Rizzuto Emanuele, Pisu Simona, Nicoletti Carmine, Del Prete Zaccaria, Musarò Antonio
Department of Mechanical and Aerospace Engineering, Sapienza University of Rome;
Institute Pasteur Cenci-Bolognetti, DAHFMO-Unit of Histology and Medical Embryology, Sapienza University of Rome.
J Vis Exp. 2017 Aug 6(126):55227. doi: 10.3791/55227.
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 SOD1 transgenic mouse, an experimental model of ALS that ubiquitously overexpresses the mutant antioxidant enzyme superoxide dismutase 1 (SOD1).
在研究运动神经元与肌肉之间通讯受损的疾病（如衰老和肌萎缩侧索硬化症（ALS））时，神经肌肉接头（NMJ）的功能起着关键作用。在此，我们描述了一种实验方案，可通过结合两种电刺激来测量NMJ功能：直接肌肉膜刺激和通过神经进行的刺激。比较肌肉对这两种不同刺激的反应有助于在功能层面确定NMJ中导致肌肉功能下降的潜在改变。离体标本适用于严格控制的研究。在此，我们描述了一种详细方案，用于测量比目鱼肌 - 坐骨神经标本和膈肌 - 膈神经标本的肌肉和NMJ功能的几个参数。该方案持续约60分钟，通过定制软件不间断地进行，该软件可测量抽搐动力学特性、肌肉和神经刺激的力 - 频率关系以及NMJ功能特有的两个参数，即神经传递失败和强直内疲劳。通过使用SOD1转基因小鼠（一种ALS实验模型，其全身过表达突变抗氧化酶超氧化物歧化酶1（SOD1）），该方法用于检测比目鱼肌和膈肌肌肉 - 神经标本中的损伤。
