Functional and Morphological Assessment of Diaphragm Innervation by Phrenic Motor Neurons

Summary

Compound muscle action potential recording quantitatively assesses functional diaphragm innervation by phrenic motor neurons. Whole-mount diaphragm immunohistochemistry assesses morphological innervation at individual neuromuscular junctions. The goal of this protocol is to demonstrate how these two powerful methodologies can be used in various rodent models of spinal cord disease.

Abstract

This protocol specifically focuses on tools for assessing phrenic motor neuron (PhMN) innervation of the diaphragm at both the electrophysiological and morphological levels. Compound muscle action potential (CMAP) recording following phrenic nerve stimulation can be used to quantitatively assess functional diaphragm innervation by PhMNs of the cervical spinal cord in vivo in anesthetized rats and mice. Because CMAPs represent simultaneous recording of all myofibers of the whole hemi-diaphragm, it is useful to also examine the phenotypes of individual motor axons and myofibers at the diaphragm NMJ in order to track disease- and therapy-relevant morphological changes such as partial and complete denervation, regenerative sprouting and reinnervation. This can be accomplished via whole-mount immunohistochemistry (IHC) of the diaphragm, followed by detailed morphological assessment of individual NMJs throughout the muscle. Combining CMAPs and NMJ analysis provides a powerful approach for quantitatively studying diaphragmatic innervation in rodent models of CNS and PNS disease.

Introduction

Amyotrophic Lateral Sclerosis (ALS) is a debilitating motor neuron disease associated with the loss of both upper and lower motor neurons and consequent muscle paralysis. Upon diagnosis, patient survival is on average only 2-5 years¹. Phrenic motor neuron (PhMN) loss is a critical component of the pathogenesis of ALS. Patients ultimately die due to loss of PhMN innervation of the diaphragm, the primary muscle of inspiration^2,3. Traumatic spinal cord injury (SCI) is also a serious problem with associated breathing difficulties. Approximately 12,000 new cases of SCI occur each year⁴ due to traumatic damage to the spinal cord. Despite disease heterogeneity with respect to location, type and severity, the majority of SCI cases involve trauma to the cervical spinal cord, which often results in debilitating and persistent respiratory compromise. In addition to ALS and SCI, other central nervous system (CNS) diseases can be associated with diaphragmatic respiratory dysfunction^5,6.

The phrenic nerve is an efferent motor nerve that innervates the ipsilateral hemi-diaphragm and that originates from PhMN cell bodies located in the C3-C5 levels of the ipsilateral cervical spinal cord. PhMN output is controlled by descending bulbospinal input from the brainstem in an area known as the rostral ventral respiratory group (rVRG)⁷. The rVRG-PhMN-diaphragm circuit is central to the control of inspiratory breathing, as well as other non-ventilatory diaphragm behaviors. Various traumatic injuries and neurodegenerative disorders that affect this circuitry can lead to a profound decline in respiratory function and patient quality of life. Descending input to PhMNs from the rVRG, PhMN survival, phrenic nerve integrity and proper innervation at the diaphragm neuromuscular junction (NMJ) are all necessary for normal diaphragm function. It is therefore important to employ techniques that can quantitatively evaluate this circuit in vivo in rodent models of ALS, SCI and other CNS diseases.

With this protocol, the goal is to describe experimental tools for assessing PhMN innervation of the diaphragm at both the electrophysiological and morphological levels. Compound muscle action potentials (CMAPs) are recorded by stimulating all efferent motor neuron axons of a given motor nerve and then analyzing the elicited depolarization response of the target myofibers. This technique can be used in vivo in anesthetized rats and mice to quantify functional innervation of the hemi-diaphragm by PhMNs⁸. Due to the fact that CMAPs represent simultaneous recording of all (or at least many/most) myofibers of the whole hemi-diaphragm, it is useful to also examine the phenotypes of individual motor axons and myofibers at the diaphragm NMJ in order to track disease- and therapy-relevant morphological changes such as partial and complete denervation, regenerative sprouting and reinnervation. This can be accomplished via whole-mount immunohistochemistry (IHC) of the diaphragm, followed by detailed morphological assessment of individual NMJs throughout the muscle⁹. Combining CMAPs and NMJ analysis provides a powerful approach for quantitatively studying diaphragmatic innervation in rodent models of CNS and PNS disease.

Protocol

Experimental procedures were approved by the Thomas Jefferson University institutional animal care and use committee and conducted in compliance with the European Communities Council Directive (2010/63/EU, 86/609/EEC and 87-848/EEC), the NIH Guide for the care and use of laboratory animals, and the Society for Neuroscience’s Policies on the Use of Animals in Neuroscience Research. 1. Compound Muscle Action Potentials (CMAPs) Preparing the animal: Anesthetize the r…

Representative Results

Adult Sprague-Dawley rats received either laminectomy only (uninjured control) or unilateral hemi-contusion SCI at the C4 spinal cord level10-12. At 5 weeks post-surgery, peak CMAP amplitude recorded from the hemi-diaphragm ipsilateral to the laminectomy/injury site was significantly reduced in SCI rats (Figure 2C) compared to laminectomy-only control (Figure 2B). All NMJs in the hemi-diaphragm were completely intact in control non-diseased wild-type rats (Figure 4A,C<…

Discussion

As respiratory function is compromised in both traumatic SCI and ALS, developing therapies that target breathing and specifically diaphragm innervation are clinically relevant^5,6. In order to comprehensively study respiratory function, a combined approach method should be used. CMAPs measure the degree of functional innervation of the diaphragm by way of external phrenic nerve stimulation, but not endogenous bulbospinal respiratory drive⁸. In addition, these recordings do not allow for examination o…

Materials

Paraformaldehyde	Fisher	T353-500	Make 10% solution first in de-ionized distilled water; make 4% with 1X PBS, adjust pH to 7.4
1X Phosphate Buffered Saline, pH 7.4	Invitrogen	10010049
2% Bovine serum albumin (2% BSA)	Sigma-Aldrich	A3059-100g	Dissolve 2g BSA into 100mL of 1X PBS
0.2% Triton X100 in 2% BSA/PBS (Blocking Buffer)	Sigma-Aldrich	T9284-100mL	Dissolve 0.2ml/100mL 2% BSA/PBS
0.1M Glycine	Sigma-Aldrich	G-7126	Add 0.185g to 25mL of 2% BSA/PBS
α-bungarotoxin	Invitrogen	T1175	Concentration 1:400
SMI-312	Sternberger Monoclonals	SMI312	Concentration 1:1,000
SV2	Developmental Studies Hybridoma Bank	SV2-Supernatant	Concentration 1:10
FITC goat anti-mouse IgG1	Roche	3117731001	Concentration 1:100
Silicone rubber	Sylgard, Dow Corning	Part # 184	Follow instructions that come with kit: can use multiple sized culture dish (30mm, 60mm, 100mm) depending on needs
Vectashield fluorescent mounting medium	Vector laboratories	H-1000	This is not a hard-set medium. You will need to secure the cover slip with clear nail polish.
Small Spring Scissors	Fine Science Tools	15002-08
Dissection forceps	Fine Science Tools	11295-51
Software for CMAP recordings	Scope 3.5.6; ADI
Disk surface electrodes	Natus neurology	019-409000
Subdermal needle electrodes	Natus neurology	019-453100
Conductive gel	Aquasonic	122-73720
Stimulator/recording system for CMAP recordings	ADI Powerlab 8SP stimulator
Amplifier for CMAP recordings	BioAMP