Name: automated gait analysis in mice with chronic constriction injury
Uploaded: 2017-10-17T14:00:00
Description: Watch this Scientific Journal Video about Automated Gait Analysis in Mice with Chronic Constriction Injury at JoVE.com

This research was supported by Chungnam National University, Korea Institute of Oriental Medicine (KIOM) and a grant of the Korea Health Technology R&#38;D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health &#38; Welfare, Republic of Korea (grant number: HI15C0007).

All experiments were conducted in accordance with the ethical guidelines of the International Association for the Study of Pain and were approved by the Institutional Animal Care and Use Committee at the Chungnam National University (Daejeon, South Korea).
1. Induction of CCI on Sciatic Nerve
<ol>
	<li>House male ICR mice weighing 20 - 25 g under a 12 h light/dark cycle at controlled room temperature (maintained at 20 - 25 &#176;C) and humidity (40 - 60%), with free access to food an...

<ol>
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P., Bories, C., De Koninck, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simplified+up-down+method+(SUDO)+for+measuring+mechanical+nociception+in+rodents+using+von+Frey+filaments.">A simplified up-down method (SUDO) for measuring mechanical nociception in rodents using von Frey filaments.</a> Mol Pain. 10, 26 (2014).</li><li>Chaplan, S. R., Bach, F. W., Pogrel, J. W., Chung, J. M., Yaksh, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+assessment+of+tactile+allodynia+in+the+rat+paw.">Quantitative assessment of tactile allodynia in the rat paw.</a> J Neurosci Methods. 53 (1), 55-63 (1994).</li><li>Chen, H., Du, J., Zhang, Y., Barnes, K., Jia, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishing+a+Reliable+Gait+Evaluation+Method+for+Rodent+Studies.">Establishing a Reliable Gait Evaluation Method for Rodent Studies.</a> J Neurosci Methods. , (2017).</li><li>Kang, D. 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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term='CatWalk'+automated+quantitative+gait+analysis+as+a+novel+method+to+assess+mechanical+allodynia+in+the+rat;+a+comparison+with+von+Frey+testing.">'CatWalk' automated quantitative gait analysis as a novel method to assess mechanical allodynia in the rat; a comparison with von Frey testing.</a> Pain. 102 (1-2), 203-209 (2003).</li><li>Martinov, T., Mack, M., Sykes, A., Chatterjea, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+changes+in+tactile+sensitivity+in+the+hind+paw+of+mice+using+an+electronic+von+Frey+apparatus.">Measuring changes in tactile sensitivity in the hind paw of mice using an electronic von Frey apparatus.</a> J Vis Exp. (82), e51212 (2013).</li><li>Ferland, C. E., Laverty, S., Beaudry, F., Vachon, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gait+analysis+and+pain+response+of+two+rodent+models+of+osteoarthritis.">Gait analysis and pain response of two rodent models of osteoarthritis.</a> Pharmacol Biochem Behav. 97 (3), 603-610 (2011).</li><li>Mogil, J. S., 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=Hypolocomotion,+asymmetrically+directed+behaviors+(licking,+lifting,+flinching,+and+shaking)+and+dynamic+weight+bearing+(gait)+changes+are+not+measures+of+neuropathic+pain+in+mice.">Hypolocomotion, asymmetrically directed behaviors (licking, lifting, flinching, and shaking) and dynamic weight bearing (gait) changes are not measures of neuropathic pain in mice.</a> Mol Pain. 6, 34 (2010).</li><li>Ferreira-Gomes, J., Adaes, S., Castro-Lopes, J. 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Currently, measurement of mechanical allodynia by using von Frey filaments is the most widely used method in pain animal models to demonstrate tactile hypersensitivity. As animal models for neuropathic pain have continued to be developed, the methodology of assessment for sensory function has been also improved8,9,10,15. In those reports, it has been suggested these modifications can provide a ...

Pathological changes of nervous system induced by trauma, metabolic dysfunction, inflammation, infection, ischemia, or autoimmune disease occasionally result in neuropathic pain, which is defined as a pain arising as a direct consequence of a lesion or disease affecting the somatosensory system1. Neuropathic pain is usually unbearable and unfortunately, conventional analgesics generally do not produce sufficient pain relief2. A major feature of neuropathic pain contains spontaneous and stimulation-evoked (i.e., allodynia and hyperalgesia) pains. Allodynia is a nociceptive response that occurs to normally non-painful stimuli, such as light touch or warm stimulation. Hyperalgesia indicates an enhanced pain response to noxious mechanical and/or thermal stimuli3. Although these two symptoms both critically impair the patient&#39;s quality of life, mechanical allodynia evoked by gentle tactile stimulation is the most aggravating symptom because soft contact is difficult to avoid in everyday life.
To investigate the underlying mechanism and new analgesics for the treatment of neuropathic pain, the precise measurement of the pain response is essential. Numerous neuropathic pain animal models have developed nociceptive responses on the hind paw area because of its high accessibility4,5,6,7. Thus, most pain response assessments have been performed on the plantar or dorsal surface of the hind paw by applying mechanical stimuli using special instruments, such as von Frey filaments. One of the most frequently used method is the up and down method described by Dixon8 and the later modified versions9,10. However, very skilled, experienced experimenters are required to perform the von Frey test and the results may be subjective.
The automated gait analysis system can investigate neurological and neuromuscular disorders by measuring various parameters of walking in freely moving rodents. In a variety of nerve injury animal models, the degree of nociception and the antinociceptive effect of several treatments can be evaluated without adding a pain stimulation11,12,13,14. This analysis system can detect static and dynamic gait parameters, such as: paw print area (the area of the complete paw print that contacts with the floor), paw intensity (the average intensity of the contacting paw area), stride length (the distance between successive placements of the same paw), stance phase (the duration of ground contact for a single hind paw), step sequence (the order in which the four paws are placed on the floor), swing (the duration of the swing phase), and swing speed (computed from stride length and swing duration and expressed as pixels per second). This paper demonstrates the use an analysis system and provides a brief comparison of data with the von Frey test using chronic constriction injury (CCI) neuropathic mice.

<table>
	<tbody>
		<tr>
			<td>0.9% Saline</td>
			<td>JW Pharmaceutical</td>
			<td>N/A</td>
			<td>Vehicle for drugs</td>
		</tr>
		<tr>
			<td>1ml syringe</td>
			<td>BD Plastipak</td>
			<td>300013</td>
			<td>Injecting device</td>
		</tr>
		<tr>
			<td>2, 2, 2-tribromoethanol (97% purity)</td>
			<td>Sigma</td>
			<td>T48402</td>
			<td>Anesthetic</td>
		</tr>
		<tr>
			<td>2-methyl-2-buthanol (99% purity)</td>
			<td>Sigma</td>
			<td>152463</td>
			<td>Solvent for 2, 2, 2-tribromoethanol</td>
		</tr>
		<tr>
			<td>Catwalk Automated gait analysis system</td>
			<td>Noldus</td>
			<td>N/A</td>
			<td>Automatic analysis software of aniaml gait</td>
		</tr>
		<tr>
			<td>Chromic catgut (4-0 thickness)</td>
			<td>AILEE</td>
			<td>C442</td>
			<td>Ligature to make chronic constriction injury on the sciatic nerve</td>
		</tr>
		<tr>
			<td>Gabapentin</td>
			<td>Sigma</td>
			<td>Y0001280</td>
			<td>Analgeisc, Used as a positive control drug in this study</td>
		</tr>
		<tr>
			<td>Graefe Forceps</td>
			<td>F.S.T</td>
			<td>11051-10</td>
			<td>Surgical instrument</td>
		</tr>
		<tr>
			<td>Heating Pad</td>
			<td>DAESHIN ELECTRONICS</td>
			<td>M-303AT</td>
			<td>Regulation of body temperature</td>
		</tr>
		<tr>
			<td>ICR Mouse</td>
			<td>Samtaco</td>
			<td>N/A</td>
			<td>Experimental animal</td>
		</tr>
		<tr>
			<td>Mersilk (3-0 thickness)</td>
			<td>ETHICON</td>
			<td>W598H</td>
			<td>Suture material for surgical closure of skin</td>
		</tr>
		<tr>
			<td>Micro-Mosquito</td>
			<td>F.S.T</td>
			<td>13010-12</td>
			<td>Surgical instrument</td>
		</tr>
		<tr>
			<td>Micro-scissors</td>
			<td>F.S.T</td>
			<td>14090-09</td>
			<td>Surgical instrument</td>
		</tr>
		<tr>
			<td>Needle holder</td>
			<td>F.S.T</td>
			<td>12002-12</td>
			<td>Surgical instrument</td>
		</tr>
		<tr>
			<td>Povidone Iodine</td>
			<td>Firson</td>
			<td>N/A</td>
			<td>Disinfectant to prevent infection after surgery</td>
		</tr>
		<tr>
			<td>Scalpel blade</td>
			<td>F.S.T</td>
			<td>10010-00 (#10)</td>
			<td>Surgical instrument to make an incision</td>
		</tr>
		<tr>
			<td>Scalpel handle</td>
			<td>F.S.T</td>
			<td>10003-12 (#3)</td>
			<td>Surgical instrument to make an incision</td>
		</tr>
		<tr>
			<td>Von-Frey filaments</td>
			<td>North Coast</td>
			<td>NC12775-99</td>
			<td>Measurement device to test sensory function for mechanical stimulation</td>
		</tr>
	</tbody>
</table>

automated gait analysis in mice with chronic constriction injury

The von Frey test is a classical method that has been widely used to examine the sensory function of neuropathic pain animals. However, it has some disadvantages such as subjective data and the requirement of a skilled, experienced experimenter. To date, a variety of modifications have improved the von Frey method, but it still has a few limitations. Recent reports have suggested that gait analysis produces more accurate and objective data from the neuropathic animals. This protocol demonstrates how to perform the automated gait analysis to determine the degree of neuropathic pain in mice. After several days of acclimation, the mice were allowed to walk freely on the glass floor to illuminate footprints. Then, quantification of the footprints and gait were performed through video clips with automatic analysis of various walking parameters, such as area of paw print, swing time, angle of paw, etc.
The main purpose of this study is to describe the methodology of automated gait analysis and briefly compare it with data from the classical sensory test using von Frey filament.

We have performed the von Frey test and automated gait analysis in CCI mice until 10 days after CCI surgery. For statistical analysis, repeated measures of two-way analysis of variance (ANOVA) determines the overall effects, and Dunnett&#39;s post-hoc analysis was performed to determine the p-value among the experimental groups.
The results shown in Figure 1 indicate the time course of the ...

Watch this Scientific Journal Video about Automated Gait Analysis in Mice with Chronic Constriction Injury at JoVE.com

Automated Gait Analysis in Mice with Chronic Constriction Injury

The precise assessment of pain response in a neuropathic animal model is critical to investigate the pathophysiology of pain diseases and develop new analgesics. We present a sensitive and objective method to determine the sensory function of the rodent hind paw by an automated gait analysis system.

The von Frey test is a classical method that has been widely used to examine the sensory function of neuropathic pain animals. However, it has some ...

The overall goal of this procedure is to analyze mechanical allodynia of mice with a chronic constriction injury using an automated gait analysis system. This method can help answer key questions in the pain research field by measuring sensory function after peripheral nerve injury in rodents. The main advantage of this technique is that the analysis system is quick and easy to use and it also automatically scores various gait parameters.For this study, house male ICR mice weighing 20 to 25 grams under standard conditions for at least one week. Select mice based on their appearance and behavior. Do not use mice that exhibit abnormal appearances or behavior such as hair loss, scarring and less movement.Using the selected mice, starting five days before the surgery collect automated gait data on a daily basis to have a baseline measurement. One day before the surgery perform the Von Frey test with a one gram filament on a hind paw to get a baseline response. Balance the responses between the injury group and the sham group.On each test day bring mice to the behavioral test room and acclimate the mice in their own home cage for at least 30 minutes before the test. Every other day after the surgery, measure mechanical allodynia using a Von Frey test. Put the mouse in a clean transparent acrylic box on a metal mesh floor and let it acclimate to the box for 30 minutes.Next, gently apply a one gram Von Frey filament to the plantar surface of the ipsilateral hind paw until the filament bends. Repeat this pattern of stimulation 10 times observing for paw withdrawals. Wait at least 10 seconds between stimulations.Record the number of withdrawals and score their frequency for analysis. Every other day, also perform the gait analysis test which must be done in the dark. Again, acclimate the mice to the test room in their home cage for at least 30 minutes.To set up the gait analysis device go to the software control. Under the setup tab set the walkway length to 30 centimeters, set the maximum run duration to five seconds and set the maximum run variation to 50%Next, select a registered camera from the setup tab of the program menu and then select open acquisition from the acquire tab. Following the status message, click the snap background button to take a background picture of the empty walkway.Now place a mouse onto the walkway and allow it to acclimate to the device for 10 minutes. The apparatus should always be washed with alcohol between mice. After the mouse has acclimated to the device, click the start acquisition button.The recording starts automatically according to the movement of the mouse. When the mouse clearly walks across the walkway, the program will automatically classify the compliant runs with a green icon. If the software fails to detect the footprints, a red icon will light up.After collecting at least five compliant runs select the classify runs option from the acquire tab of the program menu. Select which run should be analyzed and click the auto classify button. The results are automatically saved.To view the results click on view run statistics from the analyze menu. The Von Frey test and automated gait analysis were used to assess mice that underwent the CCI surgery on 10 consecutive days post-operatively. The frequency of paw withdrawal increased two days after the surgery and was sustained for 10 days.This effect could be significantly reduced by daily post-operative injections of gabapentin. Using the gait analysis software the size of the left and right hind paw prints could be compared. Normally the areas are the same which is indicated by a 50%value.The CCI surgery had a significant effect on paw print and that effect could be significantly reduced using gabapentin. The gait analysis software is also able to pick up on the change of single stance durations when the mouse is weight-bearing on just one hind paw. This is calculated and shown as a percent change.This metric shows the same effect for the CCI surgery and gabapentin treatments. After watching this video you should have a good understanding of how to operate and analyze automated gait analysis systems to investigate the pathophysiology of neuropathic pain in animal models like chronic constriction injury.

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