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.

<table><tbody><tr><td>25 G Needle</td><td>Cardinal Health</td><td>1188825100</td><td>Covidien Monoject Hypdermic Standard Needles: 25 G x 1&quot; (0.508 mm x 2.5 cm) A</td></tr><tr><td>3-0 Vicryl Violet Braided</td><td>Ethicon</td><td>J774D</td><td>3-0 Suture</td></tr><tr><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></tr><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&quot; (228 mm); Basic unit is constructed of .5&quot; (123 mm) jeweled acrylic.</td></tr><tr><td>Castroviejo Needle Holder</td><td>Fine Science Tools</td><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:&lt;br/&gt; 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></tr><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></tr><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></tr><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&amp;deg;</td></tr><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></tr><tr><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.&#039;s MP series Data Acquisition System</td></tr><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></tr><tr><td>Knife Curette</td><td>V. Mueller</td><td>VM101-4414</td><td>Tip: Sharp, Tip Diameter: 2 mm</td></tr><tr><td>Micro Dissecting Scissors</td><td>Biomedical Research Instruments, Inc.</td><td>11-2420</td><td>Length: 4&quot;, Angle: Straight, Blade Length: 23 mm</td></tr><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></tr><tr><td>PowerLab 8/35</td><td>ADInstruments</td><td>PL3508</td><td>Data acquisition system</td></tr><tr><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></tr><tr><td>Scalpel Handle #3</td><td>Fine Science Tools</td><td>10003-12</td><td>Alloy / Material: Stainless Steel, Length: 12 cm</td></tr><tr><td>Sprague Dawley Rat</td><td>Inotiv</td><td>Order code: 002</td><td>Sprague Dawley outbred rats (female and male)</td></tr><tr><td>Surgical Microscope</td><td>Olympus</td><td>SZ61</td><td>Surgical microscope&amp;nbsp;</td></tr><tr><td>Suture Cutting Scissors</td><td>George Tiemann &amp;amp; Co.</td><td>110-1250SB</td><td>Alloy / Material: Stainless Steel, Tip Shape: Straight, Tips: Sharp/Blunt, Length: 4.5&quot;</td></tr><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></tr><tr><td>Weitlaner Retractor</td><td>Codman</td><td>50-5647</td><td>Prongs: 2 x 3 Blunt, Length: 4.5&quot;</td></tr></tbody></table>

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.

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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/files/ftp_upload/66828/66828fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

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.

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 ...

assessing-functional-recovery-eupneic-diaphragm-activity-following

Nanyang Technological University

Research

JoVE Journal

Neuroscience

340 Views.  Mayo Clinic. Ons onderzoek richt zich op de neuromotorische controle van de middenrifspier tijdens de ademhaling. Phrenische motorneuronen die middenrifspiervezels innerveren, ontvangen dalende prikkelende input van de hersenstam, die voornamelijk ipsilateraal is en daarom wordt verstoord door hemisectie van het bovenste cervicale ruggenmerg of C2SH. Na C2SH is er spontaan herstel van de ipsilaterale diafragma-activiteit die neuroplasticiteit weerspiegelt.Een belan...

Video: Auteur in de schijnwerpers: Neuromotorische controle en herstel van de middenriffunctie na cervicale spinale hemisectie bij ratten

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

A Contusive Model of Unilateral Cervical Spinal Cord Injury Using the Infinite Horizon Impactor

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of spinal cord injury (SCI) in which the thoracic spinal cord is injured. Additionally, because most human cord injuries occur as the result of blunt, non-penetrating trauma (e.g. motor vehicle accident, sporting injury) where the spinal cord is violently struck by displaced bone or soft tissues, the majority of SCI researchers are of the opinion that the most clinically relevant injury models are those in which the spinal cord is rapidly contused.1 Therefore, an important step in the preclinical evaluation of novel treatments on their way to human translation is an assessment of their efficacy in a model of contusion SCI within the cervical spinal cord. Here, we describe the technical aspects and resultant anatomical and behavioral outcomes of an unilateral contusive model of cervical SCI that employs the Infinite Horizon spinal cord injury impactor.
Sprague Dawley rats underwent a left-sided unilateral laminectomy at C5. To optimize the reproducibility of the biomechanical, functional, and histological outcomes of the injury model, we contused the spinal cords using an impact force of 150 kdyn, an impact trajectory of 22.5&deg; (animals rotated at 22.5&deg;), and an impact location off of midline of 1.4 mm. Functional recovery was assessed using the cylinder rearing test, horizontal ladder test, grooming test and modified Montoya's staircase test for up to 6 weeks, after which the spinal cords were evaluated histologically for white and grey matter sparing.
The injury model presented here imparts consistent and reproducible biomechanical forces to the spinal cord, an important feature of any experimental SCI model. This results in discrete histological damage to the lateral half of the spinal cord which is largely contained to the ipsilateral side of injury. The injury is well tolerated by the animals, but does result in functional deficits of the forelimb that are significant and sustained in the weeks following injury. The cervical unilateral injury model presented here may be a resource to researchers who wish to evaluate potentially promising therapies prior to human translation.

A reliable and repeatable way to produce a cervical unilateral spinal cord injury using the Infinite Horizon impactor is described. The method takes advantage of a custom designed frame and clamp to stabilize the spine. The standardized procedure and biomechanical injury parameters result in sufficient and sustained injuries.

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of ...

Medicine

A Murine Model of Cervical Spinal Cord Injury to Study Post-lesional Respiratory Neuroplasticity

A cervical spinal cord injury induces permanent paralysis, and often leads to respiratory distress. To date, no efficient therapeutics have been developed to improve/ameliorate the respiratory failure following high cervical spinal cord injury (SCI). Here we propose a murine pre-clinical model of high SCI at the cervical 2 (C2) metameric level to study diverse post-lesional respiratory neuroplasticity. The technique consists of a surgical partial injury at the C2 level, which will induce a hemiparalysis of the diaphragm due to a deafferentation of the phrenic motoneurons from the respiratory centers located in the brainstem. The contralateral side of the injury remains intact and allows the animal recovery. Unlike other SCIs which affect the locomotor function (at the thoracic and lumbar level), the respiratory function does not require animal motivation and the quantification of the deficit/recovery can be easily performed (diaphragm and phrenic nerve recordings, whole body ventilation). This pre-clinical C2 SCI model is a powerful, useful, and reliable pre-clinical model to study various respiratory and non-respiratory neuroplasticity events at different levels (molecular to physiology) and to test diverse putative therapeutic strategies which might improve the respiration in SCI patients.

Respiratory failure is the leading cause of death following a cervical spinal cord injury. Having a reproducible, quantifiable, and reliable pre-clinical animal model of respiratory failure induced by a partial cervical injury will help to understand the subsequent respiratory and non-respiratory neuroplasticity and allow testing putative repair strategies.

A cervical spinal cord injury induces permanent paralysis, and often leads to respiratory distress. To date, no efficient therapeutics have been developed ...

Spinal Cord Transection in the Larval Zebrafish

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability to reinitiate spinal neurogenesis. However, some anamniotes including the zebrafish Danio rerio exhibit both sensory and functional recovery even after complete transection of the spinal cord. The adult zebrafish is an established model organism for studying regeneration following spinal cord injury, with sensory and motor recovery by 6&#160;weeks post-injury. To take advantage of in vivo analysis of the regenerative process available in the transparent larval zebrafish as well as genetic tools not accessible in the adult, we use the larval zebrafish to study regeneration after spinal cord transection. Here we demonstrate a method for reproducibly and verifiably transecting the larval spinal cord. After transection, our data shows sensory recovery beginning at 2&#160;days post-injury (dpi), with the C-bend movement detectable by 3 dpi and resumption of free swimming by 5 dpi. Thus we propose the larval zebrafish as a companion tool to the adult zebrafish for the study of recovery after spinal cord injury.

After spinal transection, adult zebrafish have functional recovery by six weeks post-injury. To take advantage of larval transparency and faster recovery, we present a method for transecting the larval spinal cord. After transection, we observe sensory recovery beginning at 2&#160;days post-injury, and C-bend movement by 3 days post-injury.

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

Diffusion Imaging in the Rat Cervical Spinal Cord

Magnetic resonance imaging (MRI) is the state of the art approach for assessing the status of the spinal cord noninvasively, and can be used as a diagnostic and prognostic tool in cases of disease or injury. Diffusion weighted imaging (DWI), is sensitive to the thermal motion of water molecules and allows for inferences of tissue microstructure. This report describes a protocol to acquire and analyze DWI of the rat cervical spinal cord on a small-bore animal system. It demonstrates an imaging setup for the live anesthetized animal and recommends a DWI acquisition protocol for high-quality imaging, which includes stabilization of the cord and control of respiratory motion. Measurements with diffusion weighting along different directions and magnitudes (b-values) are used. Finally, several mathematical models of the resulting signal are used to derive maps of the diffusion processes within the spinal cord tissue that provide insight into the normal cord and can be used to monitor injury or disease processes noninvasively.

The goal of this protocol is to obtain high-quality diffusion weighted magnetic resonance imaging (DWI) of the rat spinal cord for noninvasive characterization of tissue microstructure. This protocol describes optimizations of the MRI sequence, radiofrequency coil, and analysis methods to enable DWI images free from artifacts.

Magnetic resonance imaging (MRI) is the state of the art approach for assessing the status of the spinal cord noninvasively, and can be used as a ...

Functional and Morphological Assessment of Diaphragm Innervation by Phrenic Motor Neurons

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.

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.

This protocol specifically focuses on tools for assessing phrenic motor neuron (PhMN) innervation of the diaphragm at both the electrophysiological and ...

Spinal Cord Lateral Hemisection and Asymmetric Behavioral Assessments in Adult Rats

Incomplete spinal cord injury (SCI) often leads to impairments of sensorimotor functions and is clinically the most frequent type of SCI. Human Brown-S&#233;quard syndrome&#160;is a common type of incomplete SCI caused by a lesion to one half of the spinal cord which results in paralysis and loss of&#160;proprioception&#160;on the same (or ipsilesional) side as the injury, and loss of pain and temperature sensation on the opposite (or contralesional) side. Adequate methodologies for producing a spinal cord lateral hemisection (HX) and assessing neurological impairments are essential to establish a reliable animal model of Brown-S&#233;quard syndrome. Although lateral hemisection model plays a pivotal role in basic and translational research, standardized protocols for creating such a hemisection and assessing unilateralized function are lacking. The goal of this study is to describe step-by-step procedures to produce a rat spinal lateral HX at the 9th thoracic (T9) vertebral level. We, then, describe a combined behavior scale for HX (CBS-HX) that provides a simple and sensitive assessment of asymmetric neurological performance for unilateral SCI. The CBS-HX, ranging from 0 to 18, is composed of 4 individual assessments which include unilateral hindlimb stepping (UHS), coupling, contact placing, and grid walking. For CBS-HX, the ipsilateral and contralateral hindlimbs are assessed separately. We found that, after a T9 HX, the ipsilateral hindlimb showed impaired behavior function whereas the contralateral hindlimb showed substantial recovery. The CBS-HX effectively discriminated behavioral functions between ipsilateral and contralateral hindlimbs and detected temporal progression of recovery of the ipsilateral hindlimb. The CBS-HX components can be analyzed separately or in combination with other measures when needed. Although we only provided visual descriptions of the surgical procedures and behavioral assessments of a thoracic HX, the principle may be applied to other incomplete SCIs and at other levels of the injury.

Here we describe surgical procedures to produce a reliable spinal cord lateral hemisection (HX) at the 9th thoracic level in adult rats and neurobehavioral assessments designed for detecting asymmetric deficits after such a unilateral injury.

Incomplete spinal cord injury (SCI) often leads to impairments of sensorimotor functions and is clinically the most frequent type of SCI. Human ...

Thoracic Spinal Cord Hemisection Surgery and Open-Field Locomotor Assessment in the Rat

Spinal cord injury (SCI) causes disturbances in motor, sensory, and autonomic function below the level of the lesion. Experimental animal models are valuable tools to understand the neural mechanisms involved in locomotor recovery after SCI and to design therapies for clinical populations. There are several experimental SCI models including contusion, compression, and transection injuries that are used in a wide variety of species. A hemisection involves the unilateral transection of the spinal cord and disrupts all ascending and descending tracts on one side only. Spinal hemisection produces a highly selective and reproducible injury in comparison to contusion or compression techniques that is useful for investigating neural plasticity in spared and damaged pathways associated with functional recovery. We present a detailed step-by-step protocol for performing a thoracic hemisection at the T8 vertebral level in the rat that results in an initial paralysis of the hindlimb on the side of the lesion with graded spontaneous recovery of locomotor function over several weeks. We also provide a locomotor scoring protocol to assess functional recovery in the open-field. The locomotor assessment provides a linear recovery profile and can be performed both early and repeatedly after injury in order to accurately screen animals for appropriate time points in which to conduct more specialized behavioral testing. The hemisection technique presented can be readily adapted to other transection models and species, and the locomotor assessment can be used in a variety of SCI and other injury models to score locomotor function.

The rat thoracic spinal hemisection is a valuable and reproducible model of unilateral spinal cord injury to investigate the neural mechanisms of locomotor recovery and treatment efficacy. This article includes a detailed step-by-step guide to perform the hemisection procedure and to assess locomotor performance in an open-field arena.

Spinal cord injury (SCI) causes disturbances in motor, sensory, and autonomic function below the level of the lesion. Experimental animal models are ...

Performing Thoracic Spinal Cord Hemisection Surgery in Rats

After confirming a lack of response to toe pinch in an anesthetized adult rat, make a 2.5-centimeter incision in the shaved skin overlying the T6 to T10 vertebrae, and use blunt scissors to retract the skin and superficial fat. Use blunt dissection scissors and a self-retaining retractor to separate the paravertebral muscles, inserting on the dorsal aspect of the T7 to T9 vertebrae, and use fine forceps and cotton-tipped applicators to debride and clear any remaining tissue to expose the spinous processes and vertebral lamina.
Place the rat under a stereomicroscope, and use delicate bone trimmers to carefully cut the facets bilaterally on the T7 and T8 vertebrae. Use a scalpel to make a 1 millimeter superficial cut in the dorsal connective tissue between the T8 and T9 vertebral laminae, taking care not to injure the underlying cord, and use bone trimmers to remove the spinous process of the T8 vertebra.
With curved hemostatic forceps, carefully clamped on the T7 spinous process, rotate the caudal end of the T8 laminae slightly rostrally approximately 20 degrees, and insert the bone trimmers under the T8 lamina. Make a midline cut extending along the lamina, continuing the laminectomy by repeating the cuts on the left and right side of the vertebral lamina, medial to the transverse processes to expose the spinal cord.
Drip 100 microliters of 2% lidocaine into the exposed spinal canal, and use fine forceps and iridectomy scissors to remove the dura, overlaying the T8 spinal segment. Repeat the lidocaine administration to the exposed cord and identified the midline of the cord by visualization of a center line created between the spinous processes extending between the exposed T7 to T9 vertebra.
Using fine forceps to stabilize the spinal cord, use a dissecting knife to hemisect the spinal cord from the midline toward one side of the animal, taking care not to cut through the anterior spinal artery on the ventral side. Using iridectomy scissors, carefully cut through any remaining tissue on the lesion side of the spinal cord to ensure the ventrolateral quadrant is appropriately transected, and place a sterile, approximately 6 by 2-millimeter saline soaked hemostatic sponge into the exposed cavity above the spinal cord.
Then use 4-0 polyglactin 910 sutures to close muscle layers and the skin around the incision site and place the rat in a warm environment under a heat lamp with monitoring until full recovery.

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

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