Assessing Functional Recovery of Eupneic Diaphragm Activity Following Unilateral Cervical Spinal Cord Hemisection in Rats

Summary

Respiratory complications are the leading cause of death in individuals with cervical spinal cord injury (cSCI). Animal models of cSCI are essential for mechanistic evaluations and pre-clinical studies. Here, we introduce a reproducible method to assess functional recovery of diaphragm muscle (DIAm) activity following unilateral C₂ spinal hemisection (C₂SH) in rats.

Abstract

Following cSCI, activation of the DIAm can be impacted depending on the extent of the injury. The present manuscript describes a unilateral C₂ hemisection (C₂SH) model of cSCI that disrupts eupneic ipsilateral diaphragm (iDIAm) electromyographic (EMG) activity during breathing in rats. To evaluate recovery of DIAm motor control, the extent of deficit due to C₂SH must first be clearly established. By verifying a complete initial loss of iDIAm EMG during breathing, subsequent recovery can be classified as either absent or present, and the extent of recovery can be estimated using the EMG amplitude. Additionally, by measuring the continued absence of iDIAm EMG activity during breathing after the acute spinal shock period following C₂SH, the success of the initial C₂SH may be validated. Measuring contralateral diaphragm (cDIAm) EMG activity can provide information about the compensatory effects of C₂SH, which also reflects neuroplasticity. Moreover, DIAm EMG recordings from awake animals can provide vital physiological information about the motor control of the DIAm after C₂SH. This article describes a method for a rigorous, reproducible, and reliable C₂SH model of cSCI in rats, which is an excellent platform for studying respiratory neuroplasticity, compensatory cDIAm activity, and therapeutic strategies and pharmaceuticals.

Introduction

There are more than 300,000 individuals with spinal cord injury (SCI) in the United States, approximately half of whom have cervical injuries¹. These injuries result in significant loss of well-being and place a financial strain on individuals, their families, and the healthcare system. Fortunately, the majority of SCIs are incomplete—providing the potential for strengthening of spared pathways¹. This neuroplasticity may allow recovery of at least some function, including DIAm activity, which is important for ventilatory and non-ventilatory behaviors. Thus, promoting neuroplasticity is a promi....

Protocol

This protocol was approved by the Mayo Clinic Institutional Animal Care and Use Committee (Protocol Number: A00003105-17-R23). The animals in the present study were a mix of male and female Sprague-Dawley rats approximately 3 months old and weighing between 200 g to 350 g. The details of the reagents and the equipment used in the study are listed in the Table of Materials.

1. Electrode implantation

1. Preparing the electrodes
   1. Ensure t.......

Discussion

C₂ spinal hemisection
The procedure described in this article emphasizes assessments of DIAm EMG activity that serve as a validation of a C₂ spinal lesion that transects the lateral and ventral funiculi while sparing the dorsal funiculi (Figure 2A). The proposed surgical approach has two major benefits. First, it spares the dorsal funiculi, which preserves ambulatory function in rats, while still severing ipsilateral inputs to phrenic motor neuron.......

Materials

Name	Company	Catalog Number	Comments
25 G Needle	Cardinal Health	1188825100	Covidien Monoject Hypdermic Standard Needles: 25 G x 1" (0.508 mm x 2.5 cm) A
3-0 Vicryl Violet Braided	Ethicon	J774D	3-0 Suture
Adson-Brown Forceps	Fine Science Tools	11627-12	Tip Shape: Straight, Tips: Shark Teeth, Tip Width: 1.4mm, Tip Dimensions: 2 x 1.4 m, Alloy / Material: Stainless Steel, Length: 12 cm
Bowman Style Cage	Braintree Scientific	POR-530	Weight range: 250 up to 750 g; Maximum length: 9" (228 mm); Basic unit is constructed of .5" (123 mm) jeweled acrylic.
Castroviejo Needle Holder	Fine Science Tools	12565-14	Tip Shape: Straight, Tip Width: 1.5 mm, Clamping Length: 10 mm, Lock: Yes, Scissors: No, Alloy / Material: Stainless Steel, Length: 14 cm, Serrated: Yes, Feature: Tungsten Carbide
Clip Lead 1m TP Shielded	Biopac Systems, Inc	LEAD110S	Shielded lead wires for EMG
Data Acquisition Software	LabChart	LabChart 7.3.8	Data recording, visualization, and analysis software for multi-channel recordings and real-time assessments
Data Analysis Software - Matlab 2023b	Mathworks, Inc.	Version 23.2	General purpose programming language for post hoc analysis
Dissecting Knife	Fine Science Tools	10056-12	Cutting Edge: 4 mm, Thickness: 0.5 mm, Alloy / Material: Stainless Steel, Length: 12.5 cm, Blade Shape: Angled 30°
Dumont #3 Forceps	Fine Science Tools	11293-00	Style: #3, Tip Shape: Straight, Tips: Standard, Tip Dimensions: 0.17 x 0.1 mm, Length: 12 cm, Alloy / Material: Dumostar
Electromyogram Amplifier	Biopac Systems, Inc	EMG100C	EMG amplifier
Friedman Rongeur	Fine Science Tools	16000-14	Tip Shape: Curved, Cup Size: 2.5mm, Alloy / Material: Stainless Steel, Length: 13cm, Joint Action: Single
Friedman-Pearson Rongeurs	Fine Science Tools	16021-14	Alloy / Material: Stainless Steel, Length: 14cm, Joint Action: Single, Cup Size: 1mm, Tip Shape: Curved
Isolated Power Supply Module	Biopac Systems, Inc	IPS100C	Operates 100-series amplifier modules indepdent of the Biopac Systems, Inc.'s MP series Data Acquisition System
Kelly Hemostats	Fine Science Tools	13019-14	Tips: Serrated, Tip Width: 1.5mm, Clamping Length: 22mm, Alloy / Material: Stainless Steel, Length: 14cm, Tip Shape: Curved
Knife Curette	V. Mueller	VM101-4414	Tip: Sharp, Tip Diameter: 2 mm
Micro Dissecting Scissors	Biomedical Research Instruments, Inc.	11-2420	Length: 4", Angle: Straight, Blade Length: 23 mm
Multistranded stainless steel wire	Cooner Wire, Inc.	AS 631	AWG 40; Overall diameter: 0.011 mm (with insulation), 0.008 mm (without insulation).
PowerLab 8/35	ADInstruments	PL3508	Data acquisition system
Scalpel Blade #11	Fine Science Tools	10011-00	Blade Shape: Angled, Cutting Edge: 20 mm, Thickness: 0.4 mm, Alloy / Material: Carbon Steel
Scalpel Handle #3	Fine Science Tools	10003-12	Alloy / Material: Stainless Steel, Length: 12 cm
Sprague Dawley Rat	Inotiv	Order code: 002	Sprague Dawley outbred rats (female and male)
Surgical Microscope	Olympus	SZ61	Surgical microscope
Suture Cutting Scissors	George Tiemann & Co.	110-1250SB	Alloy / Material: Stainless Steel, Tip Shape: Straight, Tips: Sharp/Blunt, Length: 4.5"
Vannas Spring Scissors	Fine Science Tools	15000-08	Tips: Sharp, Cutting Edge: 2.5 mm, Tip Diameter: 0.05 mm, Length: 8 cm, Alloy / Material: Stainless Steel, Serrated: No, Tip Shape: Straight
Weitlaner Retractor	Codman	50-5647	Prongs: 2 x 3 Blunt, Length: 4.5"