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 ...

in-vivo-electrophysiological-measurement-rat-ulnar-nerve-with-axonal

Universidad de Cartagena UDEC

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 ...

Measuring Neuromuscular Junction Functionality

Neuromuscular junction (NMJ) functionality plays a pivotal role when studying diseases in which the communication between motor neuron and muscle is impaired, such as aging and amyotrophic lateral sclerosis (ALS). Here we describe an experimental protocol that can be used to measure NMJ functionality by combining two types of electrical stimulation: direct muscle membrane stimulation and the stimulation through the nerve. The comparison of the muscle response to these two different stimulations can help to define, at the functional level, potential alterations in the NMJ that lead to functional decline in muscle.
Ex vivo preparations are suited to well-controlled studies. Here we describe an intensive protocol to measure several parameters of muscle and NMJ functionality for the soleus-sciatic nerve preparation and for the diaphragm-phrenic nerve preparation. The protocol lasts approximately 60 min and is conducted uninterruptedly by means of a custom-made software that measures the twitch kinetics properties, the force-frequency relationship for both muscle and nerve stimulations, and two parameters specific to NMJ functionality, i.e. neurotransmission failure and intratetanic fatigue. This methodology was used to detect damages in soleus and diaphragm muscle-nerve preparations by using SOD1G93A transgenic mouse, an experimental model of ALS that ubiquitously overexpresses the mutant antioxidant enzyme superoxide dismutase 1 (SOD1).

A functional assessment of the neuromuscular junction (NMJ) can provide essential information on the communication between muscle and nerve. Here we describe a protocol to comprehensively evaluate both the NMJ and muscle functionality using two different muscle-nerve preparations, i.e. soleus-sciatic and diaphragm-phrenic.

测量神经肌肉接头功能

Neuromuscular junction (NMJ) functionality plays a pivotal role when studying diseases in which the communication between motor neuron and muscle is ...

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

Patients with diabetes mellitus (DM) or those experiencing the neurotoxic effects of chemotherapeutic agents may develop sensation disorders due to degeneration and injury of small-diameter sensory neurons, referred to as small fiber neuropathy. Present animal models of small fiber neuropathy affect both large- and small-diameter sensory fibers and thus create a neuropathology too complex to properly assess the effects of injured small-diameter sensory fibers. Therefore, it is necessary to develop an experimental model of pure small fiber neuropathy to adequately examine these issues. This protocol describes an experimental model of small fiber neuropathy specifically affecting small-diameter sensory nerves with resiniferatoxin (RTX), an ultrapotent agonist of transient receptor potential vanilloid type 1 (TRPV1), through a single dose of intraperitoneal injection, referred to as RTX neuropathy. This RTX neuropathy showed pathological manifestations and behavioral abnormalities that mimic the clinical characteristics of patients with small fiber neuropathy, including intraepidermal nerve fiber (IENF) degeneration, specifically injury in small-diameter neurons, and induction of thermal hypoalgesia and mechanical allodynia. This protocol tested three doses of RTX (200, 50, and 10 &#181;g/kg, respectively) and concluded that a critical dose of RTX (50 &#181;g/kg) is required for the development of typical small fiber neuropathy manifestations, and prepared a modified immunostaining procedure to investigate IENF degeneration and neuronal soma injury. The modified procedure is fast, systematic, and economic. Behavioral evaluation of neuropathic pain is critical to reveal the function of small-diameter sensory nerves. The evaluation of mechanical thresholds in experimental rodents is particularly challenging and this protocol describes a customized metal mesh that is suitable for this type of assessment in rodents. In summary, RTX neuropathy is a new and easily established experimental model to evaluate the molecular significance and intervention underlying neuropathic pain for the development of therapeutic agents.

This study establishes an experimental model of pure small fiber neuropathy with resiniferatoxin (RTX). A unique dose of RTX (50 &#181;g/kg) is optimal for developing a small fiber neuropathy model that mimics patient characteristics and could help investigate the nociceptive molecular significance underlying neuropathic pain.

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

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

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 ...

Use of In Vivo Single-fiber Recording and Intact Dorsal Root Ganglion with Attached Sciatic Nerve to Examine the Mechanism of Conduction Failure

Single-fiber recording has been a classical and effective electrophysiological technique over the last few decades because of its specific application for nerve fibers in the central and peripheral nervous systems. This method is particularly applicable to dorsal root ganglia (DRG), which are primary sensory neurons that exhibit a pseudo-unipolar structure of nervous processes. The patterns and features of the action potentials passed along axons are recordable in these neurons. The present study uses in vivo single-fiber recordings to observe the conduction failure of sciatic nerves in complete Freund&#8217;s adjuvant (CFA)-treated rats. As the underlying mechanism cannot be studied using in vivo single-fiber recordings, patch-clamp-recordings of DRG neurons are performed on preparations of intact DRG with the attached sciatic nerve. These recordings reveal a positive correlation between conduction failure and the rising slope of the after-hyperpolarization potential (AHP) of DRG neurons in CFA-treated animals. The protocol for in vivo single fiber-recordings allows the classification of nerve fibers via the measurement of conduction velocity and monitoring of abnormal conditions in nerve fibers in certain diseases. Intact DRG with attached peripheral nerve allows observation of the activity of DRG neurons in most physiological conditions. Conclusively, single-fiber recording combined with electrophysiological recording of intact DRGs is an effective method to examine the role of conduction failure during the analgesic process.

Single-fiber recording is an effective electrophysiological technique that is applicable to the central and peripheral nervous systems. Along with the preparation of intact DRG with the attached sciatic nerve, the mechanism of conduction failure is examined. Both protocols improve the understanding of the peripheral nervous system&#39;s relationship with pain.

使用 Vivo 单纤维记录和带附骨神经的固定背道根结条，以检查传导失败机制

Single-fiber recording has been a classical and effective electrophysiological technique over the last few decades because of its specific application for ...

Functional and Physiological Methods of Evaluating Median Nerve Regeneration in the Rat

The main goal of this investigation is to show how to create and repair different types of median nerve (MN) lesions in the rat. Moreover, different methods of simulating postoperative physiotherapy are presented. Multiple standardized strategies are used to assess motor and sensory recovery using an MN model of peripheral nerve lesion and repair, thus permitting easy comparison of the results. Several options are included for providing a postoperative physiotherapy-like environment to rats that have undergone MN injuries. Finally, the paper provides a method to evaluate the recovery of the MN using several noninvasive tests (i.e., grasping test, pin prick test, ladder rung walking test, rope climbing test, and walking track analysis), and physiological measurements (infrared thermography, electroneuromyography, flexion strength evaluation, and flexor carpi radialis muscle weight determination). Hence, this model seems particularly appropriate to replicate a clinical scenario, facilitating extrapolation of results to the human species.
Although the sciatic nerve is the most studied nerve in peripheral nerve research, analysis of the rat MN presents various advantages. For example, there is a reduced incidence of joint contractures and automutilation of the affected limb in MN lesion studies. Furthermore, the MN is not covered by muscle masses, making its dissection easier than that of the sciatic nerve. In addition, MN recovery is observed sooner, because the MN is shorter than the sciatic nerve. Also, the MN has a parallel path to the ulnar nerve in the arm. Hence, the ulnar nerve can be easily used as the nerve graft for repairing MN injuries. Finally, the MN in rats is located in the forelimb, akin to the human upper limb; in humans, the upper limb is the site of most peripheral nerve lesions.

Presented is a protocol to produce different types of median nerve (MN) lesions and repair in the rat. Additionally, the protocol shows how to evaluate the functional recovery of the nerve using several noninvasive behavioral tests and physiological measurements.

大鼠中位神经再生评价的功能与生理方法

The main goal of this investigation is to show how to create and repair different types of median nerve (MN) lesions in the rat. Moreover, different ...

医学

Preparation of Rat Sciatic Nerve for Ex Vivo Neurophysiology

Ex vivo preparations enable the study of many neurophysiological processes in isolation from the rest of the body while preserving local tissue structure. This work describes the preparation of rat sciatic nerves for ex vivo neurophysiology, including buffer preparation, animal procedures, equipment setup and neurophysiological recording. This work provides an overview of the different types of experiments possible with this method. The outlined method aims to provide 6 h of stimulation and recording on extracted peripheral nerve tissue in tightly controlled conditions for optimal consistency in results. Results obtained using this method are A-fibre compound action potentials (CAP) with peak-to-peak amplitudes in the millivolt range over the entire duration of the experiment. CAP amplitudes and shapes are consistent and reliable, making them useful to test and compare new electrodes to existing models, or the effects of interventions on the tissue, such as the use of chemicals, surgical alterations, or neuromodulatory stimulation techniques. Both conventional commercially available cuff electrodes with platinum-iridium contacts and custom-made conductive elastomer electrodes were tested and gave similar results in terms of nerve stimulus strength-duration response.

Preparation of Rat Sciatic Nerve for Ex Vivo Neurophysiology

This protocol describes the preparation of rat whole sciatic nerve tissue for ex vivo electrophysiological stimulation and recording in an environmentally-regulated, two-compartment, perfused saline bath.

大鼠坐骨神经用于 离体 神经生理学的制备

Ex vivo preparations enable the study of many neurophysiological processes in isolation from the rest of the body while preserving local tissue structure. ...

Terminal H-reflex Measurements in Mice

The Hoffmann reflex (H-reflex), as an electrical analog to the stretch reflex, allows electrophysiological validation of the integrity of neural circuits after injuries such as spinal cord damage or stroke. An increase of the H-reflex response, together with symptoms like non-voluntary muscle contractions, pathologically augmented stretch reflex, and hypertonia in the corresponding muscle, is an indicator of post-stroke spasticity (PSS).
In contrast to rather nerve-unspecific transcutaneous measurements, here, we present a protocol to quantify the H-reflex directly at the ulnar and median nerves of the forepaw, which is applicable, with minor modifications, to the tibial and sciatic nerve of the hindpaw. Based on the direct stimulation and the adaptation to different nerves, the method represents a reliable and versatile tool to validate electrophysiological changes in spasticity-related disease models.

The clinical evaluation of spasticity based on the Hoffmann reflex (H-reflex) and using electrical stimulation of peripheral nerves is an established method. Here, we provide a protocol for a terminal and direct nerve stimulation for H-reflex quantification in the mouse forepaw.

小鼠终末H反射测量

The Hoffmann reflex (H-reflex), as an electrical analog to the stretch reflex, allows electrophysiological validation of the integrity of neural circuits ...

生物学

In Vivo Measurement of the Compound Muscle Action Potential in a Rat

Source: Wild, B. M., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/56102">In Vivo Electrophysiological Measurement of the Rat Ulnar Nerve with Axonal Excitability Testing.</a> J. Vis. Exp. (2018)
This video demonstrates the procedure for recording compound muscle action potential (CMAP) in rats by applying electrical stimuli to the ulnar nerve, adjusting electrodes for optimal signal, and increasing stimulation to assess nerve functionality. This method enables the in vivo investigation of the peripheral nerve functions in neurological disorders.

大鼠复合肌肉动作电位的体内测量

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

在体内轴突兴奋性试验对大鼠尺神经的电生理测量

摘要

探索更多视频

此视频中的章节

化疗引起的毒性神经的兴奋性评估

在对小鼠坐骨神经体内的电测量

测量神经肌肉接头功能

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

体内运动神经元变性小鼠前肢肌作用电位的电生理测量

使用 Vivo 单纤维记录和带附骨神经的固定背道根结条，以检查传导失败机制

大鼠中位神经再生评价的功能与生理方法

大鼠坐骨神经用于离体神经生理学的制备

小鼠终末H反射测量

大鼠复合肌肉动作电位的体内测量

在体内轴突兴奋性试验对大鼠尺神经的电生理测量

摘要

探索更多视频

此视频中的章节

化疗引起的毒性神经的兴奋性评估

在对小鼠坐骨神经体内的电测量

测量神经肌肉接头功能

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

体内运动神经元变性小鼠前肢肌作用电位的电生理测量

使用 Vivo 单纤维记录和带附骨神经的固定背道根结条，以检查传导失败机制

大鼠中位神经再生评价的功能与生理方法

大鼠坐骨神经用于 离体 神经生理学的制备

小鼠终末H反射测量

大鼠复合肌肉动作电位的体内测量

大鼠坐骨神经用于离体神经生理学的制备