The authors acknowledge the NIH funding source (NIH R01HL146114).

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.&#160;Electrode implantation
<ol>
	<li>Preparing the electrodes
	<ol>
		<li>Ensure t...

Standardized Evaluation of DIAm EMG Post-C2SH in Rats to Minimize Variability

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...

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&#8212;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 promising avenue of research to help individuals with SCI2.
Rodent models of SCI have the potential to contribute substantially to the discovery of treatments to improve human health. One of the classic models of SCI used to study neuroplasticity is a unilateral transection (hemisection) of the spinal cord at C2 (C2SH), which leaves the contralateral side intact3,4,5,6,7,8,9,10,11,12,13. The effect of C2SH on phrenic output and the importance of spared contralateral pathways was first revealed over a hundred years ago by Porter12, whose seminal article laid the foundation for modern-day studies of respiratory neuroplasticity. The C2SH model interrupts descending inputs from the rostral ventral respiratory group (rVRG) in the medulla, which contains premotor neurons responsible for transmitting the output of respiratory rhythm generation14. These rVRG premotor neurons also transmit excitatory neural drive to phrenic motor neurons (Figure 1). Several investigators have taken different approaches to the C2SH model10,11,15,16, which may partly explain some of the variability in recovery across studies. Briefly, approaches vary in terms of sparing the dorsal funiculi, performing a complete hemisection, or performing a lateral partial transection that does not completely interrupt descending inputs from the ipsilateral rVRG. Generally, C2SH models are particularly useful for studying respiratory neuroplasticity due to the rates of spontaneous recovery of eupneic iDIAm electromyographic (EMG) activity over time, which can be improved by several factors, including neurotrophic signaling17,18,19,20,21. However, an initial loss of function&#8212;defined as the silencing of eupneic iDIAm EMG activity&#8212;must be first established before recovery can be clearly classified. This validation of inactivity at the time of C2SH is not done in several studies3,4,6,7,11,22,23.
Histological assessments of the excised spinal cord only provide evidence of damage to the appropriate location of ipsilateral excitatory bulbospinal pathways innervating phrenic motor neurons in the spinal cord, but histology does not substitute for physiological evidence (e.g., DIAm EMG). Furthermore, histological assessments are performed in ex vivo at terminal time points (often several weeks to months post-injury) and thus do not provide &#34;real-time&#34; information. Some investigators have noted that the magnitude of the lesion relates to the amount of functional deficit or lack thereof5,24,25,26. It is important to note that the validity of such claims is likely highly dependent on how &#34;function&#34; is classified (i.e., what the functional tasks are and how they are quantified), and the variability across studies highlights the difficulty of producing functionally identical lesions across animals. Indeed, investigators have emphasized that the relationship between the extent of injury and limb muscle locomotor function (quantified by the Basso, Beattie, and Bresnahan (BBB) score24) is not linear27,28. In previous studies, we have found no relationship between the extent of the C2SH and the extent of recovery of eupneic iDIAm EMG activity post-injury10,29,30,31, although other investigators have reported a relationship between ventilatory function and the extent of white matter sparing5. Thus, in the case of the C2SH model, an approach for functional validation of iDIAm inactivity at the time of the surgery and preferably early in the time course of chronic spinal cord injury experiments is both beneficial and necessary.
The present article underscores the use of DIAm EMG for real-time confirmation of the initial loss of DIAm EMG during breathing after the C2SH as well as subsequent confirmatory assessments at 3 days (Day 3) after the injury18,21,31,32,33. In earlier work with the C2SH model, repeated laparotomies were performed to record DIAm EMG10,13,30,34. However, more recent work has used chronic EMG electrodes, which allow the recording of EMG in anesthetized and awake rats. Additionally, chronic electrodes reduce the risk of pneumothorax and don&#39;t require repeated laparotomies, which can cause inhibition of the DIAm35,36. Although versions of the C2SH model have been used by many investigators, confirmation of the silencing of iDIAm activity was not made at the time of surgery3,4,6,7,11,22,23. Without such a confirmation of inactivity, it is difficult to know what portion of subsequent recovery to attribute to the neuroplasticity of ipsilateral versus contralateral pathways, which may have differential impacts. This is an important consideration because the inspiratory neural drive from the rVRG to phrenic motoneurons is primarily ipsilateral, with a loss of about 50% of excitatory glutamatergic inputs to phrenic motor neurons after C2SH33. However, there are remaining inspiratory excitatory inputs from the contralateral rVRG that decussate below the site of the lesion to innervate ipsilateral phrenic motor neurons and can be strengthened via neuroplasticity to promote functional recovery. By removing the predominant ipsilateral excitatory input to phrenic motor neurons, eupneic iDIAm EMG activity is lost (at least under anesthesia), while the activity of the cDIAm continues and is even enhanced. The loss of iDIAm EMG activity during breathing is thus a measure of a successful C2SH (Figure 2).
Some level of iDIAm EMG activity is present as early as 1-4 days following C2SH in awake animals23,37. Additionally, in decerebrate animals, iDIAm activity is present within minutes to hours after upper cervical hemisection and is suppressed by anesthesia38. Additionally, the success of the C2SH is validated by confirming the absence of iDIAm EMG activity during breathing (eupnea) in anesthetized rats on Day 3 post-injury. Confocal imaging studies confirmed the loss of glutamatergic synaptic inputs on phrenic motor neurons during this initial stage of injury37. At Day 3 post-injury, if there is any residual eupneic iDIAm EMG activity, this is interpreted as evidence of incomplete removal of ipsilateral descending inspiratory drive from the rVRG. The present article is divided into three sections: (1) chronic DIAm EMG recordings, (2) C2SH, and (3) EMG data acquisition in awake and anesthetized animals. This protocol describes 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.

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	<tbody>
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			<td>25 G Needle</td>
			<td>Cardinal Health</td>
			<td>1188825100</td>
			<td>Covidien Monoject Hypdermic Standard Needles: 25 G x 1&#34; (0.508 mm x 2.5 cm) A</td>
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			<td>3-0 Vicryl Violet Braided</td>
			<td>Ethicon</td>
			<td>J774D</td>
			<td>3-0 Suture</td>
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			<td>Adson-Brown Forceps</td>
			<td>Fine Science Tools</td>
			<td>11627-12</td>
			<td>Tip Shape: Straight, Tips: Shark Teeth, Tip Width: 1.4mm, Tip Dimensions: 2 x 1.4 m, Alloy / Material: Stainless Steel, Length: 12 cm</td>
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		<tr>
			<td>Bowman Style Cage</td>
			<td>Braintree Scientific</td>
			<td>POR-530</td>
			<td>Weight range: 250 up to 750 g; Maximum length: 9&#34; (228 mm); Basic unit is constructed of .5&#34; (123 mm) jeweled acrylic.</td>
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			<td>Castroviejo Needle Holder</td>
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			<td>12565-14</td>
			<td>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</td>
		</tr>
		<tr>
			<td>Clip Lead 1m TP Shielded</td>
			<td>Biopac Systems, Inc</td>
			<td>LEAD110S</td>
			<td>Shielded lead wires for EMG</td>
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		<tr>
			<td>Data Acquisition Software</td>
			<td>LabChart</td>
			<td>LabChart 7.3.8</td>
			<td>Data recording, visualization, and analysis software for multi-channel recordings and real-time assessments</td>
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		<tr>
			<td>Data Analysis Software - Matlab 2023b</td>
			<td>Mathworks, Inc.</td>
			<td>Version 23.2</td>
			<td>General purpose programming language for post hoc analysis</td>
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		<tr>
			<td>Dissecting Knife</td>
			<td>Fine Science Tools</td>
			<td>10056-12</td>
			<td>Cutting Edge: 4 mm, Thickness: 0.5 mm, Alloy / Material: Stainless Steel, Length: 12.5 cm, Blade Shape: Angled 30&#176;</td>
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		<tr>
			<td>Dumont #3 Forceps</td>
			<td>Fine Science Tools</td>
			<td>11293-00</td>
			<td>Style: #3, Tip Shape: Straight, Tips: Standard, Tip Dimensions: 0.17 x 0.1 mm, Length: 12 cm, Alloy / Material: Dumostar</td>
		</tr>
		<tr>
			<td>Electromyogram Amplifier</td>
			<td>Biopac Systems, Inc</td>
			<td>EMG100C</td>
			<td>EMG amplifier</td>
		</tr>
		<tr>
			<td>Friedman Rongeur</td>
			<td>Fine Science Tools</td>
			<td>16000-14</td>
			<td>Tip Shape: Curved, Cup Size: 2.5mm, Alloy / Material: Stainless Steel, Length: 13cm, Joint Action: Single</td>
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			<td>Friedman-Pearson Rongeurs</td>
			<td>Fine Science Tools</td>
			<td>16021-14</td>
			<td>Alloy / Material: Stainless Steel, Length: 14cm, Joint Action: Single, Cup Size: 1mm, Tip Shape: Curved</td>
		</tr>
		<tr>
			<td>Isolated Power Supply Module</td>
			<td>Biopac Systems, Inc</td>
			<td>IPS100C</td>
			<td>Operates 100-series amplifier modules indepdent of the Biopac Systems, Inc.&#39;s MP series Data Acquisition System</td>
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		<tr>
			<td>Kelly Hemostats</td>
			<td>Fine Science Tools</td>
			<td>13019-14</td>
			<td>Tips: Serrated, Tip Width: 1.5mm, Clamping Length: 22mm, Alloy / Material: Stainless Steel, Length: 14cm, Tip Shape: Curved</td>
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			<td>Knife Curette</td>
			<td>V. Mueller</td>
			<td>VM101-4414</td>
			<td>Tip: Sharp, Tip Diameter: 2 mm</td>
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			<td>Micro Dissecting Scissors</td>
			<td>Biomedical Research Instruments, Inc.</td>
			<td>11-2420</td>
			<td>Length: 4&#34;, Angle: Straight, Blade Length: 23 mm</td>
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		<tr>
			<td>Multistranded stainless steel wire</td>
			<td>Cooner Wire, Inc.</td>
			<td>AS 631</td>
			<td>AWG 40; Overall diameter: 0.011 mm (with insulation), 0.008 mm (without insulation).</td>
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			<td>PowerLab 8/35</td>
			<td>ADInstruments</td>
			<td>PL3508</td>
			<td>Data acquisition system</td>
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			<td>Scalpel Blade #11</td>
			<td>Fine Science Tools</td>
			<td>10011-00</td>
			<td>Blade Shape: Angled, Cutting Edge: 20 mm, Thickness: 0.4 mm, Alloy / Material: Carbon Steel</td>
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			<td>Scalpel Handle #3</td>
			<td>Fine Science Tools</td>
			<td>10003-12</td>
			<td>Alloy / Material: Stainless Steel, Length: 12 cm</td>
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			<td>Sprague Dawley Rat</td>
			<td>Inotiv</td>
			<td>Order code: 002</td>
			<td>Sprague Dawley outbred rats (female and male)</td>
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			<td>Surgical Microscope</td>
			<td>Olympus</td>
			<td>SZ61</td>
			<td>Surgical microscope&#160;</td>
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			<td>Suture Cutting Scissors</td>
			<td>George Tiemann &#38; Co.</td>
			<td>110-1250SB</td>
			<td>Alloy / Material: Stainless Steel, Tip Shape: Straight, Tips: Sharp/Blunt, Length: 4.5&#34;</td>
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		<tr>
			<td>Vannas Spring Scissors</td>
			<td>Fine Science Tools</td>
			<td>15000-08</td>
			<td>Tips: Sharp, Cutting Edge: 2.5 mm, Tip Diameter: 0.05 mm, Length: 8 cm, Alloy / Material: Stainless Steel, Serrated: No, Tip Shape: Straight</td>
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		<tr>
			<td>Weitlaner Retractor</td>
			<td>Codman</td>
			<td>50-5647</td>
			<td>Prongs: 2 x 3 Blunt, Length: 4.5&#34;</td>
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assessing functional recovery of eupneic diaphragm activity following unilateral cervical spinal cord hemisection in rats

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.

Author Spotlight: Neuromotor Control and Recovery of Diaphragm Function Following Cervical Spinal Hemisection in Rats

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

The approach presented in this article minimizes inter-operator variability by setting clear criteria for evaluating DIAm EMG in a rat model of C2SH. First, the cessation of eupneic iDIAm EMG activity immediately after C2SH must be observed, as shown in Figure 2. If not, a secondary transection can be performed until eupneic iDIAm activity disappears. Second, on Day 3 post-C2SH, the continued absence of eupneic iDIAm EMG must be verified while animals are ane...

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.

Following cSCI, activation of the DIAm can be impacted depending on the extent of the injury. The present manuscript describes a unilateral C2 hemisection ...

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

Abstract