Assessing Rat Diaphragm Motor Unit Connectivity Outcome Measures as Quantitative Biomarkers of Phrenic Motor Neuron Degeneration and Compensation

Summary

In this study, we present an in vivo method for estimating motor unit number and size to quantify rat diaphragm motor unit connectivity. A step-by-step approach to these techniques is described.

Abstract

Loss of ventilatory muscle function is a consequence of motor neuron injury and neurodegeneration (e.g., cervical spinal cord injury and amyotrophic lateral sclerosis, respectively). Phrenic motor neurons are the final link between the central nervous system and muscle, and their respective motor units (groups of muscle fibers innervated by a single motor neuron) represent the smallest functional unit of the neuromuscular ventilatory system. Compound muscle action potential (CMAP), single motor unit potential (SMUP), and motor unit number estimation (MUNE) are established electrophysiological approaches that enable the longitudinal assessment of motor unit integrity in animal models over time but have mostly been applied to limb muscles. Therefore, the objectives of this study are to describe an approach in preclinical rodent studies that can be used longitudinally to quantify the phrenic MUNE, motor unit size (represented as SMUP), and CMAP, and then to demonstrate the utility of these approaches in a motor neuron loss model. Sensitive, objective, and translationally relevant biomarkers for neuronal injury, degeneration, and regeneration in motor neuron injury and diseases can significantly aid and accelerate experimental research discoveries to clinical testing.

Introduction

Phrenic motor neurons (MNs), extending from C3 to C6 myotome levels, form the final link from the central nervous system (CNS) to the diaphragm muscle¹. Phrenic motor units (MUs) are comprised of a single spinal MN and its innervated diaphragm muscle fibers forming the smallest functional unit of the respiratory neuromuscular system. The ventilatory function requires adequate contraction of the diaphragm muscle achieved through coordinated activation of the phrenic MU pool^2,3. Many neurological diseases, including amyotrophic lateral sclerosis (ALS), result in severe ventilatory impairm....

Protocol

All procedures were approved and conducted in compliance with the guidelines established by the Institutional Animal Care and Use Committee at the University of Missouri. Experiments were performed on adult male Sprague-Dawley rats, aged 11 to 15 weeks. These rats were housed in pairs and kept under a 12:12 light-dark cycle, with access to standard commercial pelleted food and HCl-treated water available at all times.

1. Animal preparation and anesthesia delivery

1. W.......

Discussion

In MN degenerative diseases, such as ALS, it is crucial to assess the MUs involved in ventilation²⁸. Despite the occurrence of respiratory MN degeneration in ALS patients, the specific onset and progression of MN death remain incompletely understood^29,30,31. Recognizing the significance of this aspect, various models, both genetic-based (e.g., SOD1^2,32<.......

Materials

Name	Company	Catalog Number	Comments
2 mL Glass Syringe	Kent Scientific Corporation	SOMNO-2ML
50 mL, Model 705 RN syringe	Hamilton Company	7637-01	Utilized to conduct intrapleural injection
5008 - Formulab Diet	LabDiet	0001325
Autoclavable 26 G needles (26S RN 9.52 mm 40°)	Hamilton Company	7804-04	Utilized to conduct intrapleural injection
Cholera toxin B-subunit (CTB)	MilliporeSigma	C9903	Utilized for intrapleural injection to label surviving motor neurons
Cholera toxin B-subunit conjugated to saporin (CTB-SAP)	Advanced Targeting Systems	IT-14	Utilized for intrapleural injection to cause motor neuron death
Detachable Cable	Technomed	202845-0000	to connect the recorder electrode to the electrodiagnostic machine
Disposable 2" x 2" disc electrode with leads	Cadwell	302290-000	ground electrode
disposable monopolar needles 28 G	Technomed	202270-000	cathode and anode stimulating electrodes- recording electrodes
EMG needle cable (Amp/stim switch box)	Cadwell	190266-200	to connect monopolar electrodes to electrodiagnostic stimulator
Helping Hands alligator clip with iron base	Radio Shack	64-079	Maintaining recording electrode placement
Isoflurane (250 mL bottle)	Piramal Healthcare
monoject curved tip irrigating syringe	Covidien	81412012	utilized for application of electrode gel
PhysioSuite Physiological Monitoring System with RightTemp Homeothermic Warming	Kent Scientific Corporation	PS-RT	Includes infrared warming pad, rectal probe, and pad temperature probe
Pro trimmer Pet Grooming Kit	Oster	078577-010-003	clippers for hair removal
Saporin (SAP)	Advanced Targeting Systems	PR-01	Utilized for intrapleural injection (control agent when injected by itself)
Sierra Summit EMG system	Cadwell Industries, Inc., Kennewick, WA		portable electrodiagnostic system
SomnoSuite Low-Flow Digital Anesthesia System	Kent Scientific Corporation	SOMNO	Includes anti-spill, anti-vapor bottle top adapter; Y adapter tubing; charcoal scavenging filter
Sprague-Dawley rat	Envigo colony 208a, Indianapolis, IN
Veterinarian petroleum-based ophthalmic ointment	Puralube	26870	applied during anesthesia to avoid corneal injury