Name: terminal h-reflex measurements in mice
Uploaded: 2022-06-16T12:30:00
Description: Watch this Scientific Journal Video about Terminal H-reflex Measurements in Mice at JoVE.com

作者感谢达尔豪斯大学的T. Akay在MG访问他的实验室期间给予的支持。这项工作得到了Friebe基金会（T0498/28960/16）和德国研究基金会（DFG，德国研究基金会）-项目ID 431549029-SFB 1451的资助。

所有实验均按照欧洲和国家动物护理法律和机构指南进行，并获得了北莱茵-威斯特法伦州自然、环境、北莱茵-威斯特法伦州和韦尔布劳赫舒茨州政府的批准（Az：81-02.04.2019.A309）。该方案针对成年小鼠（约8-16周龄C57Bl / 6J小鼠）和前肢记录进行了优化。它可以通过刺激后肢的相应神经和记录后爪肌肉来轻松适应（图1B）。记录电极和刺激电极的描述添加到 材料表中</str...

<ol>
	<li>Palmieri, R. M., Ingersoll, C. D., Hoffman, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Hoffmann+reflex:+methodologic+considerations+and+applications+for+use+in+sports+medicine+and+athletic+training+research.">The Hoffmann reflex: methodologic considerations and applications for use in sports medicine and athletic training research.</a> Journal of Athletic Training. 39 (3), 268-277 (2004).</li><li>Henneman, E., Somjen, G., Carpenter, D. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excitability+and+inhibitibility+of+motoneurons+of+different+sizes.">Excitability and inhibitibility of motoneurons of different sizes.</a> Journal of Neurophysiology. 28 (3), 599-620 (1965).</li><li>Toda, T., Ishida, K., Kiyama, H., Yamashita, T., Lee, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Down-regulation+of+KCC2+expression+and+phosphorylation+in+motoneurons,+and+increases+the+number+of+in+primary+afferent+projections+to+motoneurons+in+mice+with+post-stroke+spasticity.">Down-regulation of KCC2 expression and phosphorylation in motoneurons, and increases the number of in primary afferent projections to motoneurons in mice with post-stroke spasticity.</a> PLoS ONE. 9 (12), 114328 (2014).</li><li>Lee, S., Toda, T., Kiyama, H., Yamashita, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Weakened+rate-dependent+depression+of+Hoffmann&#8217;s+reflex+and+increased+motoneuron+hyperactivity+after+motor+cortical+infarction+in+mice.">Weakened rate-dependent depression of Hoffmann&#8217;s reflex and increased motoneuron hyperactivity after motor cortical infarction in mice.</a> Cell Death &amp; Disease. 5 (1), 1007 (2014).</li><li>Knikou, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+H-reflex+as+a+probe:+Pathways+and+pitfalls.">The H-reflex as a probe: Pathways and pitfalls.</a> Journal of Neuroscience Methods. 171 (1), 1-12 (2008).</li><li>Wieters, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction+to+spasticity+and+related+mouse+models.">Introduction to spasticity and related mouse models.</a> Experimental Neurology. 335, 113491 (2020).</li><li>Pearson, K. G., Acharya, H., Fouad, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+electrode+configuration+for+recording+electromyographic+activity+in+behaving+mice.">A new electrode configuration for recording electromyographic activity in behaving mice.</a> Journal of Neuroscience Methods. 148 (1), 36-42 (2005).</li><li>Akay, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+measurement+of+muscle+denervation+and+locomotor+behavior+in+individual+wild-type+and+ALS+model+mice.">Long-term measurement of muscle denervation and locomotor behavior in individual wild-type and ALS model mice.</a> Journal of Neurophysiology. 111 (3), 694-703 (2014).</li><li>Haghighi, S. S., Green, D. K., Oro, J. J., Drake, R. K., Kracke, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Depressive+effect+of+isoflurane+anesthesia+on+motor+evoked+potentials.">Depressive effect of isoflurane anesthesia on motor evoked potentials.</a> Neurosurgery. 26 (6), 993 (1990).</li><li>Chang, H. -. Y., Havton, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+effects+of+urethane+and+isoflurane+on+external+urethral+sphincter+electromyography+and+cystometry+in+rats.">Differential effects of urethane and isoflurane on external urethral sphincter electromyography and cystometry in rats.</a> American Journal of Physiology-Renal Physiology. 295 (4), 1248-1253 (2008).</li><li>Nolan, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Section+4:+Nervous+system+(Br.+J.+Pharmacol).">Section 4: Nervous system (Br. J. Pharmacol).</a> Clinical pharmacology. , 295-310 (2012).</li><li>Struck, M. B., Andrutis, K. A., Ramirez, H. E., Battles, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+a+short-term+fast+on+ketamine-xylazine+anesthesia+in+rats.">Effect of a short-term fast on ketamine-xylazine anesthesia in rats.</a> Journal of the American Association for Laboratory Animal Science: JAALAS. 50 (3), 344-348 (2011).</li><li>Zandieh, S., Hopf, R., Redl, H., Schlag, M. 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与前面描述的小鼠6经皮H反射测量相比，我们提供了更直接和神经特异性的测量。这种新方法可以应用于前肢和后肢的神经（例如，正中神经、尺神经和桡神经，以及胫神经和坐骨神经），使该方法可作为许多疾病模型（例如卒中、多发性硬化症、肌萎缩侧索硬化症、创伤性脑损伤和脊髓损伤）的诊断工具。根据所选神经，建议验证 H 波的振幅作为刺激强度的函数。振幅可能因?...

以生理学家保罗·霍夫曼（Paul Hoffmann）命名的霍夫曼反射（H反射）可以通过对周围神经的电刺激来引起，周围神经携带来自并导致相同肌肉的感觉和运动神经元的轴突。它是单突触拉伸反射的电诱导类似物，并且共享相同的通路1。与肌肉拉伸不同，H反射是由电刺激引起的。当周围神经以低电流强度受到电刺激时，由于其轴突直径大，Ia传入纤维通常首先去极化2。它们的动作电位激发脊髓中的α运动神经元（αMN），进而引发沿着αMN轴突向肌肉移动的动作电位（图1）。这种级联产生小振幅的肌肉反应，反映在所谓的H波中。通过逐渐增加刺激强度，由于招募额外的运动单元，H波的振幅增加。从一定的刺激强度，直接引出αMN较薄轴突中的动作电位，记录为M波。该 M 波的延迟比 H 波短（图 2）。如果刺激强度进一步增加，由于募集更多的αMN轴突，M波的振幅变大，而H波逐渐变小。由于αMN轴突中动作电位的逆向传播，H波可以在高刺激强度下受到抑制。这些触发的动作电位与来自Ia刺激的动作电位碰撞，因此可以相互抵消。在超最大刺激强度下，所有 MN 轴突中都发生顺向（朝向肌肉）和逆向（朝向脊髓）动作电位;前者产生最大M波振幅（Mmax），而后者导致H反射3完全消除。
为了评估中风后痉挛（PSS）或脊髓损伤（SCI），H反射已被用于评估人类运动和痉挛的神经基础1。通过使用 H 波和 M 波的比率（H/M 比率），可以改进测量之间和受试者之间 H 反射变化的量化。或者，使用一组升频（例如，0.1、0.5、1.0、2.0 和 5.0 Hz）测量速率依赖性抑制 （RDD）。RDD反映了可能受到中风或SCI干扰的抑制回路的完整性。当所有神经回路都完好无损时，H反射受到均匀的、频率无关的抑制。然而，如果由于卒中或SCI导致神经抑制减少，则H反射的抑制随着刺激频率的增加而降低4。
使用表面电极进行正确的电生理记录可能具有挑战性，并且可能受到运动任务、抑制机制和αMN兴奋性的影响5。在啮齿动物的经皮记录中，将刺激电极放置在胫神经附近，并将记录电极放置在前爪的相关肌肉附近。然而，根据我们的经验，啮齿动物中经皮电极的正确放置（图1A）比人类的表面电极放置更加复杂和多变。这可能导致引发 H 反射所需的长度、频率和刺激强度的差异。这些方法学挑战可以解释为什么只有非常有限的H反射测量研究（例如，在实验卒中模型3，4和其他痉挛模型中6。原则上，使用围绕目标神经的植入式电极可以实现对单个神经的精确（长期）刺激和记录H反射7，8。由于具有挑战性的手术对动物有潜在的副作用和探针的潜在不稳定性，这种方法尚未成为该领域的标准。这里介绍的方法还需要一些外科专业知识。然而，它允许使用低刺激强度对体内孤立的神经进行新颖、精确的刺激和记录，从而避免同时刺激邻近神经。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Absorbent underpad</td>
			<td>VWR</td>
			<td>115-0684</td>
			<td></td>
		</tr>
		<tr>
			<td>AD converter</td>
			<td>Cambridge Electronic Design, UK</td>
			<td>CED 1401micro</td>
			<td></td>
		</tr>
		<tr>
			<td>Amplifier</td>
			<td>Workshop Zoological Institute, UoC</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital stimulator</td>
			<td>Workshop Zoological Institute, UoC</td>
			<td>MS 501</td>
			<td></td>
		</tr>
		<tr>
			<td>EMG electrodes</td>
			<td>Workshop Zoological Institute, UoC</td>
			<td></td>
			<td>Two twisted, insulated copper wires (50 &#181;m outer diameter) were soldered to a male plug and connected to a differential amplifier.</td>
		</tr>
		<tr>
			<td>Eye ointment</td>
			<td>Bayer</td>
			<td>Bepanthen</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass pipette</td>
			<td>Workshop Zoological Institute, UoC</td>
			<td>-</td>
			<td>Prepare a glass pipette bent into a simple glass hook in the flame of a Bunsen burner.</td>
		</tr>
		<tr>
			<td>Heating box</td>
			<td>MediHeat</td>
			<td>MediHeat V1200</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>WPI</td>
			<td>61840 Heating pad</td>
			<td></td>
		</tr>
		<tr>
			<td>Hook electrodes</td>
			<td>Workshop Zoological Institute, UoC</td>
			<td>-</td>
			<td>To produce the electrodes, bend stainless steel miniature pins into hooks at one end and insert into blunt cannulas to create direct mechanical contact. Solder the end of the cannula to copper wires (length approx. 50 cm), which are connected to either stimulation or recording device.</td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Pfizer</td>
			<td>Ketavet</td>
			<td></td>
		</tr>
		<tr>
			<td>Rectal probe</td>
			<td>WPI</td>
			<td>RET-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Stimulator isolation unit</td>
			<td>Workshop Zoological Institute, UoC</td>
			<td>MI 401</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterilizer</td>
			<td>CellPoint Scientific</td>
			<td>Germinator 500</td>
			<td>Routine pre- and post-operative disinfection of the surgical equipment should be done by heat sterilization. Decontaminate instruments for 15 s in the heated glass bead bath (260&#176;C).</td>
		</tr>
		<tr>
			<td>Temperature controller</td>
			<td>WPI</td>
			<td>ATC200</td>
			<td></td>
		</tr>
		<tr>
			<td>Vaseline</td>
			<td>Bayer</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Bayer</td>
			<td>Rompun</td>
			<td></td>
		</tr>
	</tbody>
</table>

terminal h-reflex measurements in mice

霍夫曼反射（H反射）作为拉伸反射的电模拟，允许在脊髓损伤或中风等损伤后对神经回路的完整性进行电生理验证。H 反射反应增加，以及非自主肌肉收缩、病理性拉伸反射增强和相应肌肉肌张力亢进等症状，是卒中后痉挛 （PSS） 的指标。
与相当神经非特异性的经皮测量相反，在这里，我们提出了一种直接量化前爪尺神经和正中神经的H反射的方案，该协议适用于后爪的胫骨和坐骨神经。基于直接刺激和对不同神经的适应，该方法是验证痉挛相关疾病模型中电生理变化的可靠且通用的工具。

从每个刺激频率和爪子的n = 15次刺激试验中，选择至少n = 10次成功的记录进行分析。有测量误差（例如，M波缺失）的试验被排除在分析之外。分别分析每个试验，并在以后生成组/时间比较的平均值。每次试验都记录刺激与M波和H波出现之间的潜伏期。根据我们的经验，由于通过脊髓的传输时间较长，M波在刺激后约2毫秒发生，H波在6-8毫秒后发生（图1A 和 图2B...

Watch this Scientific Journal Video about Terminal H-reflex Measurements in Mice at JoVE.com

Terminal H-reflex Measurements in Mice

基于霍夫曼反射（H反射）和使用周围神经电刺激的痉挛状态的临床评估是一种既定的方法。在这里，我们提供了一种用于小鼠前爪H反射定量的终末和直接神经刺激的方案。

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 ...

H反射允许对神经回路的完整性进行电生理验证。我们提出了一种终端协议，用于直接量化小鼠前爪神经的H反射。与传统的相当神经非特异性的经皮记录不同，结果在小鼠和测量日之间的变化较小。与患者的H反射记录类似，因为精确的定量允许在疾病模型中进行电生理学表征，例如脊髓损伤和中风。该方案需要扎实的实验技能，以便在小鼠中使用手术设备。如果您不确定如何将神经与结缔组织和血管分开，请先练习。演示该程序将由我实验室的博士生Frederique Wieters进行。首先，将成人麻醉的 8 至 16 周龄 C57 黑色/6J 小鼠放在有盖的加热垫上，等待小鼠平静、呼吸稳定且反射不存在。将鼠标放在其背面。然后，用胶带固定前爪。放置胶带，使测量电极易于插入前爪。要测量鼠标的温度，请插入直肠探头并用胶带固定。涂抹眼膏以防止眼睛干燥。用一把细而圆的剪刀小心地去除皮毛，同时用镊子提起皮肤。随后，用一把细小的圆形剪刀沿着前爪的腹后轴在皮肤上做一个一厘米的切口。小心地去除结缔组织，露出肌肉和神经下方。然后用镊子切除暴露的胸大肌深部肌肉，以评估正中神经。用软纸巾去除少量血液和组织液。接下来，小心地从上到下切开乳房或腋窝肌肉，露出神经束。将神经束从结缔组织和肌肉组织中释放出来，长度约为 1.5 厘米。使用弯曲的玻璃移液管小心地分离前爪的尺神经和正中神经。上神经是尺神经，下神经是正中神经。将刺激器钩电极平行排列在0.5至1毫米的距离处，并使用显微操纵器将双钩放置在靠近神经的位置。使用玻璃钩将尺神经提升到刺激钩电极上。然后，用神经拉回电极并将其与其他神经分开。将电极沿爪子的长轴放置，以减少肌肉之间的串扰。表面干燥附着在神经上的电极钩，并使用注射器施用凡士林以提供与邻近组织的电绝缘。要测量 H 反射，请将肌电图电极肌内注射在前爪。然后，将参比电极皮下放置在后肢中，由微型鳄鱼夹固定。当刺激器打开时，观察前爪的微小抽搐等成功的刺激。引发M波和前爪微小可见抽搐的最小刺激电流在10至50微安之间。进行神经刺激15次，每次0.2毫秒长的脉冲。在两组刺激之间暂停两分钟时，频率从 0.1、0.5、1.0、2.0 增加到 5 赫兹。此处显示了在通路中设置的记录图，用于测量霍夫曼反射和肌肉反应。评估了H波和M波振幅之间的比值。刺激和各自的刺激伪影设置为零毫秒，然后是直接M波和随后代表H波的较小峰值。M波在刺激后约2毫秒发生，H波在6至8毫秒后发生，因为通过脊髓的传输时间较长。除了神经分离之外，凡士林在电极上以及两个钩子之间的应用至关重要。这是一个终端实验。在我们的实验室中，这种技术为经皮H反射记录提供了新的金标准参考。

Research

JoVE Journal

Biology

A Reversible, Non-invasive Method for Airway Resistance Measurements and Bronchoalveolar Lavage Fluid Sampling in Mice

一个可逆的，非侵入性方法，气道阻力测量和支气管肺泡灌洗小鼠流体取样

Airway hyperreactivity (AHR) measurements and bronchoalveolar lavage (BAL) fluid sampling are essential to experimental asthma models, but repeated ...

科研

生物学

Transverse Aortic Constriction in Mice

在小鼠体内的横向主动脉缩窄

Transverse aortic constriction (TAC) in the mouse is a commonly used experimental model for pressure overload-induced cardiac hypertrophy and heart ...

Ambulatory ECG Recording in Mice

在小鼠体内的动态心电图记录

Telemetric ECG recording in mice is essential to understanding the mechanisms behind arrhythmias, conduction disorders, and sudden cardiac death. Although ...

Examination of Drosophila Larval Tracheal Terminal Cells by Light Microscopy

Examination of Drosophila Larval Tracheal Terminal Cells by Light Microscopy

考试<em&gt;果蝇</em幼虫气管终端细胞光镜

Cell shape is critical for cell function. However, despite the importance of cell morphology, little is known about how individual cells generate specific ...

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

在肾小管上皮细胞胞浆钙的测量流式细胞仪

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial ...

Non-Terminal Blood Sampling Techniques in Guinea Pigs

豚鼠非终端采血技术

Guinea pigs possess several biological similarities to humans and are validated experimental animal models1-3. However, the use of guinea pigs currently ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

纯化野生型和突变CFTR蛋白的功能重建和通道活性测定

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

Contractility Measurements on Isolated Papillary Muscles for the Investigation of Cardiac Inotropy in Mice

对离体乳头肌的心脏收缩力对小鼠的研究收缩尺寸

Papillary muscle isolated from adult mouse hearts can be used to study cardiac contractility during different physiological/pathological conditions. The ...

Cystometric and External Urethral Sphincter Measurements in Awake Rats with Implanted Catheter and Electrodes Allowing for Repeated Measurements

压和外尿道括约肌测量在清醒大鼠植入导管和电极允许重复测量

Lower urinary tract function is mainly assessed by means of cystometric bladder function analysis in rodents. Conventional cystometries are usually ...

Measurements of Physiological Stress Responses in C. Elegans

Measurements of Physiological Stress Responses in C. Elegans

C. Elegans生理应激反应的测量

Organisms are often exposed to fluctuating environments and changes in intracellular homeostasis, which can have detrimental effects on their proteome and ...