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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
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			<td>25 G Needle</td>
			<td>Cardinal Health</td>
			<td>1188825100</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>
		</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&#34; (228 mm); Basic unit is constructed of .5&#34; (123 mm) jeweled acrylic.</td>
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			Yes, Feature: Tungsten Carbide</td>
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		<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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		<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.&#39;s MP series Data Acquisition System</td>
		</tr>
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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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		<tr>
			<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>
		</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>
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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

Our research focuses on neuromotor control of the diaphragm muscle during breathing. Phrenic motor neurons innervating diaphragm muscle fibers receive descending excitatory input from the brainstem, which is primarily ipsilateral and therefore disrupted by upper cervical spinal cord hemisection or C2SH. Following C2SH, there is spontaneous recovery of ipsilateral diaphragm activity reflecting neuroplasticity.A major development is demonstrating a role of brain-derived neurotropic factor or BDNF signaling through its high affinity TrkB receptor in the recovery of diaphragm activity following C2SH. A second development is the use of machine learning approaches to facilitate high throughput analyses of diaphragm activity during numerous respiratory cycles. The lack of reliable, unbiased high throughput techniques to evaluate basic elements of diaphragm neuromotor control is a challenge in defining recovery of function.This becomes especially important when you're dealing with recordings from animals that are not anesthetized. Our cervical spinal hemisection intentionally leaves the ipsilateral dorsal funiculus intact, minimizing limb muscle deficits, but still causing loss of diaphragm activity. This protocol emphasizes validation of the loss of diaphragm activity at the time of surgery, thereby establishing a clear starting point for recovery of diaphragm muscle function.Further exploring the role of BDNF TrkB signaling in neuroplasticity and recovery of diaphragm neuromotor control after cervical spinal cord injury.

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-eupneic-diaphragm-activity-following

Research

JoVE Journal

Neuroscience

219 ビュー.  Mayo Clinic. 私たちの研究は、呼吸中の横隔膜筋の神経運動制御に焦点を当てています。横隔膜筋線維を神経支配する横隔膜運動ニューロンは、脳幹から下行興奮性入力を受け取りますが、これは主に同側であるため、上部頸髄半解剖またはC2SHによって破壊されます。C2SHに続いて、神経可塑性を反映した同側の横隔膜活動の自発的な回復があります。?...

ビデオ: 著者スポットライト:ラットの頸椎半球切除後の横隔膜機能の神経運動制御と回復

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...

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cervical spinal hemisection and diaphragm muscle electromyographic (emg) monitoring in rats

Cervical Spinal Hemisection and Diaphragm Muscle Electromyographic (EMG) Monitoring in Rats

spinal cord lateral hemisection and asymmetric behavioral assessments in adult rats

Spinal Cord Lateral Hemisection and Asymmetric Behavioral Assessments in Adult Rats

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...

a minimally invasive, fast spinal cord lateral hemisection technique for modeling open spinal cord injuries in rats

A Minimally Invasive, Fast Spinal Cord Lateral Hemisection Technique for Modeling Open Spinal Cord Injuries in Rats

Here, we describe a new, fast technique modeling open spinal cord injury in rats that eliminates laminectomy. Lateral hemisection is performed while viewing through a microscope. The technique is versatile and can also be used in the cervical, thoracic, and lumbar regions of the spinal cord of other...

implantation of optoelectronic devices in the rodent spinal cord

Author Spotlight: Innovative Methodology for Implanting and Securing Neural Probes in the Rodent Spinal Cord

This protocol details a surgical procedure for performing spinal cord surgery and for implanting and securing an optical shank over the spinal cord in...

Spinal Cord Surgery and Optical Shank Implantation in Rodents

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thoracic spinal cord hemisection surgery and open-field locomotor assessment in the rat

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

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...

cervical spinal cord exposure and epidural placement of an optoelectronic device in long evans rats

Cervical Spinal Cord Exposure and Epidural Placement of an Optoelectronic Device in Long Evans Rats

measuring diaphragm thickness and function using point-of-care ultrasound

Author Spotlight: Unraveling the Impact of Mechanical Ventilation on Diaphragm Function and Patient Outcomes 

Diaphragm thickness and function can be assessed in healthy individuals and critically ill patients using point-of-care ultrasound. This technique offers an accurate, reproducible, feasible, and well-tolerated method for evaluating diaphragm structure and function.

Ultrasound in Evaluating Diaphragm Dysfunction and Monitoring Treatment

https://cloudfront.jove.com/CDNSource/teasers/65431_b.jpg

assessing rat diaphragm motor unit connectivity outcome measures as quantitative biomarkers of phrenic motor neuron degeneration and compensation

Author Spotlight: Studying Neuromuscular Responses and Motor Neuron Plasticity in Neurodegenerative Diseases

In this study, we present an in vivo method for estimating motor unit number and size to quantify rat diaphragm motor unit connectivity. A step-by-step approach to these techniques is described.

Quantifying Phrenic Motor Unit Function in Rodent Models of Neurodegeneration

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enhancing the development and growth of infant cerebral palsy rats using selective spinal manipulations

Author Spotlight: Exploring the Efficacy of Selective Spinal Manipulation in Pediatric Cerebral Palsy

This study illustrates the effect of selective spinal manipulation on the growth and development of infant rats with cerebral palsy, emphasizing the specific procedure and standardized protocol. Body weight measurement, Rotarod test, Foot-fault score, other behavioral tests, and growth hormone detection were performed to evaluate the protocol.

Developmental Enhancement in Cerebral Palsy Rats via Spinal Manipulations

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assessment of cardiac function and myocardial morphology using small animal look-locker inversion recovery (salli) mri in rats

Assessment of Cardiac Function and Myocardial Morphology Using Small Animal Look-locker Inversion Recovery SALLI MRI in Rats

Contrast enhanced cardiac magnetic resonance (CMR) imaging allows comprehensive in vivo assessment of the heart in small animal models of cardiovascular disease. Here we detail the procedures to perform CMR imaging, reconstruction and analysis using Small Animal Lock-Locker Inversion Recovery (SALLI) in...

Assessment of Cardiac Function and Myocardial Morphology Using Small Animal Look-locker Inversion Recovery (SALLI) MRI in Rats

diagnostic ultrasound imaging of mouse diaphragm function

Diagnostic Ultrasound Imaging of Mouse Diaphragm Function

Diagnostic ultrasound imaging has proven to be effective in diagnosing various respiratory diseases in human and animal subjects. We demonstrate a comprehensive ultrasound protocol utilized by Dr. Zuo's lab to analyze diaphragm kinetics specifically in mouse models. This is also a non-invasive research technique which can provide quantitative information on mouse respiratory muscle...

diffusion imaging in the rat cervical spinal cord

Diffusion Imaging in the Rat Cervical Spinal Cord

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...

Measuring Diaphragm Thickness and Function Using Point-of-Care Ultrasound

a murine model of cervical spinal cord injury to study post-lesional respiratory neuroplasticity

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

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...

a radio-telemetric system to monitor cardiovascular function in rats with spinal cord transection and embryonic neural stem cell grafts

A Radio-telemetric System to Monitor Cardiovascular Function in Rats with Spinal Cord Transection and Embryonic Neural Stem Cell Grafts

We present a protocol for using a radio-telemetric system to record cardiovascular parameters in T4 spinal cord transected rats eight weeks after embryonic brainstem neural stem cell grafting into the lesion site. Telemetry is an advanced technique to accurately evaluate cardiovascular function in conscious freely moving spinal cord injured...

a contusion model of severe spinal cord injury in rats

A Contusion Model of Severe Spinal Cord Injury in Rats

A contusion model of severe spinal cord injury is described. Detailed pre-operative, operative and post-operative steps are described to obtain a consistent...

knockdown of fam83a to verify its role in cervical cancer cell growth and cisplatin sensitivity

Author Spotlight: Exploring the Role of FAM83A in Cervical Cancer 

Here, we show the procedures for FAM83A knockdown; the assays to detect its effects on proliferation, migration, and invasion of cervical cancer cells; and the sensitization of these cells to cisplatin. This study provides a promising target gene for cervical cancer and a reference for further drug...

Validation of &lt;em&gt;FAM83A&lt;/em&gt; as a Target Gene in Cervical Cancer

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synergetic use of neural precursor cells and self-assembling peptides in experimental cervical spinal cord injury

Synergetic Use of Neural Precursor Cells and Self-assembling Peptides in Experimental Cervical Spinal Cord Injury

Treating cervical spinal cord injury with both self-assembling peptides (SAP) and neural precursor cells (NPC), together with growth factors, is a promising approach to promote regeneration and recovery. A contusion/compression aneurysm clip rat model of cervical SCI and combined treatment involving SAP injection and NPC transplantation is established.

To begin, arrange the sterile surgical equipment on a surgical platform. Approximately 72 hours after the diaphragm muscle electromyographic, or EMG, electrode implantation, weigh the rat. Prior to anesthetizing the rat, place the rat in a Bowman-style cage.Carefully connect the wires, exiting the rat dorsum to a preamplifier to record bilateral diaphragm muscle EMG. Next, place the anesthetized rat in a prone position. Shave the hair from the neck at about the ear level, and down to the scapulae.Connect the exposed electrode wires from the rat's back to the amplifier. Disinfect the skin alternating with 4%chlorhexidine gluconate and isopropyl alcohol three times. Cover the rat with sterile surgical covers, except for the upper dorsum.Using a scalpel, make a four centimeter rostrocaudal incision. Retract the skin and cut the The acromiotrapezius muscle. Then dissect the rhomboid muscle to expose the spinalis muscles.Under a surgical microscope, remove the spinalis from C1 to C3.Then use rongeur to carefully perform a laminectomy at C2 without damaging major arteries or nerves. Cut and remove the dura mater at C2.While monitoring diaphragm muscle EMG, insert the angle dissecting knife just below the point where the dorsal root enters the spinal cord, and section all the way to the midpoint of the ventral surface. Then record the eupneic ipsilateral diaphragm EMG activity on the anesthetized rat with C2 spinal hemisection performed.Suture the spinal cord muscles with 3-0 sutures, and then close the skin incision. To maintain hydration, subcutaneously inject one milliliter of saline per 50 grams of animal mass. Place the rat in a clean cage with a heating pad for recovery.Set the high band-pass filter of the preamplifier to 100 hertz, and the low-pass filter to 1000 hertz. After collecting one to two minutes of left and right hemidiaphragm EMG eupneic recording, save the date in an appropriate format for further analyses. A successful example of C2 spinal hemisection showed the absence of eupneic ipsilateral diaphragm EMG activity under anesthesia on day three post-injury.Diaphragm muscle EMG activity under awake conditions showed a reduction in eupneic ipsilateral diaphragm compared to the pre-injury baseline. The contralateral diaphragm muscle EMG activity increased in both anesthetized and awake conditions.