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

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

The authors have no conflicts of interest to declare.

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 neurons. Second, by monitoring DIAm EMG, we can validate the efficacy of the C2 lesion in eliminating eupneic iDIAm EMG activity initially during surgery while the animals are anesthetized. At Day 3 post-injury, while the animals are anesthetized, we then verify that there is indeed continued silencing of eupneic iDIAm EMG activity. It was previously shown that rVRG excitatory inputs on phrenic motor neurons and NMDA receptor expression in phrenic motor neurons are reduced at 3 to 7 days post-injury and that both glutamatergic synaptic input and NMDA receptor expression increase over time after 7 days post-injury33,42. These data, combined with histological confirmation of the C2 lesion10,13,29,30,33, suggest that continued inactivity under anesthesia on Day 3 post-injury provides information about the efficacy of the initial C2SH. Across studies conducted in multiple years, relatively stable rates of spontaneous recovery under anesthesia between 30%-40% were seen at 14 days post-C2SH18,19,20,29,31,43, suggesting that this method of verifying the success of the C2SH is reproducible and reliable.
The C2SH procedure involves several critical steps. Importantly, the C2SH model proposed here spares the dorsal funiculi and thus does not lead to limb motor deficits. In full C2 spinal hemisection models involving the dorsal funiculi, limb motor deficits are considerably greater3,4,5,6,7,37. Thus, an added benefit of the C2SH model (Figure 2A) is that in addition to the silencing of eupneic iDIAm EMG activity in anesthetized C2 lesioned rats, the rats are functionally similar to sham laminectomy rats in terms of ambulation and other functions. Accordingly, they are generally able to feed and groom themselves, which reduces the caretaking burden while still allowing studies of DIAm neuromotor control and respiratory neuroplasticity. To ensure that the C2 model is implemented appropriately, great care must be taken to avoid excessive lesioning. Inserting the dissecting knife right below where the dorsal root enters the spinal cord and cutting the ventral portion sparingly are both helpful rules of thumb. The goal is to ensure that eupneic iDIAm EMG activity ceases; this can be done with multiple small cuts if needed. Indeed, in the week following the C2SH, it will become clear if there was excessive damage to the spinal cord if the animals have difficulty ambulating and reaching for food pellets.
Electrode placement and EMG recordings 
Chronic DIAm EMG electrodes have several clear benefits over other approaches. Electrodes can (and should) be implanted several days before the C2SH procedure, allowing sufficient time for recovery and not requiring a laparotomy and a C2SH during the same surgical session. This is important because it is well-accepted that laparotomy causes inhibition of the DIAm35,36. By implanting chronic DIAm electrodes, there is also no need for repeated laparotomies, which were performed in earlier work10,13; there is also a reduced risk of pneumothorax as electrodes are not being inserted into the DIAm during each session. However, several potentially serious adverse events can occur as a result of electrode placement. Although the risk of pneumothorax is reduced by avoiding repeated electrode insertions, it is not completely nullified, and indeed, pneumothorax can occur, causing either immediate death or prolonged problems. In order to reduce this risk, it is best practice to ensure that the needle that is being threaded through the DIAm does not perforate the superior surface of the DIAm. Additionally, avoid using sharp forceps that may inflict damage to the DIAm while manipulating the electrode wires. Occasionally, the rats may chew the stitches at their abdomen, potentially disemboweling themselves if left to their own devices. Rats should be observed at regular intervals after electrode placement to detect these types of behaviors early. In some cases, it may be possible to anesthetize the rats to repair damage to stitches. However, it is best if such behavior is mitigated by providing adequate pain relief during and after the surgery and by ensuring that the sterile field is not broken during surgery. In extreme cases, it may be necessary to euthanize rats that repeatedly remove their stitches.
The recording and analysis of the EMG signals is not the primary focus of the present study and is highly dependent on the particular equipment and software available in each lab. Although equipment information is provided in the Table of Materials, a wide variety of hardware options exist for amplifying and recording EMG activity, and the specifics will depend on a mixture of features, availability, and affordability for each lab. However, there are some general principles of recording EMG that are important to mention. One potential issue is the movement or destruction of implanted electrodes, which can limit quantitative assessments of DIAm EMG. If the electrodes have dislodged from the DIAm, or if the rats have managed to consume or otherwise damage the externalized wires, it may not be possible to record EMG activity, or the noise level may change. This can be avoided by confirming that the electrodes are firmly secured in the DIAm, and a signal with a high signal-to-noise ratio (SNR) can be recorded from them during the DIAm electrode placement surgery. In addition, externalizing the electrode wires high on the dorsum and cutting off excess wire such that the rats are unable to access the electrode wires are both conducive to the success of chronic electrodes. As an alternative to externalized wires, head caps37 or telemetry44,45 may be reasonable options. Both approaches can obtain recordings in awake animals more easily but may be slightly more difficult to implement than simply externalizing the multistranded wires. All nearby electronic sources can potentially be sources of noise. It is paramount that all equipment is grounded appropriately, shielded cables are used, and the rats are not touched while active recordings are taking place. In addition to turning off electronic heat pads during recording, lights, nearby equipment, electrically operated surgical tables, and other such devices may need to be temporarily turned off to achieve low-noise recordings. To minimize environmental influences, electronic equipment that does not need to be on for the DIAm EMG recordings should be turned off. When possible, recordings should be made in an electrically shielded room. Over time, tissue scarring and fibrosis around the DIAm EMG electrodes can decrease the conductivity of the electrode, reducing SNR. Additionally, even slight changes in posture can have large impacts on the DIAm EMG signal; thus, to minimize these potentially complicating issues, DIAm EMG should be recorded with the rats in the same posture across recording sessions.
Another important consideration is the filtering and sampling settings. The type of electrode (i.e., intramuscular vs. esophageal/surface) is extremely relevant when it comes to determining the frequency content of&#8212;and consequently, appropriate high- and low-pass filters for&#8212;a signal. Considerable effort has been expended to determine the optimal filters for surface/esophageal DIAm EMG46. Similar studies in the rat DIAm have not been performed, but previously, we showed that effectively, the entirety of the frequency content of the DIAm EMG recorded using chronic intramuscular electrodes in rats was below 1000 Hz, with the centroid frequency around 300 Hz47. In a direct comparison of the mean and median frequencies of the power spectrum of biceps brachialis EMG in humans obtained via both surface and intramuscular electrodes, Christensen et al.48 found that both the mean and median frequencies were approximately 3-fold higher for intramuscular electrodes compared to surface electrodes. In the DIAm, investigators have reported centroid frequencies of around 100 Hz when using bipolar esophageal electrodes49,50,51,52,53,54, which would suggest that the 300 Hz, which was previously determined for intramuscular electrodes, approximately matches the same trend shown by Christensen et al.48. Despite this, multiple published studies utilizing intramuscular DIAm EMG in rodent models have placed their high-pass filters at 300 Hz4,37,55,56 and some have even gone as high as 500 Hz57. It is uncontroversial that such a filtering approach would substantially reduce the amplitude of the DIAm EMG signal by at least one-half. We propose a far less destructive approach: (1) high-pass filtering at 100 Hz because the majority of the ECG power spectrum is below 100 Hz while comparatively little of the DIAm EMG power spectrum is below 100 Hz51,52, and (2) subsequently removing the leftover ECG by waveform matching58. Thus, the appropriate filters for DIAm EMG for most studies may be set between 100 Hz and 1000 Hz, with a sampling frequency of at least 2000 Hz to capture all the relevant features within the data. Detailed analyses of the DIAm EMG can then be performed using the techniques published in previous reports40,41,47,58.
Awake and anesthetized animals 
Notably, some studies have highlighted that C2SH models of cSCI do not lead to complete inactivity of eupneic iDIAm EMG23,37. In one sense, this is not surprising, as it has been noted previously that &#34;breakthrough&#34; activity occurs during behaviors necessitating higher drive (e.g., deep breaths and the response to airway occlusion)4,7,29,42. These data suggest that it is probably not appropriate to think of the C2SH model as a true model of continuous inactivity. Indeed, it seems that drive is sufficiently high in awake animals for eupneic iDIAm EMG activity to be present as early as four days after a complete C2SH37, although it is not present consistently at one day post-injury23. In all cases, anesthesia suppresses iDIAm activity after upper cervical spinal hemisection, as highlighted in previous reports23,38 and shown in Figure 4 and Figure 5. There are no published data available on DIAm EMG activity in awake rats with the C2SH with spared dorsal funiculi proposed in the present manuscript (Figure 2A). Future work should provide a detailed characterization of the time course of DIAm EMG activity in awake animals with this C2SH model. That said, it is still prudent to perform validation of the continued absence of eupneic iDIAm inactivity on Day 3 post-injury under anesthesia to mimic the experimental conditions during which the spinal cord was initially lesioned/transected. When eupneic iDIAm EMG activity in awake animals at Day 3 post-injury may be present, it is noted that even half-doses of anesthetics will usually lead to a complete cessation of eupneic iDIAm EMG activity. With these doses, the animals are calm and sedate. In addition to verifying the silencing of eupneic iDIAm EMG activity during C2SH and at Day 3 post-injury, it is recommended that the spinal cord should be extracted after the terminal experiment to perform histological confirmation of the site and extent of the C2SH9,29,59. Histological confirmation of the injury&#8212;when combined with functional confirmation of silenced eupneic iDIAm EMG activity&#8212;provides strong evidence of a successful C2SH.
The present article presents a C2SH of cSCI in rats that leads to the immediate silencing of eupneic iDIAm EMG activity with continued silencing under anesthesia at Day 3 post-injury. Due to the sparing of the dorsal funiculus in the C2SH model proposed in the present manuscript, limb motor function is preserved, thereby avoiding off-target effects. This allows longitudinal studies of the effects of a high-cervical lesion on DIAm neuromotor control to be performed in rats that are otherwise relatively healthy, thus adding minimal caretaking burden for investigators. The validation of inactivity under anesthesia at Day 3 post-injury ensures that a clear baseline is established for assessing the subsequent extent of recovery of eupneic iDIAm EMG. This approach provides a rigorous, reliable, and reproducible method to perform a C2SH in rats. This model has the potential to greatly improve the understanding of the time course of respiratory neuroplasticity and its intersection with potential therapeutic strategies.

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>
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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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			<td>Bowman Style Cage</td>
			<td>Braintree Scientific</td>
			<td>POR-530</td>
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			<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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			<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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			<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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			<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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			<td>Dumont #3 Forceps</td>
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		<tr>
			<td>Electromyogram Amplifier</td>
			<td>Biopac Systems, Inc</td>
			<td>EMG100C</td>
			<td>EMG amplifier</td>
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			<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>
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			<td>Alloy / Material: Stainless Steel, Length: 14cm, Joint Action: Single, Cup Size: 1mm, Tip Shape: Curved</td>
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			<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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			<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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			<td>Multistranded stainless steel wire</td>
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			<td>Data acquisition system</td>
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			<td>Scalpel Blade #11</td>
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			<td>Scalpel Handle #3</td>
			<td>Fine Science Tools</td>
			<td>10003-12</td>
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			<td>Sprague Dawley Rat</td>
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			<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>
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			<td>Vannas Spring Scissors</td>
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			<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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			<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 anesthetized. Figure 4A shows an example of a successful C2SH as determined by the continued absence of eupneic iDIAm EMG activity under anesthesia on Day 3 post-injury. Figure 4B shows DIAm EMG activity in the same animal under unanesthetized awake conditions, highlighting the reduction&#8212;but not absence&#8212;of iDIAm activity compared to the pre-injury baseline. Notably, the cDIAm EMG activity is increased in both awake and anesthetized conditions. The anesthetized recordings on Day 3 provide validation that the initial C2SH was successful, but quantitative analyses should be performed in awake animals. In awake animals, DIAm EMG activity at Day 3 post-C2SH represents the starting point for recovery. Figure 4B shows that iDIAm EMG activity is reduced, but not absent, on Day 3 in awake rats.
In some cases, C2SH is not accompanied by total cessation of eupneic iDIAm EMG activity (Figure 5A). An inadequate C2SH occurs in fewer than 5% of all cases using the approach outlined in the present manuscript; however, it is essential that an evaluation of DIAm EMG activity at Day 3 post-C2SH be performed on each rat to validate the success of the C2SH. Figure 5A shows an example of DIAm EMG activity from a rat on Day 3 post-injury, in which the C2SH was inadequate in eliminating eupneic iDIAm EMG activity by Day 3. It is important to note that validation of the efficacy of the C2SH was performed while the animal was anesthetized. Continued eupneic EMG activity suggests that at least a portion of the descending ipsilateral bulbospinal pathway was spared, which can complicate the interpretation of the results. Figure 5B shows that iDIAm EMG activity is reduced, but not absent, at Day 3 in awake rats. However, the sparing of ipsilateral descending inputs may lessen the initial deficit.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66828/66828fig01.jpg" /> 
Figure 1: Conceptual framework. The rostral ventral respiratory group (rVRG) sends descending inputs to phrenic motor neurons in the cervical spinal cord (C3-C5). The majority of these descending axons innervate the ipsilateral phrenic motor neuron pool, but a small fraction cross the midline to activate the contralateral motor neuron pool. Immediately after a C2SH, descending inputs from the rVRG to the ipsilateral phrenic motor neuron pool are disrupted. <a href="https://www.jove.com/files/ftp_upload/66828/66828fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66828/66828fig02.jpg" /> 
Figure 2: Representative C2SH and DIAm EMG activity. (A) The transverse view of the rat spinal cord and tran site (shaded red) with important structures labeled is shown. The spinal lesion is performed by inserting a dissecting knife right below the point where the dorsal root enters and cutting through to approximately the midline (shaded translucent red area). Note the severing of major motor tracts in the lateral and ventral funiculi, but the sparing of the dorsal funiculi. (B) The longitudinal view, depicting the silencing of ipsilateral motor neuron activity, complements the raw EMG traces used for real-time confirmation of a satisfactory lesion (C). As the spinal cord is lesioned, eupneic iDIAm EMG activity ceases, suggesting ipsilateral inputs to phrenic motor neurons have been severed; meanwhile, the cDIAm EMG immediately increases its activity as shown. <a href="https://www.jove.com/files/ftp_upload/66828/66828fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66828/66828fig03.jpg" /> 
Figure 3: Electrode manufacturing. The process for manufacturing DIAm EMG electrodes is shown. The basic process involved measuring and cutting an appropriate length of wire (Step 1), tying an anchoring knot and stripping way insulation to expose the electrode (Step 2), attaching a 25 gauge needle to the wire (Steps 3 and 4), bending the needle to give it a curvature similar to a surgical suture needle (Step 5), and stripping away insulation to expose a portion of the wire that will be connected to the amplifier for recording EMG (Step 6). <a href="https://www.jove.com/files/ftp_upload/66828/66828fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66828/66828fig04.jpg" /> 
Figure 4: Successful spinal hemisection. (A) DIAm EMG activity is shown in an anesthetized rat before C2SH and at Day 3 post-injury. There is no evidence of eupneic iDIAm EMG activity in this rat at day 3 post-injury, indicating that the initial C2SH was successful. Note the compensatory increase in cDIAm EMG activity and increase in respiratory rate compared to the pre-injury condition. (B) DIAm EMG activity is shown in the same rat under unanesthetized awake conditions on Day 3 post-C2SH. There is clear evidence of reduced-albeit not completely silenced-iDIAm EMG activity. Note the compensatory increase in cDIAm EMG activity and increase in respiratory rate compared to the pre-injury condition. <a href="https://www.jove.com/files/ftp_upload/66828/66828fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/66828/66828fig05.jpg" /> 
Figure 5: Unsuccessful spinal hemisection. (A) DIAm EMG activity is shown in an anesthetized rat before C2SH and at Day 3 post-injury. In this rat, eupneic iDIAm EMG activity persisted at Day 3 post-injury, indicating that the initial C2SH was inadequate. Accordingly, this rat was excluded from further analyses of functional recovery. Note the compensatory increase in the cDIAm EMG activity despite an unsuccessful C2SH, but minimal increase in respiratory rate. (B) DIAm EMG activity is shown in the same run under unanesthetized awake conditions on Day 3 post-C2SH. There is evidence of reduced iDIAm EMG, increased cDIAm EMG activity, and a slight increase in respiratory rate. <a href="https://www.jove.com/files/ftp_upload/66828/66828fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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-eupneic-diaphragm-activity-following

National Institute of Mental Health and Neurosciences

Research

JoVE Journal

Neuroscience

247 Views.  Mayo Clinic. 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.