该项目得到了 Lundbeck 基金会、诺德基金会、丹麦医学研究理事会、Ludvig 和萨拉 Elsass 基金会、神经病学研究基金会和 Jytte Kaj 基金会的支持。R. A 得到澳大利亚国家卫生和医学研究理事会 (#1091006) 早期职业博士后奖学金的支持

这里描述的所有实验程序都符合悉尼新南动物保育和伦理委员会的要求, 并按照澳大利亚国家卫生和医学研究理事会 (澳洲) 的规定进行动物实验。
1. 实验设置
注:12 周前, 雌性长埃文斯大鼠被用于此手术。
<ol><li>麻醉在感应室使用4% 异氟醚和1升每分钟2流量。确认充分的麻醉, 通过测试矫正反射, 并确保其缺席之前, 从诱导室移除动物。请注意, 各种麻醉药对神经兴奋性有不同的影响10。</li>
	<li>安全地将动物的鼻子放在鼻锥附件中, 并提供2.5% 异氟烷和1升每分钟的维护剂量 (2流率。</li>
	<li>通过捏动动物的脚趾并轻轻地触碰它的眼睛, 确认适当的麻醉, 同时检查踏板和角膜的反射。 注意: 建议使用兽医软膏, 以防止在麻醉时干燥, 但并不必要, 因为手术通常需要30分钟的动物。</li>
	<li>使用反馈控制加热垫和直肠温度计探头, 在37° c 下保持大鼠体温。将加热垫和集成体温传感器设置为40° c, 以防止在较高温度下发生任何皮肤损伤。 注意: 建议每10s 使用动物生理监测系统监测和记录生理措施 (心率、氧饱和度、体温和呼吸率)。最佳记录程序应包括局部测量的肢体温度, 因为外围温度可能比核心温度11 (图 1) 冷。</li>
	<li>戴上防腐手套, 始终使用70% 乙醇清洗过的器械, 在整个过程中保持无菌环境。</li>
</ol>2. 电生理设置
<ol><li>使用低阻抗铂脑电图 (EEG) 针电极为这个过程。</li>
	<li>通过鱼际肌肉和参考电极插入记录针电极 (图 1; 紫色表示), 通过第四位数字的背侧 (图 1; 表示橙色) 记录复合肌肉动作电位 (CMAPs)。</li>
	<li>在刺激和记录电极 (图 1; 绿色指示) 之间, 将接地电极通过皮肤放在前臂的上级方面。插入电极时要小心, 以避免肌肉组织。</li>
	<li>通过插入阴极 (图 1; 用蓝色标记) 约4毫米的远端肘管的肘部, 准备经皮刺激针电极。插入阳极 (图 1; 用红色标记) 大约1厘米下部通过 axillar 区域的皮肤。</li>
</ol><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56102/56102fig1.jpg"> 图 1: 大鼠前肢针电极放置的示意图.负极 ( 蓝色 ) 入大约 4 毫米远端对肘形隧道在弯头和阳极 ( 红色 ) 入大约 1 cm 下部通过 axillar 区域的皮肤。地面针电极 (绿色) 通过皮肤在刺激和记录电极之间的前臂的高级方面插入。记录 (紫色) 和参考 (橙色) 针电极插入通过鱼际肌肉和通过背部方面的 4th数字。温度探针 (灰色) 被放置在鱼际肌肉的优越方面。<a href="//cloudflare.jove.com/files/ftp_upload/56102/56102fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
3. 轴突兴奋性程序
<ol><li>执行啮齿动物运动神经 TRONDNF 协议使用半自动化, 计算机控制轴突兴奋性程序 (见材料表) 链接到一个恒定电流刺激器和扩音器。使用 50/60 hz 的噪声消除器去除超过50赫兹的电噪声。<ol>
<li>记录 CMAP 从鱼际肌肉, 同时可视化的 CMAP 形态学和应用 1 ms 方波脉冲到尺神经与阴极针电极。<ol>
<li>要实现最佳记录, 请仔细调整阴极的角度和/或位置, 直到达到具有恒定振幅的最佳双相响应曲线 (图 2A。一旦确定了最佳位置, 就用可电极保持器稳定阴极。 注: 使用的软件自动交付测试刺激, 下面描述, 并增加或减少当前所需的达到阈值。</li>
		</ol>
</li>
		<li>通过将 1 ms 冲量的刺激强度增量递增 1 mA 来记录刺激反应曲线, 直到达到最大响应。 注: 图2B 和2C 中的紫色和绿色线分别代表了刺激强度和自动系统的增量增长。阈值跟踪的目标振幅自动设定为刺激响应曲线上最陡斜率区域对应的最大振幅的40%。各种测试刺激所诱发的 "阈值" (即激发 40% CMAP 所需的刺激) 的变化是在该协议的其余部分中获得的变量。</li>
		<li>记录多个轴突兴奋性参数, 包括阈值 electrotonus (TE)、当前阈值 (I/V) 关系和恢复周期 (RC), 如参考12所述。 注: 计算机控制轴突兴奋性程序评估阈值 (TE) 与 100 ms 阈极化和极化电流设置在±和±40% 的控制阈值电流。在极化和极化电流后, 阈值的变化在100毫秒偏振电流和极化电流后12点自动记录在14时间点。阈值 electrotonus 间接评估间电导, 是膜电位的标志。与 200 ms 阈调节电流, 在强度从 +50% 到-100% 的控制阈值交替10% 的增量。I/V 的关系不同于 TE, 因为电流在持续时间较长, 极化强度从 +50% 逐渐改变为-100%, 并且在极化电流停止后每强度1毫秒评估阈值变化。"I/V" 关系提供了对轴突1的整流特性的洞察。RC 是用配对的脉冲范式评估的, 在这里, 最初的 supramaximal 调节刺激是应用在特定的时间间隔, 从2.5 毫秒到200毫秒的一系列测试刺激. 此参数测试在节点中发生的一系列定义良好的事件。轴突的区域在 supramaximal 刺激以后。这些事件包括, 电压门控 Na+通道失活, 这使得它更难引出一个后续的反应, 并通过耐火和相对耐火期1量化。其次是对节间的充电和由快速钾通道介导的高度兴奋期, 由 superexcitability。最后, 缓慢激活钾通道调解后期的兴奋性降低, 量化为 subexcitability。</li>
	</ol>
</li>
</ol><img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56102/56102fig2.jpg"> 图 2: 轴突兴奋性测试的原始数据.在对尺神经的刺激增量增加后, abiphasic CMAP 反应曲线。面板 B 和 C 分别代表刺激强度 (mA) 和振幅的 CMAP (mV)。B 和 C 的绿色成分描述了自动增量减少刺激强度和相关的乙状结肠形减少 CMAP 所需的阈值跟踪。
4. 后电生理程序
<ol><li>把老鼠转移到一个单独的笼子里, 直到它恢复了足够的意识来维持胸骨卧床。在完全从麻醉中恢复之前, 不要把动物单独留在其他动物的陪伴下。一旦老鼠完全从麻醉中恢复过来, 把它转移回原来的笼子。</li>
	<li>在研究完成后, 安乐鼠无疼痛的动物, 如致命的腹腔注射巴比妥 (如 lethabarb)。</li>
</ol>

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作者没有什么可透露的。

所描述的过程证明了一种简单而可靠的微创技术, 允许在短时间内对轴突的生物物理特性和膜电位进行评估。与其他更多的侵入性技术, 需要暴露的神经, 目前的方法轴突兴奋性测试诱导最小的组织损害, 从而使在体内评估, 保留了生理条件神经的兴趣, 并允许重复测量。
为了确保取得一致的结果, 有一些方法上的考虑需要加以解决。其中一个因素是麻醉的浓度和类型。以...

使用电生理学技术是一个必要的工具, 在体内研究周围神经功能的神经紊乱。传统的神经传导方法利用 supramaximal 刺激记录运动动作电位振幅和潜伏期。因此, 这些技术提供了关于导电纤维数量和最快纤维的传导速度的有用信息。一个有价值的辅助工具是轴突兴奋性测试。这种技术使用复杂的电生理刺激模式间接评估周围神经的生物物理特性, 如离子通道、能量依赖性泵、离子交换过程和膜电位的活性1。
轴突兴奋性测试通常用于临床设置, 以调查病理生理过程和治疗干预对各种神经疾病的影响。重要的是, 轴突兴奋性措施是敏感的治疗干预, 影响周围神经功能, 如静脉注射免疫球蛋白 (丙种球蛋白) 治疗2, 化疗3和磷酸抑制剂 (CNI) 治疗4. 虽然这些研究提供了重要的见解, 但临床研究往往排除了早期疾病特征的调查和新的治疗方案5。因此, 这些方法在神经紊乱动物模型中的应用最近获得了牵引6,7,8,9。事实上, 这些方法提供了一个机会, 以了解特定的神经兴奋性变化与这些疾病相关, 从而推进转化研究。
这里描述的程序是一种简单而可靠的方法来记录轴突兴奋性措施的完整大鼠尺神经。

<table>
	<tbody>
		<tr>
			<td>QTracS Program</td>
			<td>Digitimer Ltd.</td>
			<td></td>
			<td>Axonal excitability program</td>
		</tr>
		<tr>
			<td>AM-Systems 2200, Analog Stimulus Isolator, 2200V/50Hz</td>
			<td>SDR Scientific</td>
			<td>850005</td>
			<td>Stimulator</td>
		</tr>
		<tr>
			<td>High Performance AC Amplifier Model LP511</td>
			<td>Grass Technologies</td>
			<td></td>
			<td>Amplifier</td>
		</tr>
		<tr>
			<td>Humbug 50/60Hz Noise eliminator</td>
			<td>Quest Scientific Instruments</td>
			<td>726310</td>
			<td>Noise eliminator</td>
		</tr>
		<tr>
			<td>Low Impedance Platinum Monopolar Subdermal Needle Electrodes</td>
			<td>Grass Technologies</td>
			<td>F-E2-24</td>
			<td>Recording electrodes, 10 mm length, 30 gauge</td>
		</tr>
		<tr>
			<td>Low Impedance Platinum Electroencephalography Needle Electrodes</td>
			<td>Cephalon</td>
			<td>9013L0702</td>
			<td>Stimulating electrodes, 10 mm length, 30 gauge</td>
		</tr>
		<tr>
			<td>Multifunction I/O Device Model USB-6341</td>
			<td>National Instruments</td>
			<td></td>
			<td>Multifunction input/output device</td>
		</tr>
		<tr>
			<td>Iron Base Plate IP</td>
			<td>Narishige Scientific Instrument Laboratory</td>
			<td></td>
			<td>Used for holding stimulating needle electrode in place</td>
		</tr>
		<tr>
			<td>Rotating X-block X-4</td>
			<td>Narishige Scientific Instrument Laboratory</td>
			<td></td>
			<td>Used for holding stimulating needle electrode in place</td>
		</tr>
		<tr>
			<td>Magnetic Stand GJ-8</td>
			<td>Narishige Scientific Instrument Laboratory</td>
			<td></td>
			<td>Used for holding stimulating needle electrode in place</td>
		</tr>
		<tr>
			<td>Micromanipulator M-3333</td>
			<td>Narishige Scientific Instrument Laboratory</td>
			<td></td>
			<td>Used for holding stimulating needle electrode in place</td>
		</tr>
	</tbody>
</table>

in vivo electrophysiological measurement of the rat ulnar nerve with axonal excitability testing

电生理学能够客观评估周围神经功能在体内。传统的神经传导措施, 如振幅和潜伏期检测慢性轴突损失和鞘, 分别。轴突兴奋技术 "通过阈值跟踪" 扩大了这些措施, 提供有关的离子通道, 泵和交换器的活动有关的急性功能和可能之前的退化事件的信息。因此, 在神经紊乱动物模型中使用轴突兴奋性可以为评估新的治疗干预提供一个有用的体内措施。在这里, 我们描述了一个实验设置的多项措施的运动轴突兴奋技术在大鼠尺神经。
动物被麻醉与异氟醚和仔细监测, 以确保持续和充分的麻醉深度。持续监测血液中的体温、呼吸速率、心率和血氧饱和度。轴突兴奋性研究是利用经皮刺激尺神经和记录从鱼际肌肉的前肢爪。在正确的电极放置, 一个明确的复合肌肉动作电位增加振幅随着刺激强度增加记录。然后利用一个自动程序提供一系列的电脉冲, 产生5特定的兴奋性措施在以下序列: 刺激反应行为, 强度持续时间常数, 阈 electrotonus, 电流阈值关系和恢复周期。
这里提供的数据表明, 这些措施是可重复的, 并显示了在同一天评估左、右尺神经的相似性。这些技术的局限性是在麻醉条件下剂量和时间的影响。对这些变数进行仔细的监测和记录应在分析时加以考虑。

采用本方案获得了大鼠尺神经的电生理测量。图 3演示了一个12周大的雌性长埃文斯鼠左尺神经的有代表性的记录。复合肌肉动作电位与同时激活的导电纤维数量有关。supramaximal 峰值响应 (mV) (图 3A) 演示了在增量递增刺激之前达到的峰值响应 (图 2B)。
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In Vivo Electrophysiological Measurement of the Rat Ulnar Nerve with Axonal Excitability Testing

在体内轴突兴奋性试验对大鼠尺神经的电生理测量

轴突兴奋技术提供了一个强有力的工具, 以检查病理生理学和生物物理变化之前, 不可逆转的退化事件。这篇手稿展示了这些技术在麻醉大鼠尺神经上的应用。

Electrophysiology enables the objective assessment of peripheral nerve function in vivo. Traditional nerve conduction measures such as amplitude and ...

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Research

JoVE Journal

Neuroscience

In Vivo Electrophysiological Measurement of the Rat Ulnar Nerve with Axonal Excitability Testing

9.1K 次观看.  University of New South Wales. 轴突兴奋技术提供了一个强有力的工具, 以检查病理生理学和生物物理变化之前, 不可逆转的退化事件。这篇手稿展示了这些技术在麻醉大鼠尺神经上的应用。

视频: In Vivo Electrophysiological Measurement of the Rat Ulnar Nerve with Axonal Excitability Testing

Nerve Excitability Assessment in Chemotherapy-induced Neurotoxicity

Chemotherapy-induced neurotoxicity is a serious consequence of cancer treatment, which occurs with some of the most commonly used chemotherapies1,2. Chemotherapy-induced peripheral neuropathy produces symptoms of numbness and paraesthesia in the limbs and may progress to difficulties with fine motor skills and walking, leading to functional impairment. In addition to producing troubling symptoms, chemotherapy-induced neuropathy may limit treatment success leading to dose reduction or early cessation of treatment. Neuropathic symptoms may persist long-term, leaving permanent nerve damage in patients with an otherwise good prognosis3. As chemotherapy is utilised more often as a preventative measure, and survival rates increase, the importance of long-lasting and significant neurotoxicity will increase.
There are no established neuroprotective or treatment options and a lack of sensitive assessment methods. Appropriate assessment of neurotoxicity will be critical as a prognostic factor and as suitable endpoints for future trials of neuroprotective agents. Current methods to assess the severity of chemotherapy-induced neuropathy utilise clinician-based grading scales which have been demonstrated to lack sensitivity to change and inter-observer objectivity4. Conventional nerve conduction studies provide information about compound action potential amplitude and conduction velocity, which are relatively non-specific measures and do not provide insight into ion channel function or resting membrane potential. Accordingly, prior studies have demonstrated that conventional nerve conduction studies are not sensitive to early change in chemotherapy-induced neurotoxicity4-6. In comparison, nerve excitability studies utilize threshold tracking techniques which have been developed to enable assessment of ion channels, pumps and exchangers in vivo in large myelinated human axons7-9.
Nerve excitability techniques have been established as a tool to examine the development and severity of chemotherapy-induced neurotoxicity10-13. Comprising a number of excitability parameters, nerve excitability studies can be used to assess acute neurotoxicity arising immediately following infusion and the development of chronic, cumulative neurotoxicity. Nerve excitability techniques are feasible in the clinical setting, with each test requiring only 5 -10 minutes to complete. Nerve excitability equipment is readily commercially available, and a portable system has been devised so that patients can be tested in situ in the infusion centre setting. In addition, these techniques can be adapted for use in multiple chemotherapies.
In patients treated with the chemotherapy oxaliplatin, primarily utilised for colorectal cancer, nerve excitability techniques provide a method to identify patients at-risk for neurotoxicity prior to the onset of chronic neuropathy. Nerve excitability studies have revealed the development of an acute Na+ channelopathy in motor and sensory axons10-13. Importantly, patients who demonstrated changes in excitability in early treatment were subsequently more likely to develop moderate to severe neurotoxicity11. However, across treatment, striking longitudinal changes were identified only in sensory axons which were able to predict clinical neurological outcome in 80% of patients10. These changes demonstrated a different pattern to those seen acutely following oxaliplatin infusion, and most likely reflect the development of significant axonal damage and membrane potential change in sensory nerves which develops longitudinally during oxaliplatin treatment10. Significant abnormalities developed during early treatment, prior to any reduction in conventional measures of nerve function, suggesting that excitability parameters may provide a sensitive biomarker.

This abstract describes a novel method to assess the development of neurotoxicity in patients receiving chemotherapy treatment. While conventional assessment methods are limited in their ability to detect early changes in nerve function, nerve excitability techniques provide early identification of patients at risk of severe neurotoxicity and insight into pathophysiology.

化疗引起的毒性神经的兴奋性评估

Chemotherapy-induced neurotoxicity is a serious consequence of cancer treatment, which occurs with some of the most commonly used chemotherapies1,2. ...

科研

神经科学

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological techniques, in the understanding of the underlying pathophysiology1. Here we describe a method to perform electrophysiological studies on mouse sciatic nerves in vivo.
The animals are anesthetized with isoflurane in order to ensure analgesia for the tested mice and undisturbed working environment during the measurements that take about 30 min/animal. A constant body temperature of 37 &#176;C is maintained by a heating plate and continuously measured by a rectal thermo probe2. Additionally, an electrocardiogram (ECG) is routinely recorded during the measurements in order to continuously monitor the physiological state of the investigated animals.
Electrophysiological recordings are performed on the sciatic nerve, the largest nerve of the peripheral nervous system (PNS), supplying the mouse hind limb with both motoric and sensory fiber tracts. In our protocol, sciatic nerves remain in situ and therefore do not have to be extracted or exposed, allowing measurements without any adverse nerve irritations along with actual recordings. Using appropriate needle electrodes3 we perform both proximal and distal nerve stimulations, registering the transmitted potentials with sensing electrodes at gastrocnemius muscles. After data processing, reliable and highly consistent values for the nerve conduction velocity (NCV) and the compound motor action potential (CMAP), the key parameters for quantification of gross peripheral nerve functioning, can be achieved.

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Measurements of nerve conduction properties in vivo exemplify a powerful tool to characterize various animal models of neuromuscular diseases. Here, we present an easy and reliable protocol by which electrophysiological analysis on sciatic nerves of anesthetized mice can be performed.

在对小鼠坐骨神经体内的电测量

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological ...

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of the olfactory epithelium continuously give rise to new sensory neurons that extend their axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. Because of this particular feature, the olfactory system represents a unique opportunity to monitor axonal wiring and guidance, and to investigate synapse formation. Here we describe a procedure for in vivo labeling of sensory neurons and subsequent visualization of axons in the olfactory system of larvae of the amphibian Xenopus laevis. To stain sensory neurons in the olfactory organ we adopt the electroporation technique. In vivo electroporation is an established technique for delivering fluorophore-coupled dextrans or other macromolecules into living cells. Stained sensory neurons and their axonal processes can then be monitored in the living animal either using confocal laser-scanning or multiphoton microscopy. By reducing the number of labeled cells to few or single cells per animal, single axons can be tracked into the olfactory bulb and their morphological changes can be monitored over weeks by conducting series of in vivo time lapse imaging experiments. While the described protocol exemplifies the labeling and monitoring of olfactory sensory neurons, it can also be adopted to other cell types within the olfactory and other systems.

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

We describe a protocol for in vivo labeling of olfactory sensory neurons by electroporation and subsequent confocal laser-scanning or multiphoton microscopy to visualize neuronal morphology and its development over time.

嗅觉系统作为模型来研究轴突生长的模式和形态<em&gt;在体内</em

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

同时<em&gt;体外</em两个由视网膜&gt;功能测试<em&gt;在体内</em&gt;电图系统

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

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 ...

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous system. SC-selective genetic manipulation in living animals is a powerful technique for studying the molecular and cellular mechanisms of SC myelination and demyelination in vivo. While knockout/knockin and transgenic mice are powerful tools for studying SC biology, these methods are costly and time consuming. Viral vector-mediated transgene introduction into the sciatic nerve is a simpler and less laborious method. However, viral methods have limitations, such as toxicity, transgene size constraints, and infectivity restricted to certain developmental stages. Here, we describe a new method that allows selective transfection of myelinating SCs in the rodent sciatic nerve using electroporation. By applying electric pulses to the sciatic nerve at the site of plasmid DNA injection, genes of interest can be easily silenced or overexpressed in SCs in both neonatal and more mature animals. Furthermore, this in vivo electroporation method allows for highly efficient simultaneous expression of multiple transgenes. Our novel technique should enable researchers to efficiently manipulate SC gene expression, and facilitate studies on SC development and function.

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

Here, we present an in vivo technique for gene transfer to Schwann cells (SCs) in the rodent sciatic nerve. This simple technique is useful for investigating signaling mechanisms involved in the development and maintenance of myelinating SCs.

体内基因转移到雪旺细胞在啮齿动物坐骨神经电穿孔

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous ...

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. Single-molecule imaging is gaining momentum as it provides exceptional access to the visualization of individual molecules in living cells. Here, we describe a technique that we developed to perform single-particle tracking photo-activated localization microscopy (sptPALM) in Drosophila larvae. Synaptic communication relies on key presynaptic proteins that act by docking, priming, and promoting the fusion of neurotransmitter-containing vesicles with the plasma membrane. A range of protein-protein and protein-lipid interactions tightly regulates these processes and the presynaptic proteins therefore exhibit changes in mobility associated with each of these key events. Investigating how mobility of these proteins correlates with their physiological function in an intact live animal is essential to understanding their precise mechanism of action. Extracting protein mobility with high resolution in vivo requires overcoming limitations such as optical transparency, accessibility, and penetration depth. We describe how photoconvertible fluorescent proteins tagged to the presynaptic protein Syntaxin-1A can be visualized via slight oblique illumination and tracked at the motor nerve terminal or along the motor neuron axon of the third instar Drosophila larva.

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

Here we illustrate how single molecule photo-activated localization microscopy can be carried out on the motor nerve terminal of a live Drosophila melanogaster third instar larva.

在体内果蝇前运动神经末端的单分子跟踪

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. ...

In Vivo Electrophysiological Measurement of Compound Muscle Action Potential from the Forelimbs in Mouse Models of Motor Neuron Degeneration

Assessing the functionality of the nerve axon provides detailed information on the progression of neuromuscular disorders. Electrophysiological recordings provide a sensitive approach to measure nerve conduction in humans and rodent models. To broaden the technical possibilities for electromyography in mice, the measurement of compound muscle action potentials (CMAPs) from the brachial plexus nerve in the forelimb using needle electrodes is described here. CMAP recordings after stimulating the sciatic nerve in hindlimbs have been previously described. The newly introduced method here allows for the evaluation of the nerve conductivity at an additional site, and thus provides a more profound overview of the neuromuscular functionality. The technique provides information on both the relative number of functional axons and the myelination level. Thereby, this method can be applied to assess both axonal diseases as well as demyelinating conditions. This minimally invasive method does not require extraction of the nerve and therefore it is suitable for repeated measurements for longitudinal follow-up in the same animal. Similar recordings are performed in clinical setups to emphasize the translational relevance of the method.

In Vivo Electrophysiological Measurement of Compound Muscle Action Potential from the Forelimbs in Mouse Models of Motor Neuron Degeneration

The measurement of nerve conduction is a useful tool to assess mouse models of neurodegeneration but it is frequently only applied to stimulate the sciatic nerve in hindlimbs. Here, we describe a technique to measure compound muscle action potential (CMAP) in vivo in the mouse forelimb muscles innervated by the brachial plexus.

体内运动神经元变性小鼠前肢肌作用电位的电生理测量

Assessing the functionality of the nerve axon provides detailed information on the progression of neuromuscular disorders. Electrophysiological recordings ...

Screening of Axonal Degeneration in Carpal Tunnel Syndrome Using Ultrasonography and Nerve Conduction Studies

Axonal degeneration, indicative of surgical decompression, may coexist in carpal tunnel syndrome (CTS) as the disease progresses. However, the current diagnostic and severity gradation system cannot clearly indicate its coexistence, resulting in&#160;confusion of appropriate treatment prescription. There are also constraints in conventional methods for&#160;differentiation as well. This study aims at introducing an innovative, efficient, and quick screening protocol to differentiate axonal degeneration associated with CTS, using ultrasound and nerve conduction studies (NCS). It starts by using NCS to perform orthodromic stimulation at the wrist, to obtain the sensory conduction of the median and the ulnar nerves respectively. Meanwhile, the motor conduction&#160;of the median nerve is collected by stimulating the palm, wrist, and elbow, followed by the stimulation of the ulnar nerve at the wrist,&#160;below and above the elbow. Then, an ultrasound assessment is performed, using a linear array transducer, with cross-sectional area (CSA) and perimeter (P) at the wrist and at the one-third distal forearm calipered. Ratios (R-CSA, R-P) and changes from wrist to one-third distal forearm (&#916;CSA and &#916;P) are calculated according to a standard format. Potential axonal degeneration coexisting in CTS will be screened according to the criteria of NCS and cut-off values of ultrasound measurements established in a previous study. In terms of its noninvasiveness, low cost, convenience, and efficiency, it is easy to apply ultrasound complimentarily in clinical practice to prescreen patients with potential coexisting axonal degeneration. Nevertheless, the ultrasonographic imaging cannot directly reflect axonal degeneration. It still relies on conventional but invasive methods&#160;such as electromyography (EMG) and biopsy&#160;for confirmation if needed.

Here we present a protocol using nerve conduction studies and ultrasound to screen potential axonal degeneration associated with carpal tunnel syndrome. The criteria for differentiation are established. Compared to conventional approaches, this method is noninvasive, convenient, and efficient, with an overall satisfactory accuracy, sensitivity, and specificity.

超声和神经传导研究对腕管综合征轴突变性的筛查

Axonal degeneration, indicative of surgical decompression, may coexist in carpal tunnel syndrome (CTS) as the disease progresses. However, the current ...

Facial Nerve Surgery in the Rat Model to Study Axonal Inhibition and Regeneration

This protocol describes consistent and reproducible methods to study axonal regeneration and inhibition in a rat facial nerve injury model. The facial nerve can be manipulated along its entire length, from its intracranial segment to its extratemporal course. There are three primary types of nerve injury used for the experimental study of regenerative properties: nerve crush, transection, and nerve gap. The range of possible interventions is vast, including surgical manipulation of the nerve, delivery of neuroactive reagents or cells, and either central or end-organ manipulations. Advantages of this model for studying nerve regeneration include simplicity, reproducibility, interspecies consistency, reliable survival rates of the rat, and an increased anatomic size relative to murine models. Its limitations involve a more limited genetic manipulation versus the mouse model and the superlative regenerative capability of the rat, such that the facial nerve scientist must carefully assess time points for recovery and whether to translate results to higher animals and human studies. The rat model for facial nerve injury allows for functional, electrophysiological, and histomorphometric parameters for the interpretation and comparison of nerve regeneration. It thereby boasts tremendous potential toward furthering the understanding and treatment of the devastating consequences of facial nerve injury in human patients.

This protocol describes a reproducible approach to facial nerve surgery in the rat model, including descriptions of various inducible patterns of injury.

大鼠模型中的面部神经外科研究同氧抑制与再生

This protocol describes consistent and reproducible methods to study axonal regeneration and inhibition in a rat facial nerve injury model. The facial ...