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<s...

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>Chemical reagent</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&#8217;-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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Primary Antisera</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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Secondary Antisera</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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Material</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/?...

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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

Research

JoVE Journal

Neuroscience

9.4K 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

Application of a NMDA Receptor Conductance in Rat Midbrain Dopaminergic Neurons Using the Dynamic Clamp Technique

Neuroscientists study the function of the brain by investigating how neurons in the brain communicate. Many investigators look at changes in the electrical activity of one or more neurons in response to an experimentally-controlled input. The electrical activity of neurons can be recorded in isolated brain slices using patch clamp techniques with glass micropipettes. Traditionally, experimenters can mimic neuronal input by direct injection of current through the pipette, electrical stimulation of the other cells or remaining axonal connections in the slice, or pharmacological manipulation by receptors located on the neuronal membrane of the recorded cell.
Direct current injection has the advantages of passing a predetermined current waveform with high temporal precision at the site of the recording (usually the soma). However, it does not change the resistance of the neuronal membrane as no ion channels are physically opened. Current injection usually employs rectangular pulses and thus does not model the kinetics of ion channels. Finally, current injection cannot mimic the chemical changes in the cell that occurs with the opening of ion channels.
Receptors can be physically activated by electrical or pharmacological stimulation. The experimenter has good temporal precision of receptor activation with electrical stimulation of the slice. However, there is limited spatial precision of receptor activation and the exact nature of what is activated upon stimulation is unknown. This latter problem can be partially alleviated by specific pharmacological agents. Unfortunately, the time course of activation of pharmacological agents is typically slow and the spatial precision of inputs onto the recorded cell is unknown.
The dynamic clamp technique allows an experimenter to change the current passed directly into the cell based on real-time feedback of the membrane potential of the cell (Robinson and Kawai 1993, Sharp et al., 1993a,b; for review, see Prinz et al. 2004). This allows an experimenter to mimic the electrical changes that occur at the site of the recording in response to activation of a receptor. Real-time changes in applied current are determined by a mathematical equation implemented in hardware.
We have recently used the dynamic clamp technique to investigate the generation of bursts of action potentials by phasic activation of NMDA receptors in dopaminergic neurons of the substantia nigra pars compacta (Deister et al., 2009; Lobb et al., 2010). In this video, we demonstrate the procedures needed to apply a NMDA receptor conductance into a dopaminergic neuron.

In this video, we demonstrate how to apply a conductance into a dopaminergic neuron recorded in the whole cell configuration in rat brain slices. This technique is called the dynamic clamp.

一个NMDA受体电导的大鼠中脑多巴胺能神经元的动态钳技术中的应用

Neuroscientists study the function of the brain by investigating how neurons in the brain communicate. Many investigators look at changes in the ...

科研

神经科学

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life span of the animal. They can potentially replace cells that are lost in the aging process or in the process of injury and disease. The existence of neural stem cells (NSCs) was first described by Reynolds and Weiss (1992) in the adult mammalian central nervous system (CNS) using a novel serum&#8208;free culture system, the neurosphere assay (NSA). Using this assay, it is also feasible to isolate and expand NSCs from different regions of the embryonic CNS. These in vitro expanded NSCs are multipotent and can give rise to the three major cell types of the CNS. While the NSA seems relatively simple to perform, attention to the procedures demonstrated here is required in order to achieve reliable and consistent results. This video practically demonstrates NSA to generate and expand NSCs from embryonic day 14-mouse brain tissue and provides technical details so one can achieve reproducible neurosphere cultures. The procedure includes harvesting E14 mouse embryos, brain microdissection to harvest the ganglionic eminences, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in NSA culture. After 5-7 days in culture, the resulting primary neurospheres are passaged to further expand the number of the NSCs for future experiments.

This video protocol demonstrates the application of the neurosphere assay for the isolation and expansion of neural stem cells from the ganglionic eminences of embryonic day 14-mouse brain.

建立小鼠胚胎神经干细胞培养神经球含量

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life ...

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled samples with little to no tissue damage enables us to analyze cell morphology and to compare individual samples to each other, hence allowing the identification of mutant anomalies.
In the demonstrated dissection method the nervous system remains mostly inside the adult fly. Through a dorsal incision, the abdomen and thorax are opened and most of the internal organs are removed. Only the dorsal side of the ventral nerve cord (VNC) and the cervical connective 
(CvC) containing the big axons of the giant fibers (GFs)1 are exposed, while the brain containing the GF cell body and dendrites remains2 in the 
intact head. In this preparation most nerves of the VNC should remain attached to their muscles. 
Following the dissection, the intracellular filling of the giant fiber (GF) with a fluorescent dye is demonstrated. In the CvC the GF axons are located at the dorsal surface and thus can be easily visualized under a microscope with differential interference contrast (DIC) optics. This allows the injection of the GF axons with dye at this site to label the entire GF including the axons and their terminals in the VNC. This method results in reliable and strong staining of the GFs allowing the neurons to be imaged immediately after filling with an epifluorescent microscope. Alternatively, the fluorescent signal can be enhanced using standard immunohistochemistry procedures3 suitable for high resolution confocal microscopy.

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

An in vivo dissection of the adult Drosophila ventral nerve cord (VNC) is demonstrated. This particular dissection method causes little damage to the VNC allowing the subsequent labeling of the giant fiber neurons with fluorescent dye for high resolution imaging.

成人整装准备果蝇巨纤维染料注射腹侧神经索

To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled ...

Trypsin Digest Protocol to Analyze the Retinal Vasculature of a Mouse Model

Trypsin digest is the gold standard method to analyze the retinal vasculature 1-5. It allows visualization of the entire network of complex three-dimensional retinal blood vessels and capillaries by creating a two-dimensional flat-mount of the interconnected vascular channels after digestion of the non-vascular components of the retina. This allows one to study various pathologic vascular changes, such as microaneurysms, capillary degeneration, and abnormal endothelial to pericyte ratios. However, the method is technically challenging, especially in mice, which have become the most widely available animal model to study the retina because of the ease of genetic manipulations 6,7. In the mouse eye, it is particularly difficult to completely remove the non-vascular components while maintaining the overall architecture of the retinal blood vessels. To date, there is a dearth of literature that describes the trypsin digest technique in detail in the mouse. This manuscript provides a detailed step-by-step methodology of the trypsin digest in mouse retina, while also providing tips on troubleshooting difficult steps.

Trypsin digest is one of the most commonly used methods to analyze retinal vasculature. This manuscript describes the method in detail, including key alterations to optimize the technique and remove the non-vascular tissue while preserving the overall architecture of the vessels.

胰蛋白酶精华协议分析视网膜血管小鼠模型

Trypsin digest is the gold standard method to analyze the retinal vasculature 1-5. It allows visualization of the entire network of complex ...

Visual Evoked Potential Recording in a Rat Model of Experimental Optic Nerve Demyelination

The visual evoked potential (VEP) recording is widely used in clinical practice to assess the severity of optic neuritis in its acute phase, and to monitor the disease course in the follow-up period. Changes in the VEP parameters closely correlate with pathological damage in the optic nerve. This protocol provides a detailed description about the rodent model of optic nerve microinjection, in which a partial demyelination lesion is produced in the optic nerve. VEP recording techniques are also discussed. Using skull implanted electrodes, we are able to acquire reproducible intra-session and between-session VEP traces. VEPs can be recorded on individual animals over a period of time to assess the functional changes in the optic nerve longitudinally. The optic nerve demyelination model, in conjunction with the VEP recording protocol, provides a tool to investigate the disease processes associated with demyelination and remyelination, and can potentially be employed to evaluate the effects of new remyelinating drugs or neuroprotective therapies.

Focal demyelination is induced in the optic nerve using lysolecithin microinjection. Visual evoked potentials are recorded via skull electrodes implanted over the visual cortex to examine the signal conduction along the visual pathway in vivo. This protocol details the surgical procedures underlying electrode implantation and optic nerve microinjection.

在实验视神经脱髓鞘模型大鼠视觉诱发电位记录

The visual evoked potential (VEP) recording is widely used in clinical practice to assess the severity of optic neuritis in its acute phase, and to ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

小容积回收-灌注-浸没式室系统中维持的急性海马切片的突触可塑性记录

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

Optogenetics Identification of a Neuronal Type with a Glass Optrode in Awake Mice

It is a major concern in neuroscience how different types of neurons work in neural circuits. Recent advances in optogenetics have enabled the identification of the neuronal type in in vivo electrophysiological experiments in broad brain regions. In optogenetics experiments, it is critical to deliver the light to the recording site. However, it is often hard to deliver the stimulation light to the deep brain regions from the brain&#39;s surface. Especially, it is difficult for the stimulation light to reach the deep brain regions when the optical transparency of the brain surface is low, as is often the case with recordings from awake animals. Here, we describe a method to record spike responses to the light from an awake mouse using a custom-made glass optrode. In this method, the light is delivered through the recording glass electrode so that it is possible to reliably stimulate the recorded neuron with light in the deep brain regions. This custom-made optrode system consists of accessible and inexpensive materials and is easy to assemble.

This work introduces a method to perform an optogenetic single-unit recording reliably from an awake mouse using a custom-made glass optrode.

光遗传学玻璃光纤化学传感在醒鼠中的神经型鉴定

It is a major concern in neuroscience how different types of neurons work in neural circuits. Recent advances in optogenetics have enabled the ...

Methods for the Discovery of Novel Compounds Modulating a Gamma-Aminobutyric Acid Receptor Type A Neurotransmission

This manuscript presents a step-by-step protocol for screening compounds at gamma-aminobutyric acid type A (GABAA) receptors and its use towards the identification of novel molecules active in preclinical assays from an in vitro recombinant receptor to their pharmacological effects at native receptors in rodent brain slices. For compounds binding at the benzodiazepine site of the receptor, the first step is to set up a primary screen that consists of developing radioligand binding assays on cell membranes expressing the major GABAA subtypes. Then, taking advantage of the heterologous expression of rodent and human GABAA receptors in Xenopus oocytes or HEK 293 cells, it is possible to explore, in electrophysiological assays, the physiological properties of the different receptor subtypes and the pharmacological properties of the identified compounds. The Xenopus oocyte system will be presented here, starting with the isolation of the oocytes and their microinjection with different mRNAs, up to the pharmacological characterization using two-electrode voltage clamps. Finally, recordings conducted in rodent brain slices will be described that are used as a secondary physiological test to assess the activity of molecules at their native receptors in a well-defined neuronal circuit. Extracellular recordings using population responses of multiple neurons are demonstrated together with the drug application.

Here, we present protocols to discover compounds active at GABAA receptors, from the binding to the physiology and pharmacology.

新化合物调制伽玛-丁酸受体 a 型神经传递的发现方法

This manuscript presents a step-by-step protocol for screening compounds at gamma-aminobutyric acid type A (GABAA) receptors and its use towards the ...

Rabies Necropsy Techniques in Large and Small Animals

The New York State Department of Health (NYSDOH) Rabies Laboratory receives between 6,000 to 9,000 specimens annually and performs rabies testing for the entire state, with the exception of New York City. The Rabies laboratory necropsies a variety of animals ranging in size from bats to bovids. Most of these specimens are animals exhibiting neurological signs, however, less than 10% actually test positive for rabies; implying trauma, lesions or other infectious agents as the cause of these symptoms. Due to the risk of aerosolizing undiagnosed infectious agents, the Rabies Laboratory does not use power tools or saws. Three necropsy techniques will be presented for animals whose skulls are impenetrable with scissors. The laboratory has implemented these techniques to decrease potential exposure to infectious agents, eliminate unnecessary manipulation of the specimen and reduce processing time. The advantages of a preferred technique opposed to another is subject to the trained individual processing the specimen.

The goal of this protocol is to demonstrate safe necropsy techniques in small and large animals to obtain satisfactory tissue samples for rabies testing.

大、小动物狂犬病的解剖技术

The New York State Department of Health (NYSDOH) Rabies Laboratory receives between 6,000 to 9,000 specimens annually and performs rabies testing for the ...

Subretinal Transplantation of Human Embryonic Stem Cell-Derived Retinal Tissue in a Feline Large Animal Model

Retinal degenerative (RD) conditions associated with photoreceptor loss such as age-related macular degeneration (AMD), retinitis pigmentosa (RP) and Leber Congenital Amaurosis (LCA) cause progressive and debilitating vision loss. There is an unmet need for therapies that can restore vision once photoreceptors have been lost. Transplantation of human pluripotent stem cell (hPSC)-derived retinal tissue (organoids) into the subretinal space of an eye with advanced RD brings retinal tissue sheets with thousands of healthy mutation-free photoreceptors and has a potential to treat most/all blinding diseases associated with photoreceptor degeneration with one approved protocol. Transplantation of fetal retinal tissue into the subretinal space of animal models and people with advanced RD has been developed successfully but cannot be used as a routine therapy due to ethical concerns and limited tissue supply. Large eye inherited retinal degeneration (IRD) animal models are valuable for developing vision restoration therapies utilizing advanced surgical approaches to transplant retinal cells/tissue into the subretinal space. The similarities in globe size, and photoreceptor distribution (e.g., presence of macula-like region area centralis) and availability of IRD models closely recapitulating human IRD would facilitate rapid translation of a promising therapy to the clinic. Presented here is a surgical technique of transplanting hPSC-derived retinal tissue into the subretinal space of a large animal model allowing assessment of this promising approach in animal models.

Presented here is a surgical technique for transplanting human pluripotent stem cell (hPSC)-derived retinal tissue into the subretinal space of a large animal model.

猫大型动物模型中人类胚胎干细胞来源的视网膜组织的视网膜下移植

Retinal degenerative (RD) conditions associated with photoreceptor loss such as age-related macular degeneration (AMD), retinitis pigmentosa (RP) and ...