This research if funded by the Canadian Institute of Health Research and the Heart and Stroke Foundation of BC and Yukon.&#160;We would like to acknowledge Mr. Rayshad Gopaul and Dr. Shelly McErlane for technical support and expertise in animal care.

Male Wistar (Hsd: WI Wistar) rats at 7 weeks of age and weighing 300-350 g were used in this experiment. All rats were maintained on a 12&#160;hr light/dark cycle and received standard laboratory rat chow and water ad libitum. All experimental procedures conformed with the guide to the Care and Use of Experimental Animals established by the Canadian Council on Animal Care and granted ethics approval by the University of British Columbia. Surgery and animal care were conducted according to standard procedures in our laboratory (Ramsey et al. 2010)17.
1. Preparation of the Animals: Surgical Procedures
<ol>
	<li>Implantation of the Telemetry Device 
	Note: Protocol was implemented according to the manufacturer&#39;s specifications and our previous experience with the implantation of the device: 16
	<ol>
		<li>Pre-treat the animals with enrofloxacin, once a day for 3 days (10 mg/kg, s.c.) pre-operatively.</li>
		<li>Remove solid foods from cage 9-13 hr&#160;before the surgery. Soft diet nutritional meal replacement may be provided during this period.</li>
		<li>Anaesthetize the animals using an induction dose of 4% isoflurane in an induction chamber and a maintenance dose of approximately 1.5% isoflurane (with 1-1.5 L/min Oxygen) using a Bain circuit. Use the toe pinch reflex and corneal reflexes to assess whether animals are at surgical plane of anesthesia. Respiration must be monitored to assess if the depth of anesthesia is too high (i.e., very slow respiration rate and gasping, pale mucus membranes) or too low (i.e., rapid, shallow respirations indicating the animal may not be at a surgical plane of anesthesia). Repeat monitoring of surgical plane of anesthesia approximately every 5 min&#160;during the surgery and adjust isoflurane dose as necessary. 
		Note: All surgeries are conducted using aseptic technique: specifically, surgeons wear surgical scrubs, cap, mask, and sterile gloves. Open gloving procedure is observed, autoclaved surgical pack containing surgical instruments is opened using sterile technique and the animal is draped before beginning surgery. Surgical tools are tip-sterilized in hot beads between surgeries (for up to five surgeries). Surgical sponges and gauze are autoclaved prior to use. During surgery, animals are kept on a water-circulating blanket maintained at 37 &#176;C.</li>
		<li>Administer buprenorphine (0.02 mg/kg; s.c.) immediately before surgery. Further, administer ketoprofen (5&#160;mg/kg; s.c.) prior to surgery to provide multimodal analgesia. Administer bupivacaine (5 mg/kg; s.c.) immediately prior to surgery to provide a local line block. Upon shaving the abdomen, apply alternating swabs of chlorhexidine and 70% ethanol (three times each), followed by povidone iodine for at least 1&#160;min, and remove with 70% ethanol (each time with a newly opened alcohol wipe). Swabbing of the skin is made in concentric fashion beginning at the center of the incision line and circling outward (so as not to contaminate the area by &#34;back/forth&#34; movements). Use sterile gauze swabs and discard between each application. This process is repeated three times before starting animal surgery. Whilst clipped and surgically scrubbed, keep animals on a nose cone connected to a Bain non-rebreathing circuit under approximately 1.5% maintenance dose of isoflurane. Adjust isoflurane level as required to maintain the animal at a surgical plan of anesthesia.</li>
		<li>Make a 5 cm long midline abdominal incision cutting through the muscles and peritoneum exposing the intestines.</li>
		<li>Retract the intestines (this will expose the descending aorta at the posterior abdominal wall), cover the gut with sterile surgical gauze moistened with 2-3 ml&#160;of Ringers solution and keep moist with additional Ringers as often as necessary to prevent tissue desiccation through the procedure.</li>
		<li>Blunt dissect around the aorta and separate the aorta from the inferior vena cava utilizing an iris spatulae.</li>
		<li>Place ligatures (8-0 monofilament sutures) around the rostral and caudal ends of the outermost adventitial layer of aorta, just distal to the renal artery.</li>
		<li>Lift the ligatures briefly occluding blood flow and puncture the aorta at the level of 1-2 mm anterior to the iliac bifurcation with a 25 gauge needle bent at 90&#176; at the tip.</li>
		<li>Guide the tip of the catheter of the telemetry device into the aorta using a 20-Gauge curved needle. Advance rostrally to make sure that the tip is just distal to the renal arteries
		<ol>
			<li>Fix it using a small amount of tissue adhesive. Remove ligatures and ensure there is no blood flow occlusion due to the catheter or tissue adhesive.</li>
		</ol>
		</li>
		<li>Secure the body of the telemetry device into the abdominal wall using 4-0 silk sutures. Use 4-0 Vicryl and 4-0 Prolene sutures to close the muscle and skin, respectively.</li>
	</ol>
	</li>
	<li>Telemetry Implantation Post-Surgical Care 
	Note: For the first 14 days post-surgery animals should be checked at least 3 times daily, in addition to a detailed monitoring assessment once a day. Assess animals in 7 categories: body weight, physical appearance (pain is assessed based on signs such as increased porphyrin production seen around eyes, nose and front paws, and facial grimace signs), behavior/activity, defecation, skin (including hunching, piloerection, incision healing and seroma), hydration and breathing.
	<ol>
		<li>Feed the animals with fruit, assortment of cereals, soft diet nutritional meal replacement, and provide water ad libitum, for 6 days after telemetry device implantation. Animals should have regular solid rodent chow withheld for the first day post operatively. Inject animals with warmed Lactated Ringers solution (5 ml) subcutaneously for the first 3 days after surgery and upon appearance of symptoms of dehydration. Administer Ringers solution (5 ml) as necessary upon assessment of the animals (signs of skin tent, pale ears/mucus membranes) during morning and evening checks.</li>
		<li>Administer enrofloxacin (10 mg/kg, s.c.) and ketoprofen (5 mg/kg, s.c.) once daily, for 3 days post-operatively. Administer buprenorphine (0.02 mg/kg, s.c.) during the morning and evening (i.e., every 12 hr) for the following 3 days after surgery. A mid-day dose of buprenorphine (0.02 mg/kg, s.c) may be administered if signs of pain/distress are noted.</li>
	</ol>
	</li>
	<li>Complete Transection of the Spinal Cord 
	Note: Allow animals to recover from telemetry implantation surgery for at least 14 days before T3 complete transection surgery.
	<ol>
		<li>Pre-treat animals with prophylactic enrofloxacin (10 mg/kg, s.c.) for three days before SCI surgery.</li>
		<li>Anaesthetize animals under an induction dose of 4% isoflurane as described previously. Further, inject animals with ketamine hydrochloride (70 mg/kg, i.p.) and dexmedetomidine hydrochloride (0.5 mg/kg, i.p.). 
		Note: For complete transection surgeries, the vasodilation caused by isoflurane quite often presents problems with hemostasis once the cord is transected. This problem may be prevented by using ketamine/dexmedetomidine cocktail. The animals should be anesthetized for 20-25 min, and reversed using atipamezole (1 mg/kg, s.c.) following the spinal cord transection surgery.</li>
		<li>Pre-operatively administer subcutaneously buprenorphine (0.02 mg/kg), and ketoprofen (5 mg/kg). Administer bupivacaine (5 mg/kg; s.c.) immediately prior to surgery to provide a local line block. Upon shaving the abdomen, alternating brushes of chlorhexidine and 70% ethanol (three times each), followed by povidone iodine should be applied for at least 1&#160;min, and removed with 70% ethanol (each time with a newly opened alcohol wipe). Brushes are to be made in concentric fashion beginning at the center of the incision line and circling outward (so as not to contaminate the area by &#34;back/forth&#34; movements). Use sterile gauze and discard between each application. Repeat the process three times before starting animal surgery. Whilst clipped and surgically scrubbed, keep animals on a nose cone under approximately 1.5% maintenance dose of isoflurane (adjusted as required to maintain surgical plane of anesthesia). Throughout the duration of the surgery animals are kept on water-circulating blanket maintained at 37 &#176;C.</li>
		<li>Make a dorsal midline incision, 2 cm in length, in the superficial muscle overlying the C8-T3 vertebrae.</li>
		<li>Blunt dissect in order to reveal the dura layer.</li>
		<li>Pierce the dura layer with microscissors or with the tip of a 25 gauge needle.</li>
		<li>Open the dura at the T2-T3 intervertebral gap and perform a complete transection using microscissors. Open the microscissors but not laterally enough to burst vertebral arteries and veins. In swift motion transect the spinal cord and search for an exaggerated twitch in the animal hind limbs. Confirm a complete transection via visual separation of the rostral and caudal spinal cord stumps.</li>
		<li>After confirmation, place gelfoam between the stumps to achieve hemostasis. Apply sufficient gelfoam to stop bleeding and to separate the stumps of the spinal cord.</li>
		<li>Use 4-0 Vicryl and 4-0 Prolene sutures to close the muscle and skin, respectively. After SCI surgery, give animals pre-warmed Lactated Ringer&#39;s solution (5 ml, s.c. at 30 &#176;C) and allow them to recover in a temperature-controlled environment.</li>
	</ol>
	</li>
	<li>Spinal Cord Transection Post-Surgical Care 
	Note: For the first 14 days post-surgery animals should be checked 3 times daily, in addition to a detailed monitoring assessment once a day. After 14 days detailed monitoring assessment may be repeated every other day. Assess animals in 7 categories: body weight, physical appearance (pain is assessed based on signs such as porphyrin and facial grimace), behavior/activity, defecation, skin (including hunching, piloerection, incision healing and seroma), hydration and breathing. For the first 5-7 days bladders should be checked and expressed at least 3 times per day.
	<ol>
		<li>Feed the animals with fruit, assortment of cereals, nutritional soft diet meal replacement, and provide water ad libitum, for 8-14 days after spinal cord transection surgery. Inject animals with warmed Lactated Ringer&#39;s solution (5 ml) subcutaneously for the first 3 days after surgery and upon symptoms of dehydration. Administer Ringer&#39;s solution (5 ml) as necessary upon assessment of the animals during checks. 
		Note: After spinal cord transection, rats exhibit paralysis of the hind limbs, and ambulate using flexion and extension of the forelimbs, which is preserved in this injury model. The rats lack trunk and tail support. To facilitate their movement in the cage, a rubber grid should be placed under rolled oat bedding. This allows rats to grasp the grid with the forepaws, and move readily in the cage to access food and water. The bedding should be changed daily. At day 7 after spinal cord transection, the oat bedding should be replaced by their original wood chip bedding. The rolled oat bedding is used since rats given buprenorphine can develop pica in which they eat bedding and this can cause esophageal impactions. Rolled oats are absorbent impaction of the esophagus with rolled oats has not been observed.</li>
		<li>Administer enrofloxacin (10 mg/kg, s.c.) and ketoprofen (5 mg/kg, s.c.) once daily for 3 days post-operatively. Administer buprenorphine (0.02 mg/kg, s.c.) during the morning and evening (i.e., every 12 hr) for the following 3 days after surgery. A mid-day dose of buprenorphine may be administered if signs of pain/distress are noted.</li>
	</ol>
	</li>
</ol>
2. Telemetry Monitoring of Hemodynamic Parameters
<ol>
	<li>Collect beat-by-beat arterial blood pressure (ABP) and core body temperature every day for 24 hr, pre and post SCI. Sample the arterial blood pressure and core body temperature at 1,000 Hz in 24 hr&#160;blocks using the telemetry device.</li>
	<li>Place a SmartPad under the animal cage for wireless recharging of the transducer and for receiving of digital telemeter signals from the transducers. 
	Note: The output voltage is subsequently passed on to a Configurator connected to a computer, which organizes the data flow from each channel. The voltage is converted to continuous arterial blood pressure and temperature recordings. Visualize the sampled data utilizing data acquisition software, such as LabChart.</li>
	<li>Discard the telemetry data collected during animal monitoring, as well as the 10 min&#160;immediately prior to monitoring and 10 min after monitoring.</li>
</ol>
3. Assessment of Spontaneous Incidences of Autonomic Dysreflexia (AD)
Note: The frequency, severity, and duration of spontaneous AD events were assessed using an algorithm developed for our own novel AD Detection JAVA platform software (Figure 4). A novel algorithm has been developed to automatically detect spontaneous AD events based on 24 hr&#160;SBP and HR telemetry recordings before and after SCI utilizing parameters specified in Figure 2.
<ol>
	<li>Extract SBP and HR values from raw telemetry data over period of interest using acquisition software. 
	Note: Acquisition software detects and determines the value of the SBP peaks from continuous ABP recordings. Heart rate is extrapolated by determining the duration distance between adjacent SBP peaks.</li>
	<li>Upload a CSV file of the telemetry recordings with interbeat interval in column A, SBP values in column B, MAP values in column C and time of day in column D (Figure 4A).</li>
	<li>Using the software (Figure 4B-D), specify the relevant physiological HR range (i.e., R to R interval) for the SBP recordings between 180 bpm and 625 bpm. Specify the relevant HR range on the software under the Heart Rate Minimum and Heart Rate Maximum panels.</li>
	<li>Create a threshold for SBP and HR through a moving average window of 240 sec. Indicate the window length of the moving average threshold for the analysis in the AD Duration Threshold panel.</li>
	<li>Set a SBP transposed threshold at 20 mmHg above the moving average baseline. Indicate the transposition value in the Moving Average Threshold Transposition BP panel.</li>
	<li>Isolate SBP peak clusters that exceed the transposed threshold with a peak to peak interval less than 2 sec&#160;and for a duration of greater than 10 sec. Specify the peak to peak interval using the Interpeak Interval BP and the duration interval using the Peak Cluster Interval BP panels.</li>
	<li>Group SBP peak clusters that are within 120 sec&#160;of each other. 
	Note: Detected potential AD events that are within 120 sec of each other would be grouped as a single event. Specify the maximum duration allowed between consecutive SBP clusters to differentiate separate AD events in the AD Duration Threshold panel.</li>
	<li>Confirm if group of SBP peak clusters are associated with a potential spontaneous AD event by detecting a drop in HR of 40 bpm or greater.
	<ol>
		<li>Average 10% of the HR values upon onset of the potential event (specify this percentage in the upper heart rate drop average range panel). Average 75% of the heart rate values from the end of the potential event (specify this percentage in the lower heart rate drop average range panel). </li>
		<li>Subtract the lower heart rate threshold from the higher heart rate threshold, in order to ensure a corresponding drop of 40 bpm or greater. Specify the heart rate drop restriction using the heart rate drop restriction panel.</li>
	</ol>
	</li>
	<li>Once the panels have been filled, press OK. A graphical presentation of the detected AD events are presented, which include the spikes in SBP and associated HR data (Figure 4C-D). An output excel file is also generated with the pressor response (mmHg), duration (sec), max systolic blood pressure (mmHg), minimum HR (bpm) and HR drop (bpm) of each detected AD event.</li>
</ol>
4. Colorectal Distension to Intentionally Elicit AD
Note: The severity of induced AD can be determined through colorectal distension (CRD), a clinically relevant stimulus that mimics the bowel routine3,18,19.
<ol>
	<li>Verify the balloon of a French catheter (straight, 10 French, 3 ml, 35 cm, latex free) is not leaking by inflating it with 2 ml&#160;of air.</li>
	<li>Restrain the animal by wrapping in a towel and allow BP and HR to stabilize for half an hour.</li>
	<li>Place lubricant on the tip of the catheter. Place a mark on the catheter with a permanent marker, 2cm from the middle of the balloon. The catheter is inserted via the anus into the colon until the mark is just visible at the opening. Secure the catheter to the tail with surgical tape. Re-place the wrapped animal in his home cage placed on the receiver SmartPad matching the implanted transducer inside the animal. Allow the animal&#39;s BP to normalize for 10 min.</li>
	<li>After insertion of the catheter and the associated acclimation period (i.e., 10 min), infuse the balloon with 2 ml&#160;of air over 10 sec, allow BP and HR to stabilize. Maintain distension for 1 min. Make sure the tip of the catheter remains inside the rectum. It is important not to over-inflate the balloon (i.e., 3 ml+) in order to avoid rupture of the colon. 
	Note: Distension for a duration of 1 min&#160;allows appropriate time for the development of the pressor response and the baroreceptor mediated HR drop associated with an induced episode of AD. In addition, this 1 min&#160;period also provides sufficient time for normalization of cardiovascular indices.</li>
	<li>Repeat distension 3 times per trial, repeating trials daily with a minimum interval of 10 min between trials. Once assessment is complete, remove the Foley catheter from the animal.</li>
	<li>Average beat-by-beat data over 1 sec intervals and report the maximum increase in SBP and maximum decrease in HR over multiple trials.</li>
</ol>

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The authors have nothing to disclose.

The protocol describes a detailed implementation of a novel JAVA platform AD Detection software which would be combined with a telemetry device, for a long-term thorough analysis of ABP in SCI-animals (Figure&#160;1B). This is the first software that allows for the characterization of ABP patterns to detect spontaneous AD events as they occur sporadically throughout the duration of the day. A well-characterized T3 SCI animal model can illustrate the functional capacity of the software to detect the frequ...

Autonomic dysreflexia (AD) is a life-threatening emergency in individuals after acute or chronic spinal cord injury (SCI) at cervical or high-thoracic segments and is usually characterized by episodes of persistent hypertension and bradycardia1. AD is principally caused by disruption of descending spinal pathways that usually provide input from supraspinal centers to the spinal sympathetic preganglionic neurons that control sympathetic activity and vascular tone1-4. AD episodes are characterized by a spike in systolic blood pressure (SBP) up to 300 mmHg and if left untreated may lead to seizures, intracranial hemorrhage, myocardial infarction, and even death5-8. A variety of noxious and non-noxious stimuli act as a trigger of AD, including bowel and bladder distension, spasms, pressure sores, urinary bladder catheterization or iatrogenic procedures9-12.
The temporal development of AD in response to SCI has been investigated in both human9 and animal models13,14. Typically these studies have used an &#8216;induced AD&#8217; method (i.e., urodynamics, penile vibrostimulations in humans or colorectal distension in animals) to determine the temporal development of AD. Such an approach is limited by the need for repeated assessments at isolated time-points that may preclude an accurate determination of the temporal development of AD. The use of 24-hr blood pressure monitoring in humans allows serial blood pressure measurements to be made at pre-determined intervals. This technique has recently been employed to monitor spontaneously occurring AD in patients with chronic SCI. In animal models, solid-state pressure transducers are being increasingly used to chronically monitor beat-by-beat arterial blood pressure. Recently, Rabchesvky et al. (2012), developed an algorithm that extracted one second averages of mean arterial pressure (MAP) and compared against a moving average threshold15. Spontaneous AD events were characterized based on MAP peaks that are 10 mmHg or greater above threshold concurrently with a HR drop of 10 bpm or greater.
Here a novel JAVA software that has a built in AD Detection Algorithm is presented. This algorithm works by detecting pre-determined patterns in arterial blood pressure (ABP) and heart rate (HR) that are indicative of a spontaneously occurring AD event. The user is able to manually adjust all input variables to the software such that the &#8216;detection algorithm&#8217; can be easily customized to the specific parameters of interest. The software is also able to dichotomize ABP and HR into a given epoch such that diurnal rhythmicity of hemodynamic parameters can be analyzed16. In the present manuscript, a detailed explanation is given of the surgical technique that is used to implant the telemetry devices and conduct the SCI surgery. Examples are also provided with respect to&#160;the post-processing capabilities of the AD Detection software and how cardiovascular function is altered post-SCI. For comparative purposes, the methodology and results obtained from a method of induced AD known as colorectal distension (CRD) is also illustrated.

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development of an algorithm to perform a comprehensive study of autonomic dysreflexia in animals with high spinal cord injury using a telemetry device

Spinal cord injury (SCI) is a debilitating neurological condition characterized by somatic and autonomic dysfunctions. In particular, SCI above the mid-thoracic level can lead to a potentially life-threatening hypertensive condition called autonomic dysreflexia (AD) that is often triggered by noxious or non-noxious somatic or visceral stimuli below the level of injury. One of the most common triggers of AD is the distension of pelvic viscera, such as during bladder and bowel distension or evacuation. This protocol presents a novel pattern recognition algorithm developed for a JAVA platform software to study the fluctuations of cardiovascular parameters as well as the number, severity and duration of spontaneously occurring AD events. The software is able to apply a pattern recognition algorithm on hemodynamic data such as systolic blood pressure (SBP) and heart rate (HR) extracted from telemetry recordings of conscious and unrestrained animals before and after thoracic (T3) complete transection. With this software, hemodynamic parameters and episodes of AD are able to be detected and analyzed with minimal experimenter bias.

Using telemetry, arterial blood pressure is sampled at a frequency of 1,000 Hz continuously for 24 hr. An illustrative recording of arterial blood pressure (ABP) using LabChart is shown in Figure 1B. The sample ABP was monitored by a solid state pressure sensor inserted into the descending aorta. The novel JAVA platform AD Detection software is able to extract relevant SBP (mmHg) peaks (Figure&#160;1C). We may also extract the HR (bpm) from the time interval between adjacent SBP peaks (<...

Watch this Scientific Journal Video about Development of an Algorithm to Perform a Comprehensive Study of Autonomic Dysreflexia in Animals with High Spinal Cord Injury Using a Telemetry Device at JoVE

Development of an Algorithm to Perform a Comprehensive Study of Autonomic Dysreflexia in Animals with High Spinal Cord Injury Using a Telemetry Device

The catheter of a telemetry device is implanted into the abdominal aorta in order to continuously collect beat-by-beat hemodynamic data from animals pre and post-high thoracic spinal cord transection. A novel JAVA software was employed to analyze hemodynamic parameters as well as frequency and intensity of spontaneous episodes of autonomic dysreflexia.

Spinal cord injury (SCI) is a debilitating neurological condition characterized by somatic and autonomic dysfunctions. In particular, SCI above the ...

development-an-algorithm-to-perform-comprehensive-study-autonomic

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

7.8K Views.  Faculty of Medicine, University of British Columbia. The catheter of a telemetry device is implanted into the abdominal aorta in order to continuously collect beat-by-beat hemodynamic data from animals pre and post-high thoracic spinal cord transection. A novel JAVA software was employed to analyze hemodynamic parameters as well as frequency and intensity of spontaneous episodes of autonomic dysreflexia.

Video: Development of an Algorithm to Perform a Comprehensive Study of Autonomic Dysreflexia in Animals with High Spinal Cord Injury Using a Telemetry Device

Acute and Chronic Tactile Sensory Testing after Spinal Cord Injury in Rats

Spinal cord injury (SCI) impairs sensory systems causing allodynia1-8. To identify cellular and molecular causes of allodynia, sensitive and valid sensory testing in rat SCI models is needed. However, until recently, no single testing approach had been validated for SCI so that standardized methods have not been implemented across labs. Additionally, available testing methods could not be implemented acutely or when severe motor impairments existed, preventing studies of the development of SCI-induced allodynia3. Here we present two validated sensory testing methods using von Frey Hair (VFH) monofilaments which quantify changes in tactile sensory thresholds after SCI4-5. One test is the well-established Up-Down test which demonstrates high sensitivity and specificity across different SCI severities when tested chronically5. The other test is a newly-developed dorsal VFH test that can be applied acutely after SCI when allodynia develops, prior to motor recovery4-5. Each VFH monofilament applies a calibrated force when touched to the skin of the hind paw until it bends. In the up-down method, alternating VFHs of higher or lower forces are used on the plantar L5 dermatome to delineate flexor withdrawal thresholds. Successively higher forces are applied until withdrawal occurs then lower force VFHs are used until withdrawal ceases. The tactile threshold reflects the force required to elicit withdrawal in 50% of the stimuli. For the new test, each VFH is applied to the dorsal L5 dermatome of the paw while the rat is supported by the examiner. The VFH stimulation occurs in ascending order of force until at least 2 of 3 applications at a given force produces paw withdrawal. Tactile sensory threshold is the lowest force to elicit withdrawal 66% of the time. Acclimation, testing and scoring procedures are described. Aberrant trials that require a retest and typical trials are defined. Animal use was approved by Ohio State University Animal Care and Use Committee.

We describe two tactile sensory testing methods for acute or chronic periods of spinal cord injury in rats. These validated procedures can detect the development and maintenance of allodynia-like sensations.

Spinal cord injury (SCI) impairs sensory systems causing allodynia1-8. To identify cellular and molecular causes of allodynia, sensitive and valid sensory ...

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 ...

A Contusion Model of Severe Spinal Cord Injury in Rats

The translational potential of novel treatments should be investigated in severe spinal cord injury (SCI) contusion models. A detailed methodology is described to obtain a consistent model of severe SCI. Use of a stereotactic frame and computer controlled impactor allows for creation of reproducible injury. Hypothermia and urinary tract infection pose significant challenges in the post-operative period. Careful monitoring of animals with daily weight recording and bladder expression allows for early detection of post-operative complications. The functional results of this contusion model are equivalent to transection models. The contusion model can be utilized to evaluate the efficacy of both neuroprotective and neuroregenerative approaches.

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

The translational potential of novel treatments should be investigated in severe spinal cord injury (SCI) contusion models. A detailed methodology is ...

Evaluation of Respiratory Muscle Activation Using Respiratory Motor Control Assessment (RMCA) in Individuals with Chronic Spinal Cord Injury

During breathing, activation of respiratory muscles is coordinated by integrated input from the brain, brainstem, and spinal cord. When this coordination is disrupted by spinal cord injury (SCI), control of respiratory muscles innervated below the injury level is compromised1,2 leading to respiratory muscle dysfunction and pulmonary complications. These conditions are among the leading causes of death in patients with SCI3. Standard pulmonary function tests that assess respiratory motor function include spirometrical and maximum airway pressure outcomes: Forced Vital Capacity (FVC), Forced Expiratory Volume in one second (FEV1), Maximal Inspiratory Pressure (PImax) and Maximal Expiratory Pressure (PEmax)4,5. These values provide indirect measurements of respiratory muscle performance6. In clinical practice and research, a surface electromyography (sEMG) recorded from respiratory muscles can be used to assess respiratory motor function and help to diagnose neuromuscular pathology. However, variability in the sEMG amplitude inhibits efforts to develop objective and direct measures of respiratory motor function6. Based on a multi-muscle sEMG approach to characterize motor control of limb muscles7, known as the voluntary response index (VRI)8, we developed an analytical tool to characterize respiratory motor control directly from sEMG data recorded from multiple respiratory muscles during the voluntary respiratory tasks. We have termed this the Respiratory Motor Control Assessment (RMCA)9. This vector analysis method quantifies the amount and distribution of activity across muscles and presents it in the form of an index that relates the degree to which sEMG output within a test-subject resembles that from a group of healthy (non-injured) controls. The resulting index value has been shown to have high face validity, sensitivity and specificity9-11. We showed previously9 that the RMCA outcomes significantly correlate with levels of SCI and pulmonary function measures. We are presenting here the method to quantitatively compare post-spinal cord injury respiratory multi-muscle activation patterns to those of healthy individuals.

Evaluation of Respiratory Muscle Activation Using Respiratory Motor Control Assessment RMCA in Individuals with Chronic Spinal Cord Injury

The purpose of this publication is to present our original work on a multi-muscle surface electromyographic approach to quantitatively characterize respiratory muscle activation patterns in individuals with chronic spinal cord injury using vector-based analysis.

During breathing, activation of respiratory muscles is coordinated by integrated input from the brain, brainstem, and spinal cord. When this coordination ...

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 ...

Controlled Cortical Impact Model for Traumatic Brain Injury

Every year over a million Americans suffer a traumatic brain injury (TBI). Combined with the incidence of TBIs worldwide, the physical, emotional, social, and economical effects are staggering. Therefore, further research into the effects of TBI and effective treatments is necessary. The controlled cortical impact (CCI) model induces traumatic brain injuries ranging from mild to severe. This method uses a rigid impactor to deliver mechanical energy to an intact dura exposed following a craniectomy. Impact is made under precise parameters at a set velocity to achieve a pre-determined deformation depth. Although other TBI models, such as weight drop and fluid percussion, exist, CCI is more accurate, easier to control, and most importantly, produces traumatic brain injuries similar to those seen in humans. However, no TBI model is currently able to reproduce pathological changes identical to those seen in human patients. The CCI model allows investigation into the short-term and long-term effects of TBI, such as neuronal death, memory deficits, and cerebral edema, as well as potential therapeutic treatments for TBI.

Traumatic brain injuries (TBIs) remain a serious health problem. Using the controlled cortical impact surgery model, research on the effects of TBI and possible treatment methods may be performed.

Every year over a million Americans suffer a traumatic brain injury (TBI).  Combined with the incidence of TBIs worldwide, the physical, emotional, ...

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

High thoracic or cervical spinal cord injury (SCI) can lead to cardiovascular dysfunction. To monitor cardiovascular parameters, we implanted a catheter connected to a radio transmitter into the femoral artery of rats that underwent a T4 spinal cord transection with or without grafting of embryonic brainstem-derived neural stem cells expressing green fluorescent protein. Compared to other methods such as cannula insertion or tail-cuff, telemetry is advantageous to continuously monitor blood pressure and heart rate in freely moving animals. It is also capable of long term multiple data acquisitions. In spinal cord injured rats, basal cardiovascular data under unrestrained condition and autonomic dysreflexia in response to colorectal distension were successfully recorded. In addition, cardiovascular parameters before and after SCI can be compared in the same rat if a transmitter is implanted before a spinal cord transection. One limitation of the described telemetry procedure is that implantation in the femoral artery may influence the blood supply to the ipsilateral hindlimb.

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 rats.

High thoracic or cervical spinal cord injury (SCI) can lead to cardiovascular dysfunction. To monitor cardiovascular parameters, we implanted a catheter ...

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

Spinal cord injuries (SCI) cause serious neurological impairment and psychological, economic, and social consequences for patients and their families. Clinically, more than 50% of SCI affect the cervical spine1. As a consequence of the primary injury, a cascade of secondary mechanisms including inflammation, apoptosis, and demyelination occur finally leading to tissue scarring and development of intramedullary cavities2,3. Both represent physical and chemical barriers to cell transplantation, integration, and regeneration. Therefore, shaping the inhibitory environment and bridging cavities to create a supportive milieu for cell transplantation and regeneration is a promising therapeutic target4. Here, a contusion/compression model of cervical SCI using an aneurysm clip is described. This model is more clinically relevant than other experimental models, since complete transection or ruptures of the cord are rare. Also in comparison to the weight drop model, which in particular damage the dorsum columns, circumferential compression of the spinal cord appears advantageous. Clip closing force and duration can be adjusted to achieve different injury severity. A ring spring facilitates precise calibration and constancy of clip force. Under physiological conditions, synthetic self-assembling peptides (SAP) self-assemble into nanofibers and thus, are appealing for application in SCI5. They can be injected directly into the lesion minimizing damage to the cord. SAPs are biocompatible structures erecting scaffolds to bridge intramedullary cavities and thus, equip the damaged cord for regenerative treatments. K2(QL)6K2 (QL6) is a novel SAP introduced by Dong et al.6 In comparison to other peptides, QL6 self-assembles into &#946;-sheets at neutral pH6.14 days after SCI, after the acute stage, SAPs are injected into the center of the lesion and neural precursor cells (NPC) are injected into adjacent dorsal columns. In order to support cell survival, transplantation is combined with continuous subdural administration of growth factors by osmotic micro pumps for 7 days.

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.

Spinal cord injuries (SCI) cause serious neurological impairment and psychological, economic, and social consequences for patients and their families. ...

Calibrated Forceps Model of Spinal Cord Compression Injury

Compression injuries of the murine spinal cord are valuable animal models for the study of spinal cord injury (SCI) and spinal regenerative therapy. The calibrated forceps model of compression injury is a convenient, low cost, and very reproducible animal model for SCI. We used a pair of modified forceps in accordance with the method published by Plemel et al. (2008) to laterally compress the spinal cord to a distance of 0.35 mm. In this video, we will demonstrate a dorsal laminectomy to expose the spinal cord, followed by compression of the spinal cord with the modified forceps. In the video, we will also address issues related to the care of paraplegic laboratory animals. This injury model produces mice that exhibit impairment in sensation, as well as impaired hindlimb locomotor function. Furthermore, this method of injury produces consistent aberrations in the pathology of the SCI, as determined by immunohistochemical methods. After watching this video, viewers should be able to determine the necessary supplies and methods for producing SCI of various severities in the mouse for studies on SCI and/or treatments designed to mitigate impairment after injury.

Spinal cord injury models should be highly reproducible. We demonstrate that the calibrated forceps compression model of spinal cord injury is an easy to use surgical method for generating reproducible injuries to the murine spinal cord.

Compression injuries of the murine spinal cord are valuable animal models for the study of spinal cord injury (SCI) and spinal regenerative therapy. The ...

Facial Nerve Axotomy in Mice: A Model to Study Motoneuron Response to Injury

The goal of this surgical protocol is to expose the facial nerve, which innervates the facial musculature, at its exit from the stylomastoid foramen and either cut or crush it to induce peripheral nerve injury. Advantages of this surgery are its simplicity, high reproducibility, and the lack of effect on vital functions or mobility from the subsequent facial paralysis, thus resulting in a relatively mild surgical outcome compared to other nerve injury models. A major advantage of using a cranial nerve injury model is that the motoneurons reside in a relatively homogenous population in the facial motor nucleus in the pons, simplifying the study of the motoneuron cell bodies. Because of the symmetrical nature of facial nerve innervation and the lack of crosstalk between the facial motor nuclei, the operation can be performed unilaterally with the unaxotomized side serving as a paired internal control. A variety of analyses can be performed postoperatively to assess the physiologic response, details of which are beyond the scope of this article. For example, recovery of muscle function can serve as a behavioral marker for reinnervation, or the motoneurons can be quantified to measure cell survival. Additionally, the motoneurons can be accurately captured using laser microdissection for molecular analysis. Because the facial nerve axotomy is minimally invasive and well tolerated, it can be utilized on a wide variety of genetically modified mice. Also, this surgery model can be used to analyze the effectiveness of peripheral nerve injury treatments. Facial nerve injury provides a means for investigating not only motoneurons, but also the responses of the central and peripheral glial microenvironment, immune system, and target musculature. The facial nerve injury model is a widely accepted peripheral nerve injury model that serves as a powerful tool for studying nerve injury and regeneration.

We present a surgical protocol detailing how to perform a cut or crush axotomy on the facial nerve in the mouse. The facial nerve axotomy can be employed to study the physiological response to nerve injury and test therapeutic techniques.

The goal of this surgical protocol is to expose the facial nerve, which innervates the facial musculature, at its exit from the stylomastoid foramen and ...