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

Denne forskning blev støttet af Chungnam National University, Korea Institut for orientalsk medicin (KIOM) og et tilskud af Korea sundhed teknologi R &#38; D projekt gennem den Korea sundhed industri udvikling Institute (KHIDI), finansieret af Ministeriet for sundhed &#38; Velfærd, Republikken Korea (giver nummer: HI15C0007).

alle eksperimenter blev udført i overensstemmelse med de etiske retningslinjer for den internationale sammenslutning for studiet af smerte og blev godkendt af det institutionelle Animal Care og brug udvalg på Chungnam National University ( Daejeon, Sydkorea).
 1. induktion af CCI på iskiasnerven <ol><li>hus mandlige ICR mus vejer 20-25 g under en 12t lys/mørke cyklus på kontrollerede rumtemperaturen (opretholdt ved 20-25 ° C) og fugtighed (40-60%), med fri adgang til mad og va...

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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+peripheral+mononeuropathy+in+rat+that+produces+disorders+of+pain+sensation+like+those+seen+in+man.">A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man.</a> Pain. 33 (1), 87-107 (1988).</li><li>Dixon, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+analysis+of+experimental+observations.">Efficient analysis of experimental observations.</a> Annu Rev Pharmacol Toxicol. 20, 441-462 (1980).</li><li>Bonin, R. 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. W., 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=Antinociceptive+Profile+of+Levo-tetrahydropalmatine+in+Acute+and+Chronic+Pain+Mice+Models:+Role+of+spinal+sigma-1+receptor.">Antinociceptive Profile of Levo-tetrahydropalmatine in Acute and Chronic Pain Mice Models: Role of spinal sigma-1 receptor.</a> Sci Rep. 6, 37850 (2016).</li><li>Huehnchen, P., Boehmerle, W., Endres, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+paclitaxel+induced+sensory+polyneuropathy+with+&amp;#34;Catwalk&amp;#34;+automated+gait+analysis+in+mice.">Assessment of paclitaxel induced sensory polyneuropathy with &amp;#34;Catwalk&amp;#34; automated gait analysis in mice.</a> PLoS One. 8 (10), e76772 (2013).</li><li>Vrinten, D. H., Hamers, F. 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. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+movement-evoked+pain+in+osteoarthritis+by+the+knee-bend+and+CatWalk+tests:+a+clinically+relevant+study.">Assessment of movement-evoked pain in osteoarthritis by the knee-bend and CatWalk tests: a clinically relevant study.</a> J Pain. 9 (10), 945-954 (2008).</li></ol>

Måling af mekanisk allodyni ved hjælp af von Frey filamenter er i øjeblikket mest udbredte metode i smerte dyremodeller for at demonstrere taktile overfølsomhed. Som dyremodeller for neuropatiske smerter har fortsat skal udvikles, har metode for vurdering for sensorisk funktion været også forbedret8,9,10,15. I disse rapporter, er det blevet foreslået, disse ændringer kan give en mere pr...

Patologiske forandringer af nervesystemet induceret af traume, metabolisk dysfunktion, betændelse, infektion, iskæmi, eller autoimmun sygdom resultere lejlighedsvis i neuropatiske smerter, som er defineret som en smerte, der opstår som en direkte konsekvens af en læsion eller sygdom påvirker somatosensoriske system1. Neuropatiske smerter er normalt uudholdelig og desværre konventionelle analgetika generelt producere ikke tilstrækkelig smertelindring2. Et væsentligt træk ved neuropatiske smerter indeholder spontan og stimulation-fremkaldt (dvs., allodyni og hyperalgesia) smerter. Allodyni er en nociceptive respons, der opstår normalt ikke-smertefulde stimuli, såsom let berøring eller varme stimulation. Hyperalgesia angiver en øget smerte svar til skadelige mekaniske og/eller termisk stimuli3. Selv om disse to symptomer både kritisk forringe patientens livskvalitet, er mekanisk allodyni fremkaldt af blid taktil stimulation den mest skærpende symptom fordi blød kontakt er svært at undgå i hverdagen.
At undersøge de underliggende mekanisme og nye analgetika til behandling af neuropatiske smerter, den præcise måling af smerter reaktion er afgørende. Talrige neuropatiske smerter dyremodeller har udviklet nociceptive svar på hind paw område på grund af sin høje tilgængelighed4,5,6,7. Således, de fleste smerter reaktion vurderinger har udført på bagben pote plantar eller dorsale overflade ved at anvende mekaniske stimuli ved hjælp af særlige instrumenter, som von Frey filamenter. En af de hyppigst anvendte metode er op og ned metode beskrevet af Dixon8 og de senere ændrede versioner9,10. Dog meget dygtige, erfarne eksperimentatorer er forpligtet til at udføre von Frey test og resultaterne kan være subjektive.
Automatiseret gangart analysesystem kan undersøge neurologiske og neuromuskulære lidelser ved at måle forskellige parametre af at gå i frit flytte gnavere. I en række nerve skade dyremodeller, kan graden af nociception og den antinociceptive virkning af flere behandlinger vurderes uden at tilføje en smerte stimulation11,12,13, 14. denne analysesystem kan registrere statiske og dynamiske gangart parametre, såsom: paw udskriftsområde (arealet af den komplette pote print, at kontakt med gulvet), paw intensitet (den gennemsnitlige intensitet af området kontakte pote), skridtlængde længden ( afstanden mellem successive placeringer af den samme pote), holdning fase (varighed af jorden kontakt for en enkelt bagben pote), skridt sekvens (den rækkefølge, hvori de fire paws er placeret på gulvet), swing (varighed af swing fase) og swing speed (beregnet fra skridtlængde længde og svinge varighed og udtrykt som pixel pr. sekund). Dette papir viser brug en analysesystem og giver en kort sammenligning af data med von Frey test ved hjælp af kronisk konstriktion skade (CCI) neuropatiske mus.

<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

Von Frey test er en klassisk metode, der har været meget anvendt til at undersøge den sensoriske funktion af neuropatiske smerter dyr. Det har dog nogle ulemper såsom subjektive data og kravet om en dygtige, erfarne eksperimentator. Til dato, en række ændringer har forbedret metoden von Frey, men det har stadig et par begrænsninger. Nylige rapporter har antydet, at ganganalyse producerer mere præcise og objektive data fra neuropatiske dyrene. Denne protokol demonstrerer, hvordan til at udføre automatiseret gangart analyse for at bestemme graden af neuropatisk smerte i mus. Efter flere dages akklimatisering, fik musene lov at gå frit på glas gulvet til at belyse fodspor. Derefter, kvantificering af fodspor og gangart blev udført gennem videoklip med automatisk analyse af forskellige omvandrende parametre, såsom område pote print, swing tid, vinkel på pote, osv.
Det vigtigste formål med denne undersøgelse er at beskrive for automatiseret gangart analyse og kortvarigt sammenligne det med data fra den klassiske sensoriske test ved hjælp af von Frey glødetråd.

Vi har udført von Frey-testen og automatiseret ganganalyse i CCI mus indtil 10 dage efter CCI kirurgi. Til statistisk analyse, gentagne foranstaltninger af to-vejs variansanalyse (ANOVA) bestemmer de samlede virkninger og Dunnett's post-hoc analyse blev udført for at bestemme p-værdien blandt de eksperimentelle grupper.
Resultaterne vist i figur 1 viser tidsforløb klassisk von Frey test...

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

Den præcise vurdering af smerter reaktion i en neuropatisk dyremodel er kritisk at undersøge Patofysiologi af smerte sygdomme og udvikle nye analgetika. Vi præsenterer en følsom og objektiv metode til at bestemme den sensoriske funktion af gnaver hind pote af en automatiseret gangart analysesystem.

Automatiseret ganganalyse hos mus med kronisk konstriktion skade

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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Comprehensive Understanding of Inactivity-Induced Gait Alteration in Rodents

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Electromagnetic Controlled Closed-Head Model of Mild Traumatic Brain Injury in Mice

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Spinal cord injury (SCI) due to traumatic injuries such as car accidents and falls is associated with permanent spinal cord dysfunction. Creation of ...