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Behavior
电电机组数估计(MUNE)在小鼠后肢肌肉测量复合肌肉动作电位(CMAP)
电电机组数估计(MUNE)在小鼠后肢肌肉测量复合肌肉动作电位(CMAP)
JoVE Journal
Behavior
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JoVE Journal Behavior
Electrophysiological Motor Unit Number Estimation (MUNE) Measuring Compound Muscle Action Potential (CMAP) in Mouse Hindlimb Muscles

电电机组数估计(MUNE)在小鼠后肢肌肉测量复合肌肉动作电位(CMAP)

Full Text
22,363 Views
09:07 min
September 25, 2015

DOI: 10.3791/52899-v

W. David Arnold1,2,3, Kajri A. Sheth1, Christopher G. Wier4, John T. Kissel1,3, Arthur H. Burghes1,3,4, Stephen J. Kolb1,3,4

1Department of Neurology,The Ohio State University Wexner Medical Center, 2Department of Physical Medicine and Rehabilitation,The Ohio State University, 3Department of Neuroscience,The Ohio State University Wexner Medical Center, 4Department of Biochemistry and Pharmacology,The Ohio State University Wexner Medical Center

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Please note that some of the translations on this page are AI generated. Click here for the English version.

Overview

This article presents refined protocols for in vivo monitoring of motor unit function in mice, specifically focusing on the sciatic nerve's innervation of hind limb muscles. Techniques for measuring compound muscle action potential (CMAP) and motor unit number estimation (MUNE) are detailed.

Key Study Components

Area of Science

  • Neuroscience
  • Electrophysiology
  • Motor unit analysis

Background

  • Understanding motor unit function is crucial for investigating peripheral nervous system disorders.
  • The study utilizes a mouse model to estimate functional motor units.
  • Previous methods of MUNE measurement have limitations that this study aims to address.
  • The technique is minimally invasive and applicable to both neonatal and adult mice.

Purpose of Study

  • To provide a reliable method for estimating the number of functional motor units in mouse hind limb muscles.
  • To enhance the understanding of motor unit maintenance and health.
  • To facilitate the investigation of preclinical therapies for peripheral nerve injuries.

Methods Used

  • Measurement of maximal CMAP amplitude from tricep muscles.
  • Incremental stimulation to obtain average single motor unit action potential amplitude.
  • Electrophysiological recordings using ring and monopolar electrodes.
  • Comparison of motor unit function before and after sciatic nerve crush.

Main Results

  • The technique allows for accurate estimation of functional motor units.
  • Significant reduction in MUNE observed following sciatic nerve crush.
  • Control mice exhibited a higher number of functional motor units compared to those with nerve injury.
  • Demonstrated reproducibility and reliability of the method for longitudinal studies.

Conclusions

  • The refined protocols enhance the assessment of motor unit integrity.
  • Minimally invasive techniques improve translational research potential.
  • Findings contribute to the understanding of motor neuron diseases and peripheral nerve injuries.

Frequently Asked Questions

What is the significance of measuring CMAP?
CMAP measurements provide insights into the functional status of motor units and the integrity of the peripheral nervous system.
How does this method compare to traditional MUNE techniques?
This method is less invasive and allows for repeated assessments over time, improving reliability.
Can this technique be used in both neonatal and adult mice?
Yes, the protocols are applicable to both age groups, making them versatile for various studies.
What are the main advantages of this approach?
The main advantages include minimal invasiveness, reproducibility, and the ability to conduct longitudinal studies.
What conditions can this technique help investigate?
It can be used to study motor neuron diseases and peripheral nerve injuries, aiding in the development of preclinical therapies.

我们提出精致的协议,允许在电动机单元功能的小鼠体内监测。技术测量复合肌肉动作电位(CMAP)和电动机单元数估计(MUNE)在坐骨神经支配鼠标后肢肌肉进行说明。

以下实验的总体目标是估计体内小鼠中支配肱三头肌 Siri 肌肉的功能性运动单位的数量。这是通过首先测量肱三头肌 Siri 肌肉的最大 cmap 振幅来实现的。作为第二步,获得 10 个次最大增量响应,然后将其平均以给出平均单个运动单位动作电位幅度。

接下来,将 cmap 振幅除以平均 SUP 振幅。为了计算 muni,结果提供了支配肱三头肌 sury hin 肢体肌肉的功能性运动单位数量的体内估计。电生理运动单位数估计技术或 muni 可用于研究周围神经系统疾病或损伤的临床前治疗。

我们在检查脊髓性肌萎缩症的 Delta seven 小鼠模型时首次想到了这种方法。在这个模型中,它表现出严重的表型,因此我们必须检查新生小鼠。因此,该技术既可用于新生小鼠,也可用于成年小鼠。

这种方法

的视觉演示对于获得可重复的增量响应以允许准确估计功能性运动单位的数量至关重要。与其他 muni 测量方法和小鼠方法(例如那些依赖于增量力测量的方法)相比,该技术的主要优点是该技术是微创的,并且可以随着时间的推移而重复。在运动神经元疾病和周围神经损伤模型中对运动单位完整性进行临床相关、可靠和重复评估的能力增加了这些模型的转化潜力,并增加了我们对运动单位维持和健康的决定因素的理解。

要进行 cmap 和 muni 记录,请将有源环形电极放在皮肤上,覆盖在麻醉小鼠后肢的近端部分、胃肌上。然后将参比环形电极放在足部跖骨中部上方的皮肤上,以减少阻抗,用凝胶涂覆环形电极下方的皮肤和残留的毛发,以最大限度地接触电极皮肤。避免过度使用电极凝胶,因为这可能会导致电极之间出现电桥,并妨碍准确记录刺激坐骨神经。

将绝缘的 28 号单极针作为阴极插入近端后肢。避免将刺激电极过度靠近坐骨神经或两根深处插入,因为它可能会伤害坐骨神经或其他结构。将另一个绝缘的 28 号单极针作为阳极插入,更靠近覆盖骶骨的皮下组织之后,将一次性表面电极作为接地电极放在对侧后肢或尾部。

在此过程中,通过用 0.1 毫秒的方波脉冲刺激坐骨神经来获得坐骨神经 cmap 反应。期间。将刺激强度从 1 毫安增加到 10 毫安,直到响应的幅度不再增加,从而获取 cmap 响应。为了确保超最大刺激,将刺激增加到用于获得最大响应和附加响应的刺激强度的 120% 左右。

如果 cmap 大小没有进一步增加,则将此响应记录为最大 cmap。然后以毫伏为单位记录基线到峰值和峰到峰值的 cmap 幅度。要确定平均 SUP 大小,请使用增量刺激技术,通过提供次最大刺激,同时以 0.03 毫安的增量增加强度,以获得最小的全或无响应。

如果在 0.21 毫安和 0.70 毫安之间的刺激强度之间没有发生初始反应,请调整刺激阴极位置,使其更靠近或更远离大腿近端坐骨神经的位置。获取此处显示的初始增量响应。确保增量响应的负峰值的延迟与先前获得的最大 cmap 响应的负峰值大致一致。

通过实时观察三个一致的增量响应,确保响应稳定且无分馏,并确保振幅至少为 25 微伏。接下来,获得第二个增量,通过实时叠加三个增量响应来确保增量稳定且无分馏。第二个增量应在视觉上清晰,并且至少比前一个响应大 25 微伏。

然后获取第三个增量响应,确保增量稳定且无分馏。与前面显示的第二个增量响应类似,第三个增量应在视觉上清晰,并且幅度至少比第二个响应大 25 微伏。继续以这种方式获取增量响应,直到总共记录了 10 个增量响应。

评估增量以确保每个增量响应的幅度小于所有 10 个增量之和的三分之一。接下来,使用 10 个增量值给出平均 SUP 振幅。将显示 10 个增量响应的计算值。

平均 SUP 大小是通过平均 10 个增量或简单地将最终增量除以 10 来确定的。然后通过将最大峰峰值 cmap 振幅除以平均 SM 振幅来计算 muni。为了说明这些技术的应用,我们研究了坐骨神经挤压对运动单位功能的影响。

在该图中,比较了坐骨神经挤压后 11 周成年对照小鼠和成年小鼠的反应,记录之间的灵敏度没有差异。坐骨神经挤压后,与对照小鼠中 278 个功能性运动单位的正常发现相比,在估计的 50 个功能性运动单位时,muni 严重减少。相反,由于侧支发芽,与对照相比,粉碎动物的 cmap 振幅仅显示出轻微的降低。

看完这个视频,你应该对如何在小鼠后肢、肌肉和体内进行运动单位数估计有一个很好的了解一旦掌握了,如果作得当,这项技术通常可以在不到 20 分钟的时间内完成。我们希望该协议的这种视觉演示将导致这项重要技术的广泛采用,我们相信这将提高实验室间的可靠性,并将实验室中的发现有效地转化为临床中的有效疗法。

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