Name: development of an algorithm to perform a comprehensive study of autonomic dysreflexia in animals with high spinal cord injury using a telemetry device
Uploaded: 2016-07-29T13:00:00
Description: 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

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 labor...

<ol>
	<li>Krassioukov, A., Claydon, V. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+clinical+problems+in+cardiovascular+control+following+spinal+cord+injury:+an+overview.">The clinical problems in cardiovascular control following spinal cord injury: an overview.</a> Progress in brain research. 152, 223-229 (2006).</li><li>Krassioukov, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autonomic+function+following+cervical+spinal+cord+injury.">Autonomic function following cervical spinal cord injury.</a> Respiratory physiology &amp; neurobiology. 169, 157-164 (2009).</li><li>Teasell, R. W., Arnold, J. M. O., Krassioukov, A., Delaney, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiovascular+consequences+of+loss+of+supraspinal+control+of+the+sympathetic+nervous+system+after+spinal+cord+injury.">Cardiovascular consequences of loss of supraspinal control of the sympathetic nervous system after spinal cord injury.</a> Archives of physical medicine and rehabilitation. 81, 506-516 (2000).</li><li>Alan, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recurrent+autonomic+dysreflexia+exacerbates+vascular+dysfunction+after+spinal+cord+injury.">Recurrent autonomic dysreflexia exacerbates vascular dysfunction after spinal cord injury.</a> The Spine Journal. 10, 1108-1117 (2010).</li><li>Eltorai, I., Kim, R., Vulpe, M., Kasravi, H., Ho, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fatal+cerebral+hemorrhage+due+to+autonomic+dysreflexia+in+a+tetraplegic+patient:+case+report+and+review.">Fatal cerebral hemorrhage due to autonomic dysreflexia in a tetraplegic patient: case report and review.</a> Spinal Cord. 30, 355-360 (1992).</li><li>Pan, S. -. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracerebral+hemorrhage+secondary+to+autonomic+dysreflexia+in+a+young+person+with+incomplete+C8+tetraplegia:+A+case+report.">Intracerebral hemorrhage secondary to autonomic dysreflexia in a young person with incomplete C8 tetraplegia: A case report.</a> Archives of physical medicine and rehabilitation. 86, 591-593 (2005).</li><li>Ho, C., Krassioukov, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autonomic+dysreflexia+and+myocardial+ischemia.">Autonomic dysreflexia and myocardial ischemia.</a> Spinal cord. 48, 714-715 (2010).</li><li>Wan, D., Krassioukov, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Life-threatening+outcomes+associated+with+autonomic+dysreflexia:+A+clinical+review.">Life-threatening outcomes associated with autonomic dysreflexia: A clinical review.</a> The journal of spinal cord medicine. 37, 2-10 (2014).</li><li>Mathias, C. J., Frankel, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiovascular+control+in+spinal+man.">Cardiovascular control in spinal man.</a> Annual review of physiology. 50, 577-592 (1988).</li><li>Hubli, M., Krassioukov, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ambulatory+Blood+Pressure+Monitoring+in+Spinal+Cord+Injury:+Clinical+Practicability.">Ambulatory Blood Pressure Monitoring in Spinal Cord Injury: Clinical Practicability.</a> Journal of neurotrauma. 31, 789-797 (2014).</li><li>Liu, N., Fougere, R., Zhou, M., Nigro, M., Krassioukov, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autonomic+dysreflexia+severity+during+urodynamics+and+cystoscopy+in+individuals+with+spinal+cord+injury.">Autonomic dysreflexia severity during urodynamics and cystoscopy in individuals with spinal cord injury.</a> Spinal cord. 51, 863-867 (2013).</li><li>Phillips, A. A., Elliott, S. L., Zheng, M. M., Krassioukov, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+alpha+adrenergic+antagonist+reduces+severity+of+transient+hypertension+during+sexual+stimulation+after+spinal+cord+injury.">Selective alpha adrenergic antagonist reduces severity of transient hypertension during sexual stimulation after spinal cord injury.</a> Journal of neurotrauma. , (2014).</li><li>Maiorov, D. N., Fehlings, M. G., Weaver, L. C., Krassioukov, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+between+severity+of+spinal+cord+injury+and+abnormalities+in+neurogenic+cardiovascular+control+in+conscious+rats.">Relationship between severity of spinal cord injury and abnormalities in neurogenic cardiovascular control in conscious rats.</a> Journal of neurotrauma. 15, 365-374 (1998).</li><li>Maiorov, D. N., Weaver, L. C., Krassioukov, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+between+sympathetic+activity+and+arterial+pressure+in+conscious+spinal+rats.">Relationship between sympathetic activity and arterial pressure in conscious spinal rats.</a> American Journal of Physiology-Heart and Circulatory Physiology. 41, H625 (1997).</li><li>Rabchevsky, A. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+gabapentin+on+muscle+spasticity+and+both+induced+as+well+as+spontaneous+autonomic+dysreflexia+after+complete+spinal+cord+injury.">Effects of gabapentin on muscle spasticity and both induced as well as spontaneous autonomic dysreflexia after complete spinal cord injury.</a> Frontiers in physiology. 3, (2012).</li><li>Mayorov, D. N., Adams, M. A., Krassioukov, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telemetric+blood+pressure+monitoring+in+conscious+rats+before+and+after+compression+injury+of+spinal+cord.">Telemetric blood pressure monitoring in conscious rats before and after compression injury of spinal cord.</a> Journal of neurotrauma. 18, 727-736 (2001).</li><li>Ramsey, J. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Care+of+rats+with+complete+high-thoracic+spinal+cord+injury.">Care of rats with complete high-thoracic spinal cord injury.</a> Journal of neurotrauma. 27, 1709-1722 (2010).</li><li>Krassioukov, A. V., Furlan, J. C., Fehlings, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autonomic+dysreflexia+in+acute+spinal+cord+injury:+an+under-recognized+clinical+entity.">Autonomic dysreflexia in acute spinal cord injury: an under-recognized clinical entity.</a> Journal of neurotrauma. 20, 707-716 (2003).</li><li>Krogh, K., Mosdal, C., Laurberg, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gastrointestinal+and+segmental+colonic+transit+times+in+patients+with+acute+and+chronic+spinal+cord+lesions.">Gastrointestinal and segmental colonic transit times in patients with acute and chronic spinal cord lesions.</a> Spinal cord. 38, 615-621 (2000).</li><li>Krassioukov, A. V., Johns, D. G., Schramm, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitivity+of+sympathetically+correlated+spinal+interneurons,+renal+sympathetic+nerve+activity,+and+arterial+pressure+to+somatic+and+visceral+stimuli+after+chronic+spinal+injury.">Sensitivity of sympathetically correlated spinal interneurons, renal sympathetic nerve activity, and arterial pressure to somatic and visceral stimuli after chronic spinal injury.</a> Journal of neurotrauma. 19, 1521-1529 (2002).</li><li>Braga, V. A., Prabhakar, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Refinement+of+telemetry+for+measuring+blood+pressure+in+conscious+rats.">Refinement of telemetry for measuring blood pressure in conscious rats.</a> Journal of the American Association for Laboratory Animal Science: JAALAS. 48, 268 (2009).</li><li>Whitesall, S. E., Hoff, J. B., Vollmer, A. P., D'Alecy, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+simultaneous+measurement+of+mouse+systolic+arterial+blood+pressure+by+radiotelemetry+and+tail-cuff+methods.">Comparison of simultaneous measurement of mouse systolic arterial blood pressure by radiotelemetry and tail-cuff methods.</a> American Journal of Physiology-Heart and Circulatory Physiology. 286, H2408-H2415 (2004).</li></ol>

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.

<table border="0" cellpadding="0" cellspacing="0" width="955">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:327px;">11 Male Wistar Rats -Hsd-WI (250-300g)</td>
			<td style="width:171px;">Envigo (formerly Harlan Laboratories)&#160;</td>
			<td align="right" style="width:119px;">141</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:327px;">Lab Chart (PowerLab&#174; Data Acquisition System)</td>
			<td style="width:171px;">AD Instruments&#160;</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:327px;">Pressure Telemeter&#160;</td>
			<td style="width:171px;">Millar Inc.</td>
			<td>RP-TRM54P</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Configurator</td>
			<td style="width:171px;">Millar Inc.</td>
			<td>TR190</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SmartPad</td>
			<td style="width:171px;">Millar Inc.</td>
			<td>TR180</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:327px;">Isoflurane (Aerrane)</td>
			<td style="width:171px;">Baxter Corp.</td>
			<td style="width:119px;">DIN: 02225875</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:327px;">Enrofloxacin (Baytril)</td>
			<td style="width:171px;">Bayer Healthcare</td>
			<td style="width:119px;">DIN: 02169428</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">5-0 Silk Sutures</td>
			<td style="width:171px;">Ethicon</td>
			<td style="width:119px;">S182</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">4-0 Vicryl Subcuticular</td>
			<td style="width:171px;">Ethicon</td>
			<td style="width:119px;">J496G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Buprenorphine (Temgesic)</td>
			<td style="width:171px;">Reckitt Benckiser</td>
			<td style="width:119px;">DIN: 0281250</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Bupivicaine Hydrochloride (Marcaine 0.5%)</td>
			<td style="width:171px;">Hospira Healthcare Corp.&#160;</td>
			<td style="width:119px;">DIN: 02305909</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">Ketoprofen (Anafen)</td>
			<td style="width:171px;">Merial</td>
			<td style="width:119px;">DIN: 02150999</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ketamine Hydrochloride (Vetalar)</td>
			<td style="width:171px;">Bioniche</td>
			<td style="width:119px;">DIN: 01989529</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dexmedetomidine Hydrochloride (Domitor)</td>
			<td style="width:171px;">Pfizer</td>
			<td style="width:119px;">DIN: 02333929</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:327px;">Lactated Ringer&#39;s Solution&#160;</td>
			<td style="width:171px;">Braun Medical Inc.</td>
			<td style="width:119px;">DIN: 01931636</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:327px;">Gelfoam #12</td>
			<td style="width:171px;">Pharmacia &#38; Upjohn Company</td>
			<td style="width:119px;">03603-14-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:327px;">Microscissors</td>
			<td style="width:171px;">Fine Science Tools</td>
			<td style="width:119px;">15003-008</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:327px;">Iris Spatulae</td>
			<td style="width:171px;">Fine Science Tools</td>
			<td style="width:119px;">10094-13</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:327px;">10 French 35cm Foley Catheter&#160;</td>
			<td>Coloplast&#160;</td>
			<td style="width:119px;">AA6110</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:327px;">Dietgel&#174;&#160;</td>
			<td style="width:171px;">Clear H2O, Westbrook, ME</td>
			<td style="width:119px;">76A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LabDiet Rodent Diet 5001</td>
			<td style="width:171px;">Purina Mills (PMI&#174;)</td>
			<td align="right" style="width:119px;">5001</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:327px;">Chlorhexadine (Hibitane)</td>
			<td style="width:171px;">Wyeth Animal Health, Guelph, Ontario</td>
			<td>DIN 00245097</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Atipamezole Hydrochloride(Antisedan)</td>
			<td style="width:171px;">Orion Pharma</td>
			<td style="width:119px;">DIN: 02237744</td>
			<td></td>
		</tr>
	</tbody>
</table>

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 ...

The overall goal of this procedure is to characterize the temporal development and progression of autonomic dysreflexia, or AD, using AD detection software in a rodent spinal cord injury model. This is accomplished by first implanting a telemetry device into the abdominal aorta to monitor beat by beat continuous arterial blood pressure and heart rate. 14 days later, the T3 spinal cord segment is completely transected.Next, AD detection software is used to detect and characterize the spontaneous AD episodes. Alternatively, AD can be induced non-invasively with a Foley catheter, followed by real time monitoring of the induced AD episodes. Ultimately, the episodic hypertension and onset of brachycardia that occurs as a result of the spontaneous or induced AD incidents can be evaluated by the AD detection software.Allowing sufficient time for implantation of telemetry device prior to spinal cord injury is crucial for animal survival, as mechanical obstruction by telemetry device cause insufficient blood flow to the hind limbs of animals. We used the high thoracic T3 complete spinal cord injury in our protocol, because it induces a number of cardiovascular dysfunctions, including autonomic dysreflexia that resemble the clinical reality of spinal cord injury. The AD detection software that we have developed works by identifying patterns within continuous blood pressure recordings that resemble autonomic dysreflexia, suggesting a possible use for the software in the monitoring of patients with spinal cord injury.Prior to data collection, implant the catheter of the telemetry device into the abdominal aorta of the animal according to the text protocol. After the telemetry surgery, sample the beat by beat continuous arterial blood pressure of the animal at 1000 hertz in 24 hour blocks to monitor the pre-spinal cord injury hemodynamic parameters. Two weeks later, transect the spinal cord at the T3 segment according to established protocols, followed by monitoring of the arterial blood pressure to assess the post-spinal cord injury response.To assess the incidence of spontaneous autonomic dysreflexia, or AD, use the appropriate acquisition software to extract the interbeat interval, systolic blood pressure, mean arterial pressure, and time of day from the raw telemetry data over the period of interest. Next, upload a CSV file of the extracted data, including the interbeat interval, the systolic blood and mean arterial pressure values, and the time of day recorded. Then create a threshold for the systolic blood pressure using a moving average window of 240 seconds.Set the systolic blood pressure transposed threshold at 20 millimeters of mercury above the moving average baseline. Next, identify the systolic blood pressure peak clusters that exceed the transposed threshold with an interpeak interval of less than two seconds, and a peak cluster interval of greater than 10 seconds. Set the AD duration threshold interval to within 120 seconds of each other.To confirm these peak clusters are associated with a spontaneous AD event, set a minimum heart rate drop restriction associated with the first 10%of the heart rate values upon the onset of the potential event, and 75%of the heart rate values from the end of the potential event. Then subtract the lower heart rate threshold from the higher heart rate threshold to ensure a corresponding drop of 40 BPM or greater, taking care to indicate the relevant physiological range for the systolic blood pressure and heart rate recordings. When all of the panels have been filled click OK.A graphical representation of the detected AD events will be presented, including the spikes in the systolic blood pressure and the associated heart rate data.A spreadsheet file with the pressure response, duration, maximum systolic blood pressure, minimum heart rate, and heart rate drop of each detected AD event will also be generated. To assess the severity of an induced AD episode, lubricate the tip of a Foley catheter and insert the deflated balloon tip into the rectum of the animal two centimeters from the anal opening. Animals with spinal cord injury have no sensation below the injury, however, they could experience spasmodic and jerky movements during the catheter incretion due to spasticity.Secure the catheter to the tail with surgical tape and place a custom tube around the outside of the catheter to prevent the rat from biting or pulling out the catheter. Then infuse the balloon with two milliliters of air over 10 seconds. Allow the blood pressure and heart rate to stabilize.Maintain distension for one minute. After a 10 minute rest, repeat the distention and 10 minute recovery period three more times, and then analyze the severity of induced AD using the AD detection software. Here, a representative recording of the continuous beat by beat arterial blood pressure sampled by the telemetry transducer and extracted by the data acquisition software as just demonstrated is shown.As these data from continuous arterial blood pressure monitoring demonstrate, spontaneous AD is associated with an increase in the systolic blood pressure greater than 20 millimeters of mercury accompanied by a decrease in the heart rate of 40 BPM or greater upon the onset of the AD event. In these graphs, the rapid and persistent increase in systolic blood pressure accompanied by pronounced brachycardia that occurs after induction of AD by colorectal distention as just demonstrated can be observed. By combining distention with detection and characterization of these spontaneous autonomic dysreflexia events, as just demonstrated, we can study the temporal characteristics of this life threatening condition.

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

Research

JoVE Journal

Medicine

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 ...

Contrast Enhanced Ultrasound Imaging for Assessment of Spinal Cord Blood Flow in Experimental Spinal Cord Injury

Reduced spinal cord blood flow (SCBF) (i.e., ischemia) plays a key role in traumatic spinal cord injury (SCI) pathophysiology and is accordingly an important target for neuroprotective therapies. Although several techniques have been described to assess SCBF, they all have significant limitations. To overcome the latter, we propose the use of real-time contrast enhanced ultrasound imaging (CEU). Here we describe the application of this technique in a rat contusion model of SCI. A jugular catheter is first implanted for the repeated injection of contrast agent, a sodium chloride solution of sulphur hexafluoride encapsulated microbubbles. The spine is then stabilized with a custom-made 3D-frame and the spinal cord dura mater is exposed by a laminectomy at ThIX-ThXII. The ultrasound probe is then positioned at the posterior aspect of the dura mater (coated with ultrasound gel). To assess baseline SCBF, a single intravenous injection (400 &#181;l) of contrast agent is applied to record its passage through the intact spinal cord microvasculature. A weight-drop device is subsequently used to generate a reproducible experimental contusion model of SCI. Contrast agent is re-injected 15 min following the injury to assess post-SCI SCBF changes. CEU allows for real time and in-vivo assessment of SCBF changes following SCI. In the uninjured animal, ultrasound imaging showed uneven blood flow along the intact spinal cord. Furthermore, 15 min post-SCI, there was critical ischemia at the level of the epicenter while SCBF remained preserved in the more remote intact areas. In the regions adjacent to the epicenter (both rostral and caudal), SCBF was significantly reduced. This corresponds to the previously described &#8220;ischemic penumbra zone&#8221;. This tool is of major interest for assessing the effects of therapies aimed at limiting ischemia and the resulting tissue necrosis subsequent to SCI.

Contrast Enhanced Ultrasound imaging is a reliable in-vivo tool for quantifying spinal cord blood flow in an experimental rat spinal cord injury model. This paper contains a comprehensive protocol for application of this technique in association with a contusion model of thoracic spinal cord injury.

Reduced spinal cord blood flow (SCBF) (i.e., ischemia) plays a key role in traumatic spinal cord injury (SCI) pathophysiology and is accordingly an ...