8001</td><td></td></tr><tr><td>avidin-biotin complex</td><td>Vector</td><td>PK-6100</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Primary Antisera&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Peripherin</td><td>Chemicon</td><td>MAB-1527</td><td></td></tr><tr><td>ATF3</td><td>Santa Cruz</td><td>SC-188</td><td></td></tr><tr><td>PGP9.5</td><td>UltraClone</td><td>RA95101</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Secondary Antisera&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Biotinylated goat anti-rabbit IgG</td><td>Vector</td><td>BA-1000</td><td></td></tr><tr><td>Texas Red-conjugated goat anti-mouse</td><td>Jackson ImmunoResearch</td><td>115-075-146</td><td></td></tr><tr><td>Isothiocyanate (FITC)-conjugated donkey anti-rabbit</td><td>Jackson ImmunoResearch</td><td>711-095-152</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Hot plate</td><td>IITC</td><td>Model 39</td><td></td></tr><tr><td>von Frey filament</td><td>Somedic Sales AB</td><td>10-600-0001</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Material&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Shandon coverplate</td><td>Thermo scientific</td><td>72110017</td><td></td></tr><tr><td>Slide rack</td><td>Thermo scientific</td><td>73310017</td><td></td></tr></tbody></table>

establishing a mouse model of a pure small fiber neuropathy with the ultrapotent agonist of transient receptor potential vanilloid type 1

糖尿病患者 (DM) 或那些经历了化疗药物的神经毒性作用的人可能会产生感觉障碍, 由于退化和损伤小直径感觉神经元, 称为小纤维神经病变。目前小纤维神经病变的动物模型对大直径和小口径感觉纤维都有影响, 从而造成神经病理学太复杂, 无法正确评估小直径感觉纤维受损的影响。因此, 有必要建立一个纯小纤维神经病变实验模型, 以充分研究这些问题。该协议描述了小纤维神经病变的实验模型, 具体影响小直径感觉神经与树酯毒素 (RTX), ultrapotent 激动剂的瞬态受体电位辣椒类型 1 (TRPV1), 通过单一剂量的腹腔注射, 称为 RTX 神经病。这种 RTX 神经病变的病理表现和行为异常, 模仿小纤维神经病变患者的临床特点, 包括 intraepidermal 神经纤维 (IENF) 变性, 特别是损伤小直径神经元, 并诱导热 hypoalgesia 和机械痛觉。该协议分别测试了三剂量的 RTX (200, 50 和10µg/千克), 并得出结论认为, 关键剂量的 RTX (50 µg/千克) 是必要的发展典型的小纤维神经病变的表现, 并准备了一个改进的染色程序, 以研究 IENF 变性和神经元躯体损伤。修改后的程序快速、系统、经济。神经性疼痛的行为评价对于揭示小直径感觉神经的功能至关重要。对实验性啮齿动物的机械阈值的评估特别具有挑战性, 该协议描述了一种适合于啮齿类动物评估的定制金属网格。总之, RTX 神经病变是一种新的、容易建立的实验模型, 用于评价神经性疼痛的分子意义和干预作用, 为治疗药物的发展提供依据。

该协议描述了一种新的 RTX 神经病变的小鼠模型, 它特别影响到与感官紊乱相关的微小的神经元, 包括 IENF 变性 (图 2)。根据本文所述的协议, 动物在 RTX 注射 D7 后表现出热 hypoalgesia 和机械痛觉。为了建立这种小纤维神经病变模型, 三剂量的 RTX: 200, 50, 10 µg/公斤由 ip 路线管理。RTX 剂量 (50 µg/千克) 被认为是至关重要的, 初步研究表明, 高剂量 RTX (200 µg/千克) 导致了大鼠杀伤力 (图 3)。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56651/56651fig2.jpg"> 图2。树酯毒素 (RTX) 诱导小纤维神经病变的鼠模型方案。该方案显示了建立的 RTX 诱导小纤维神经病变的协议。对系统评价、行为评价和病理检查, 分别包括热板和弗雷试验、双标染色研究。<a href="//cloudflare.jove.com/files/ftp_upload/56651/56651fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56651/56651fig3.jpg"> 图3。树酯毒素 (RTX) 对动物致死性和行为功能障碍的剂量效应。(A) 通过腹腔 (ip) 注射, 对不同剂量的 RTX 进行了管理。剂量效应的致死率为剂量依赖性;例如, 高剂量的 RTX (200 µg/千克) 导致100% 致命性。(B, C)用热板 (B) 和冯弗雷灯丝测试 (C) 分别对热延迟和机械阈值进行了评估。与车辆和10µg/千克管理组相比, 50 µg/千克剂量的 RTX 诱发的热 hypoalgesia 和机械痛觉。开放广场, 车辆;开圈, 50 µg/千克;开钻, 10 µg/公斤。虚线 (B), 热板测试的截止时间点。p < 0.001。<a href="//cloudflare.jove.com/files/ftp_upload/56651/56651fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
病理上, 有 IENF 变性和显着 ATF3 诱导。双标记研究表明, 受损神经元是专门感光 (+) 小直径神经元。相比之下, 低剂量的 RTX (10 µg/千克) 没有建立小纤维神经病变, 包括 IENF 神经支配没有改变 (图 4) 和没有神经元损伤 (ATF3 感应) (图 5)。因此, 本议定书认为50µg/千克剂量对建立小纤维神经病变鼠模型至关重要。
总之, 系统 RTX 管理与50µg/千克剂量专门影响小神经纤维。例如, 它导致神经元躯体损伤和周围 IENF 变性, 这与感官紊乱有关。
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56651/56651fig4.jpg"> 图4。intraepidermal 神经纤维变性 (IENFs) 树酯毒素 (RTX) 神经病变。(-c)从小鼠的都安皮肤组织切片 immunostained 与抗蛋白基因产品 9.5 (PGP 9.5) 血清在车辆 (A), 50 µg/千克-(B) 和10µg/千克管理 (C) 组。PGP 9.5 (+) IENFs 出现从联合应用疱神经丛与典型的静脉曲张出现。PGP 9.5 (+) IENFs 明显减少50µg/千克, 但不在10µg/千克组。(D) IENFs 是根据-c的免疫组化结果量化的。开放广场, 车辆;开圈, 50 µg/千克;开钻, 10 µg/公斤。p < 0.001 与车辆组比较。缩放条, 50 µm.<a href="//cloudflare.jove.com/files/ftp_upload/56651/56651fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56651/56651fig5.jpg"> 图5。树酯毒素 (RTX) 神经病变小直径神经元损伤的特异性。(-c)用抗活化转录 factor-3 (ATF3) 进行双标记荧光染色;在车辆 (a)、50µg/千克 (B) 和10µg/千克管理的 (C) 组中, -c、绿色) 和感光 (-c、红色)。(D) 图表明了 ATF3 (+) 神经元的密度变化。ATF3 (+) 神经元增加50µg/千克, 但不在车辆和10µg/公斤组。开放广场, 车辆;开圈, 50 µg/千克;开钻, 10 µg/公斤。p < 0.001 与车辆组比较。缩放条, 25 µm.<a href="//cloudflare.jove.com/files/ftp_upload/56651/56651fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Establishing a Mouse Model of a Pure Small Fiber Neuropathy with the Ultrapotent Agonist of Transient Receptor Potential Vanilloid Type 1 at JoVE.com

Establishing a Mouse Model of a Pure Small Fiber Neuropathy with the Ultrapotent Agonist of Transient Receptor Potential Vanilloid Type 1

利用瞬态受体电位辣椒1型 Ultrapotent 激动剂建立单纯小纤维神经病变的小鼠模型

本研究建立了树酯毒素 (RTX) 纯小纤维神经病变的实验模型。独特剂量的 RTX (50 µg/千克) 是最佳的发展一个小纤维神经病变模型, 模仿病人的特点, 并可以帮助调查的伤害性分子意义下的神经病理性疼痛。

Patients with diabetes mellitus (DM) or those experiencing the neurotoxic effects of chemotherapeutic agents may develop sensation disorders due to ...

establishing-mouse-model-pure-small-fiber-neuropathy-with-ultrapotent

Nanyang Technological University

Research

JoVE Journal

Neuroscience

9.5K 次观看.  Kaohsiung Medical University. 本研究建立了树酯毒素 (RTX) 纯小纤维神经病变的实验模型。独特剂量的 RTX (50 µg/千克) 是最佳的发展一个小纤维神经病变模型, 模仿病人的特点, 并可以帮助调查的伤害性分子意义下的神经病理性疼痛。

视频: Establishing a Mouse Model of a Pure Small Fiber Neuropathy with the Ultrapotent Agonist of Transient Receptor Potential Vanilloid Type 1

The Sciatic Nerve Cuffing Model of Neuropathic Pain in Mice

Neuropathic pain arises as a consequence of a lesion or a disease affecting the somatosensory system. This syndrome results from maladaptive changes in injured sensory neurons and along the entire nociceptive pathway within the central nervous system. It is usually chronic and challenging to treat. In order to study neuropathic pain and its treatments, different models have been developed in rodents. These models derive from known etiologies, thus reproducing peripheral nerve injuries, central injuries, and metabolic-, infectious- or chemotherapy-related neuropathies. Murine models of peripheral nerve injury often target the sciatic nerve which is easy to access and allows nociceptive tests on the hind paw. These models rely on a compression and/or a section. Here, the detailed surgery procedure for the &#34;cuff model&#34; of neuropathic pain in mice is described. In this model, a cuff of PE-20 polyethylene tubing of standardized length (2 mm) is unilaterally implanted around the main branch of the sciatic nerve. It induces a long-lasting mechanical allodynia, i.e., a nociceptive response to a normally non-nociceptive stimulus that can be evaluated by using von Frey filaments. Besides the detailed surgery and testing procedures, the interest of this model for the study of neuropathic pain mechanism, for the study of neuropathic pain sensory and anxiodepressive aspects, and for the study of neuropathic pain treatments are also discussed.

Neuropathic pain is a consequence of a lesion or disease affecting the somatosensory system. The &#8220;cuff model&#8221; of neuropathic pain in mice consists of the implantation of a polyethylene cuff around the main branch of the sciatic nerve. Mechanical allodynia is tested using von Frey filaments.

神经病理性疼痛小鼠坐骨神经掴型号

Neuropathic pain arises as a consequence of a lesion or a disease affecting the somatosensory system. This syndrome results from maladaptive changes in ...

科研

医学

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 ...

神经科学

A Simple Approach to Induce Experimental Autoimmune Neuritis in C57BL/6 Mice for Functional and Neuropathological Assessments

Experimental autoimmune neuritis (EAN) is a well-appreciated experimental model of autoimmune peripheral demyelinating diseases. EAN disease is induced by immunizing mice with neurogenic peptides to direct an inflammatory attack toward components of the peripheral nervous system (PNS). Recent advances have enabled the induction of EAN in the relatively resistant C57BL/6 mouse line using myelin protein zero (P0)106-125 or P0180-199 peptides delivered in adjuvant combined with the injection of pertussis toxin. The ability to induce EAN in the C57BL/6 strain allows for the use of the numerous genetic tools that exist on this mouse background, and thus allows the sophisticated study of disease pathogenesis and interrogation of the mechanistic action of novel therapeutics in combination with transgenic approaches. In this study, we demonstrate a simple approach to successfully induce EAN using the P0180-199 peptide in C57BL/6 mice. We also outline a protocol for the assessment of functional deficits that occur in this model, accompanied by an array of neuropathological features. Thus, this model is a powerful experimental model to study the pathogenesis of human peripheral demyelinating neuropathies, and to determine the efficacy of potential therapies that aim to promote myelin repair and protect against nerve damage in autoimmune neuritis.

This report outlines a simple approach to successfully induce experimental autoimmune neuritis (EAN) using the myelin protein zero (P0)180-199 peptide in combination with Freund&#39;s complete adjuvant and pertussis toxin. We present a sophisticated paradigm capable of accurately assessing the extent of functional deficits and neuropathology that occur in this EAN.

C57BL/6 小鼠自身免疫性神经炎的功能和病理评价的简易方法

Experimental autoimmune neuritis (EAN) is a well-appreciated experimental model of autoimmune peripheral demyelinating diseases. EAN disease is induced by ...

Intracerebroventricular Treatment with Resiniferatoxin and Pain Tests in Mice

The transient receptor potential vanilloid type 1 (TRPV1), a thermosensitive cation channel, is known to trigger pain in the peripheral nerves. In addition to its peripheral function, its involvement in brain functions has also been suggested. Resiniferatoxin (RTX), an ultrapotent TRPV1 agonist, has been known to induce long-term desensitization of TRPV1, and this desensitization has been an alternative approach for investigating the physiological relevance of TRPV1-expressing cells. Here we describe a protocol for intracerebroventricular (i.c.v.) treatment with RTX in mice. Procedures are described for testing nociception to peripheral TRPV1 stimulation (RTX test) and mechanical stimulation (tail pressure test) then follow. Although the nociceptive responses of mice that had been administered RTX i.c.v. were comparable to those of the control groups, RTX-i.c.v.-administered mice were insensitive to the analgesic effect of acetaminophen, suggesting that i.c.v. RTX treatment can induce supraspinal-selective TRPV1 desensitization. This mouse model can be used as a convenient experimental system for studying the role of TRPV1 in brain/supraspinal function. These techniques can also be applied to studies of the central actions of other drugs.

The transient receptor potential vanilloid type 1 (TRPV1) in the supraspinal region has been suggested to play some roles in the brain function. Described here is a protocol for intracerebroventricular injection of resiniferatoxin for supraspinal TRPV1 desensitization in mice. Procedures for some pain tests are also presented.

小鼠用树脂叶毒素和疼痛测试进行脑血管内治疗

The transient receptor potential vanilloid type 1 (TRPV1), a thermosensitive cation channel, is known to trigger pain in the peripheral nerves. In ...

Mouse Footpad Inoculation Model to Study Viral-Induced Neuroinflammatory Responses

This protocol describes a footpad inoculation model used to study the initiation and development of neuroinflammatory responses during alphaherpesvirus infection in mice. As alphaherpesviruses are main invaders of the peripheral nervous system (PNS), this model is suitable to characterize the kinetics of viral replication, its spread from the PNS to CNS, and associated neuroinflammatory responses. The footpad inoculation model allows virus particles to spread from a primary infection site in the footpad epidermis to sensory and sympathetic nerve fibers that innervate the epidermis, sweat glands, and dermis. The infection spreads via the sciatic nerve to the dorsal root ganglia (DRG) and ultimately through the spinal cord to the brain. Here, a mouse footpad is inoculated with pseudorabies virus (PRV), an alphaherpesvirus closely related to herpes simplex virus (HSV) and varicella-zoster virus (VZV). This model demonstrates that PRV infection induces severe inflammation, characterized by neutrophil infiltration in the footpad and DRG. High concentrations of inflammatory cytokines are subsequently detected in homogenized tissues by ELISA. In addition, a strong correlation is observed between PRV gene and protein expression (via qPCR and IF staining) in DRG and the production of pro-inflammatory cytokines. Therefore, the footpad inoculation model provides a better understanding of the processes underlying alphaherpesvirus-induced neuropathies and may lead to the development of innovative therapeutic strategies. In addition, the model can guide research on peripheral neuropathies, such as multiple sclerosis and associated viral-induced damage to the PNS. Ultimately, it can serve as a cost-effective in vivo tool for drug development.

The footpad inoculation model is a valuable tool for characterizing viral-induced neuroinflammatory responses in vivo. In particular, it provides a clear assessment of viral kinetics and associated immunopathological processes initiated in the peripheral nervous system.

小鼠脚垫接种模型，研究病毒引起的神经炎反应

This protocol describes a footpad inoculation model used to study the initiation and development of neuroinflammatory responses during alphaherpesvirus ...

免疫与感染

Modified Spared Nerve Injury Surgery Model of Neuropathic Pain in Mice

Spared nerve injury (SNI) is an animal model that mimics the cardinal symptoms of peripheral nerve injury for studying the molecular and cellular mechanism of neuropathic pain in mice and rats. Currently, there are two types of SNI model, one to cut and ligate the common peroneal and the tibial nerves with intact sural nerve, which is defined as SNIs in this study, and another to cut and ligate the common peroneal and the sural nerves with intact tibial nerve, which is defined as SNIt in this study. Because the sural nerve is purely sensory whereas the tibial nerve contains both motor and sensory fibers, the SNIt model has much less motor deficit than the SNIs model. In the traditional SNIt mouse model, the common peroneal and the sural nerves are cut and ligated separately. Here a modified SNIt surgery method is described to damage both common peroneal and sural nerves with only one ligation and one cut with a shorter procedure time, which is easier to perform and reduces the potential risk of stretching the sciatic or tibial nerves, and produces similar mechanical hypersensitivity as the traditional SNIt model.

<meta charset="utf8"></head><body>改良的小鼠神经性疼痛备用神经损伤手术模型

The modified surgery is a simplified method for mouse or rat spared nerve injury model that requires only one ligation and one cut to injure both common peroneal and sural nerves.

改良的小鼠神经性疼痛备用神经损伤手术模型

Spared nerve injury (SNI) is an animal model that mimics the cardinal symptoms of peripheral nerve injury for studying the molecular and cellular ...

Open-Source Real-Time Closed-Loop Electrical Threshold Tracking for Translational Pain Research

Nociceptors are a class of primary afferent neurons that signal potentially harmful noxious stimuli. An increase in nociceptor excitability occurs in acute and chronic pain conditions. This produces abnormal ongoing activity or reduced activation thresholds to noxious stimuli. Identifying the cause of this increased excitability is required for the development and validation of mechanism-based treatments. Single-neuron electrical threshold tracking can quantify nociceptor excitability. Therefore, we have developed an application to allow such measurements and demonstrate its use in humans and rodents. APTrack provides real-time data visualization and action potential identification using a temporal raster plot. Algorithms detect action potentials by threshold crossing and monitor their latency after electrical stimulation. The plugin then modulates the electrical stimulation amplitude using an up-down method to estimate the electrical threshold of the nociceptors. The software was built upon the Open Ephys system (V0.54) and coded in C++ using the JUCE framework. It runs on Windows, Linux, and Mac operating systems. The open-source code is available (<a data-saferedirecturl="https://www.google.com/url?q=https://github.com/Microneurography/APTrack&amp;source=gmail&amp;ust=1680789829274000&amp;usg=AOvVaw0nMk9J_EI2jUn8SlmC5JJm" href="https://github.com/Microneurography/APTrack" target="_blank">https://github.com/Microneurography/APTrack</a>). The electrophysiological recordings were taken from nociceptors in both a mouse skin-nerve preparation using the teased fiber method in the saphenous nerve and in healthy human volunteers using microneurography in the superficial peroneal nerve. Nociceptors were classified by their response to thermal and mechanical stimuli, as well as by monitoring the activity-dependent slowing of the conduction velocity. The software facilitated the experiment by simplifying the action potential identification through the temporal raster plot. We demonstrate real-time closed-loop electrical threshold tracking of single-neuron action potentials during&#160;in vivo&#160;human microneurography, for the first time, and during&#160;ex vivo&#160;mouse electrophysiological recordings of C-fibers and A&#948;-fibers. We establish proof of principle by showing that the electrical threshold of a human heat-sensitive C-fiber nociceptor is reduced by heating the receptive field. This plugin enables the electrical threshold tracking of single-neuron action potentials and allows the quantification of changes in nociceptor excitability.

APTrack is a software plugin developed for the Open Ephys platform that enables real-time data visualization and the closed-loop electrical threshold tracking of neuronal action potentials. We have successfully used this in microneurography for human C-fiber nociceptors and mouse C-fiber and A&#948;-fiber nociceptors.

用于转化疼痛研究的开源实时闭环电阈值跟踪

Nociceptors are a class of primary afferent neurons that signal potentially harmful noxious stimuli. An increase in nociceptor excitability occurs in ...

Partial Sciatic Nerve Ligation: A Mouse Model of Chronic Neuropathic Pain to Study the Antinociceptive Effect of Novel Therapies

Management of chronic pain remains challenging to this day, and current treatments are associated with adverse effects, including tolerance and addiction. Chronic neuropathic pain results from lesions or diseases in the somatosensory system. To investigate potential therapies with reduced side effects, animal pain models are the gold standard in preclinical studies. Therefore, well-characterized and well-described models are crucial for the development and validation of innovative therapies. 
Partial ligation of the sciatic nerve (pSNL) is a procedure that induces chronic neuropathic pain in mice, characterized by mechanical and thermal hypersensitivity, ongoing pain, and changes in limb temperature, making this model a great fit to study neuropathic pain preclinically. pSNL is an advantageous model to study neuropathic pain as it reproduces many symptoms observed in humans with neuropathic pain. Furthermore, the surgical procedure is relatively fast and straightforward to perform. Unilateral pSNL of one limb allows for comparison between the ipsilateral and contralateral paws, as well as evaluation of central sensitization. 
To induce chronic neuropathic hypersensitivity, a 9-0 non-absorbable nylon thread is used to ligate the dorsal third of the sciatic nerve. This article describes the surgical procedure and characterizes the development of chronic neuropathic pain through multiple commonly used behavioral tests. As a plethora of innovative therapies are now being investigated to treat chronic pain, this article provides crucial concepts for standardization and an accurate description of surgeries required to induce neuropathic pain.

Partial sciatic nerve ligation induces long-lasting chronic neuropathic pain, characterized by exaggerated responses to thermal and mechanical stimuli. This mouse model of neuropathic pain is commonly used to study innovative therapies for pain management. This article describes in detail the surgical procedure to improve standardization and reproducibility.

部分坐骨神经结扎术:慢性神经性疼痛的小鼠模型，用于研究新疗法的抗伤害感受作用

Management of chronic pain remains challenging to this day, and current treatments are associated with adverse effects, including tolerance and addiction. ...

<meta charset="utf8"></head><body>建立三叉神经性疼痛的小鼠模型

Chronic Constriction Injury of the Distal Infraorbital Nerve (DIoN-CCI) in Mice to Study Trigeminal Neuropathic Pain

Animal models remain necessary tools to study neuropathic pain. This manuscript describes the distal infraorbital nerve chronic constriction injury (DIoN-CCI) model to study trigeminal neuropathic pain in mice. This includes the surgical procedures to perform the chronic constriction injury and the postoperative behavioral tests to evaluate the changes in spontaneous and evoked behavior that are signs of ongoing pain and mechanical allodynia. The methods and behavioral readouts are similar to the infraorbital nerve chronic constriction injury (IoN-CCI) model in rats. However, important changes are necessary for the adaptation of the IoN-CCI model to mice. First, the intra-orbital approach is replaced by a more rostral approach with an incision between the eye and the whisker pad. The IoN is thus ligated distally outside the orbital cavity. Secondly, due to the higher locomotor activity in mice, allowing rats to move freely in small cages is replaced by placing mice in custom-designed and constructed restraining devices. After DIoN ligation, mice exhibit changes in spontaneous behavior and in response to von Frey hair stimulation that are similar to those in IoN-CCI rats, i.e., increased directed face grooming and hyperresponsiveness to von Frey hair stimulation of the IoN territory.

<meta charset="utf8"></head><body>作者聚焦:利用小鼠眶下神经结扎术研究三叉神经性疼痛和治疗策略

Chronic constriction injury of the distal infraorbital nerve in mice induces changes in spontaneous behavior (increased face grooming activity) and nocifensive behavior in response to tactile stimulation (hyperresponsiveness to von Frey hair stimulation) that are signs of ongoing pain and allodynia and serves as a model for trigeminal neuropathic pain.

小鼠远端眶下神经慢性缩窄损伤 （DIoN-CCI） 研究三叉神经性疼痛

Animal models remain necessary tools to study neuropathic pain. This manuscript describes the distal infraorbital nerve chronic constriction injury ...

利用瞬态受体电位辣椒1型 Ultrapotent 激动剂建立单纯小纤维神经病变的小鼠模型

摘要

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此视频中的章节

神经病理性疼痛小鼠坐骨神经掴型号

测定冷灵敏度和适应小鼠一种简单廉价的方法

C57BL/6 小鼠自身免疫性神经炎的功能和病理评价的简易方法

小鼠用树脂叶毒素和疼痛测试进行脑血管内治疗

小鼠脚垫接种模型，研究病毒引起的神经炎反应

改良的小鼠神经性疼痛备用神经损伤手术模型

改良的小鼠神经性疼痛备用神经损伤手术模型

用于转化疼痛研究的开源实时闭环电阈值跟踪

部分坐骨神经结扎术:慢性神经性疼痛的小鼠模型，用于研究新疗法的抗伤害感受作用

小鼠远端眶下神经慢性缩窄损伤（DIoN-CCI）研究三叉神经性疼痛

利用瞬态受体电位辣椒1型 Ultrapotent 激动剂建立单纯小纤维神经病变的小鼠模型

摘要

探索更多视频

此视频中的章节

神经病理性疼痛小鼠坐骨神经掴型号

测定冷灵敏度和适应小鼠一种简单廉价的方法

C57BL/6 小鼠自身免疫性神经炎的功能和病理评价的简易方法

小鼠用树脂叶毒素和疼痛测试进行脑血管内治疗

小鼠脚垫接种模型，研究病毒引起的神经炎反应

改良的小鼠神经性疼痛备用神经损伤手术模型

改良的小鼠神经性疼痛备用神经损伤手术模型

用于转化疼痛研究的开源实时闭环电阈值跟踪

部分坐骨神经结扎术:慢性神经性疼痛的小鼠模型，用于研究新疗法的抗伤害感受作用

小鼠远端眶下神经慢性缩窄损伤 （DIoN-CCI） 研究三叉神经性疼痛

小鼠远端眶下神经慢性缩窄损伤（DIoN-CCI）研究三叉神经性疼痛