Name: sagittal plane kinematic gait analysis in c57bl/6 mice subjected to mog35-55 induced experimental autoimmune encephalomyelitis
Uploaded: 2017-11-04T13:00:00
Description: Watch this Scientific Journal Video about Sagittal Plane Kinematic Gait Analysis in C57BL/6 Mice Subjected to MOG35-55 Induced Experimental Autoimmune Encephalomyelitis at JoVE.com

Nós gostaríamos de reconhecer Sid Chedrawe para sua assistência técnica com as filmagens. Este trabalho foi financiado por fundos da MS Society do Canadá (EGID 2983).

este protocolo está de acordo com o Conselho canadense sobre as orientações de cuidado Animal e foi aprovado pelo Comitê de Universidade de Dalhousie, em animais de laboratório.
 1. construir marcadores reflexivos: <ol><li>usando um furador Hand-Held, socar o número desejado de pequenos círculos de uma folha de papel reflexivo. Cada animal requer 5 marcadores para uma única gravação; dois grandes e três pequenos marcadores.</li>
	<li>Com uma tesoura bem, fazer um corte reto, este...

<ol>
	<li>Giladi, N., Horak, F. B., Hausdorff, 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=Classification+of+gait+disturbances:+distinguishing+between+continuous+and+episodic+changes.">Classification of gait disturbances: distinguishing between continuous and episodic changes.</a> Mov Disord. 28 (11), 1469-1473 (2013).</li><li>Kiehn, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decoding+the+organization+of+spinal+circuits+that+control+locomotion.">Decoding the organization of spinal circuits that control locomotion.</a> Nat Rev Neurosci. 17 (4), 224-238 (2016).</li><li>Akay, T., Tourtellotte, W. G., Arber, S., Jessell, T. 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(91), e51785 (2014).</li><li>Hetze, S., Romer, C., Teufelhart, C., Meisel, A., Engel, O. <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+as+a+method+for+assessing+neurological+outcome+in+a+mouse+model+of+stroke.">Gait analysis as a method for assessing neurological outcome in a mouse model of stroke.</a> J Neurosci Methods. 206 (1), 7-14 (2012).</li><li>Preisig, D. F., 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=High-speed+video+gait+analysis+reveals+early+and+characteristic+locomotor+phenotypes+in+mouse+models+of+neurodegenerative+movement+disorders.">High-speed video gait analysis reveals early and characteristic locomotor phenotypes in mouse models of neurodegenerative movement disorders.</a> Behav Brain Res. 311, 340-353 (2016).</li><li>Leblond, H., L'Esperance, M., Orsal, D., Rossignol, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treadmill+locomotion+in+the+intact+and+spinal+mouse.">Treadmill locomotion in the intact and spinal mouse.</a> J Neurosci. 23 (36), 11411-11419 (2003).</li><li>Ueno, M., Yamashita, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinematic+analyses+reveal+impaired+locomotion+following+injury+of+the+motor+cortex+in+mice.">Kinematic analyses reveal impaired locomotion following injury of the motor cortex in mice.</a> Exp Neurol. 230 (2), 280-290 (2011).</li><li>Zorner, 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=Profiling+locomotor+recovery:+comprehensive+quantification+of+impairments+after+CNS+damage+in+rodents.">Profiling locomotor recovery: comprehensive quantification of impairments after CNS damage in rodents.</a> Nat Methods. 7 (9), 701-708 (2010).</li><li>Pearson, K. 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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+phenotyping+of+mouse+models+of+Parkinson's+disease.">Behavioral phenotyping of mouse models of Parkinson's disease.</a> Behav Brain Res. 211 (1), 1-10 (2010).</li><li>Chen, K., 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=Differential+Histopathological+and+Behavioral+Outcomes+Eight+Weeks+after+Rat+Spinal+Cord+Injury+by+Contusion,+Dislocation,+and+Distraction+Mechanisms.">Differential Histopathological and Behavioral Outcomes Eight Weeks after Rat Spinal Cord Injury by Contusion, Dislocation, and Distraction Mechanisms.</a> J Neurotrauma. 33 (18), 1667-1684 (2016).</li><li>de Bruin, N. M., 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=Multiple+rodent+models+and+behavioral+measures+reveal+unexpected+responses+to+FTY720+and+DMF+in+experimental+autoimmune+encephalomyelitis.">Multiple rodent models and behavioral measures reveal unexpected responses to FTY720 and DMF in experimental autoimmune encephalomyelitis.</a> Behav Brain Res. 300, 160-174 (2016).</li><li>Steinman, L., Zamvil, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+successfully+apply+animal+studies+in+experimental+allergic+encephalomyelitis+to+research+on+multiple+sclerosis.">How to successfully apply animal studies in experimental allergic encephalomyelitis to research on multiple sclerosis.</a> Ann Neurol. 60 (1), 12-21 (2006).</li><li>Emerson, M. R., Gallagher, R. J., Marquis, J. G., LeVine, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancing+the+ability+of+experimental+autoimmune+encephalomyelitis+to+serve+as+a+more+rigorous+model+of+multiple+sclerosis+through+refinement+of+the+experimental+design.">Enhancing the ability of experimental autoimmune encephalomyelitis to serve as a more rigorous model of multiple sclerosis through refinement of the experimental design.</a> Comp Med. 59 (2), 112-128 (2009).</li><li>Bittner, S., Afzali, A. M., Wiendl, H., Meuth, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myelin+oligodendrocyte+glycoprotein+(MOG35-55)+induced+experimental+autoimmune+encephalomyelitis+(EAE)+in+C57BL/6+mice.">Myelin oligodendrocyte glycoprotein (MOG35-55) induced experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice.</a> J Vis Exp. (86), (2014).</li><li>Beeton, C., Garcia, A., Chandy, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+and+clinical+scoring+of+chronic-relapsing+experimental+autoimmune+encephalomyelitis.">Induction and clinical scoring of chronic-relapsing experimental autoimmune encephalomyelitis.</a> J Vis Exp. (5), e224 (2007).</li><li>Barthelmes, J., 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=Induction+of+Experimental+Autoimmune+Encephalomyelitis+in+Mice+and+Evaluation+of+the+Disease-dependent+Distribution+of+Immune+Cells+in+Various+Tissues.">Induction of Experimental Autoimmune Encephalomyelitis in Mice and Evaluation of the Disease-dependent Distribution of Immune Cells in Various Tissues.</a> J Vis Exp. (111), (2016).</li><li>Shaw, M. K., Zhao, X. Q., Tse, H. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overcoming+unresponsiveness+in+experimental+autoimmune+encephalomyelitis+(EAE)+resistant+mouse+strains+by+adoptive+transfer+and+antigenic+challenge.">Overcoming unresponsiveness in experimental autoimmune encephalomyelitis (EAE) resistant mouse strains by adoptive transfer and antigenic challenge.</a> J Vis Exp. (62), e3778 (2012).</li><li>Stromnes, I. M., Goverman, 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=Passive+induction+of+experimental+allergic+encephalomyelitis.">Passive induction of experimental allergic encephalomyelitis.</a> Nat Protoc. 1 (4), 1952-1960 (2006).</li><li>Stromnes, I. M., Goverman, 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=Active+induction+of+experimental+allergic+encephalomyelitis.">Active induction of experimental allergic encephalomyelitis.</a> Nat Protoc. 1 (4), 1810-1819 (2006).</li><li>Fiander, M. D., Stifani, N., Nichols, M., Akay, T., Robertson, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinematic+gait+parameters+are+highly+sensitive+measures+of+motor+deficits+and+spinal+cord+injury+in+mice+subjected+to+experimental+autoimmune+encephalomyelitis.">Kinematic gait parameters are highly sensitive measures of motor deficits and spinal cord injury in mice subjected to experimental autoimmune encephalomyelitis.</a> Behav Brain Res. 317, 95-108 (2017).</li><li>Jones, M. V., 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=Behavioral+and+pathological+outcomes+in+MOG+35-55+experimental+autoimmune+encephalomyelitis.">Behavioral and pathological outcomes in MOG 35-55 experimental autoimmune encephalomyelitis.</a> J Neuroimmunol. 199 (1-2), 83-93 (2008).</li><li>van den Berg, R., Laman, J. D., van Meurs, M., Hintzen, R. Q., Hoogenraad, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rotarod+motor+performance+and+advanced+spinal+cord+lesion+image+analysis+refine+assessment+of+neurodegeneration+in+experimental+autoimmune+encephalomyelitis.">Rotarod motor performance and advanced spinal cord lesion image analysis refine assessment of neurodegeneration in experimental autoimmune encephalomyelitis.</a> J Neurosci Methods. 262, 66-76 (2016).</li><li>Sasaki, M., Lankford, K. L., Brown, R. J., Ruddle, N. H., Kocsis, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focal+experimental+autoimmune+encephalomyelitis+in+the+Lewis+rat+induced+by+immunization+with+myelin+oligodendrocyte+glycoprotein+and+intraspinal+injection+of+vascular+endothelial+growth+factor.">Focal experimental autoimmune encephalomyelitis in the Lewis rat induced by immunization with myelin oligodendrocyte glycoprotein and intraspinal injection of vascular endothelial growth factor.</a> Glia. 58 (13), 1523-1531 (2010).</li><li>Merkler, D., Ernsting, T., Kerschensteiner, M., Bruck, W., Stadelmann, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+focal+EAE+model+of+cortical+demyelination:+multiple+sclerosis-like+lesions+with+rapid+resolution+of+inflammation+and+extensive+remyelination.">A new focal EAE model of cortical demyelination: multiple sclerosis-like lesions with rapid resolution of inflammation and extensive remyelination.</a> Brain. 129 (Pt 8), 1972-1983 (2006).</li><li>Franco-Pons, N., Torrente, M., Colomina, M. 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Em camundongos com EAE, os dois métodos mais comuns de medir o déficit motor estão marcando clínicos em cair a latência de um rotarod27,28. Estas técnicas têm várias limitações. Apesar de conveniente e amplamente utilizado, marcando clínica é limitada pela rendendo apenas ordinais dados nível, significando que a magnitude das diferenças entre os escores clínicos não são conhecidos. Clínica de Pontuação também sofre de ser incapaz de fornecer ...

Marcha é uma série de movimentos repetitivos dos membros utilizados para alcançar a locomoção. Marcha é composta por ciclos de passo, que são divididos em duas fases: a fase de postura, que é quando o pé está se movendo para trás no chão para impulsionar o corpo para a frente; e a fase de balanço, onde o pé está fora o chão e se movendo para a frente. Distúrbios da marcha são características da marca registrada de muitas doenças neurodegenerativas, tais como a lesão da medula espinhal (SCI), esclerose múltipla (em), esclerose lateral amiotrófica (ela), doença de Parkinson (PD) e acidente vascular cerebral; modelos pré-clínicos de roedores destes transtornos frequentemente recapitular sua marcha respectivas imparidades1. Os mecanismos de controle básico de locomoção em camundongos têm sido intensamente estudados2,3. Além disso, existem modelos de rato de muitos distúrbios neurológicos humana4. Análise de marcha em ratos é, portanto, uma abordagem atraente para medir vários aspectos dos défices de motor que tem conhecido correlações anatômicas. O estudo da marcha em modelos do rato pode fornecer insights sobre as bases neuropatológicas dos défices locomotoras em doenças neurodegenerativas e possibilitam a identificação de potenciais tratamentos.
Algumas técnicas que foram usadas para medir a marcha em roedores incluem inspeção visual (por exemplo, o Basso rato escala5 e teste de campo aberto6) e análise da marcha da ventral avião7. Mais recentemente, métodos para medir sagital cinemática dos movimentos do membro posterior ganharam popularidade, porque eles fornecem mais informações sobre a execução do movimento e, consequentemente, são mais sensíveis às mudanças sutis em marcha8, 9 , 10 , 11. cinemáticas técnicas desenvolvidas para estudar o movimento do membro posterior no plano sagital durante a caminhada em uma esteira,9,12 têm sido muito estudadas no contexto da SCI, ALS, lesões traumáticas corticais, acidente vascular cerebral, e A doença de Huntington8,9,10,11,13,14,15,16. Em contraste, estas técnicas têm visto o uso limitado no estudo do aparelho locomotoras défices para modelos de rato de esclerose múltipla17.
Encefalomielite auto-imune experimental (EAE) é o modelo do MS18rato mais comumente usados. Os dois principais métodos de induzir EAE é através de inoculação de ativa ou passiva. Em EAE ativo, os ratos são imunizados com antígenos da mielina, causando neuroinflammation de mediada por células T auto-reativas e desmielinização na medula espinhal e cerebelo. EAE passiva, por outro lado, é induzida pela transferência de células auto-reativas T de um mouse com EAE ativo para um ingênuo rato19. Conforme descrito em outro lugar, o curso da doença e neuropatologia são influenciados por antígenos do sistema nervoso central (SNC) e rato Coe20,21,22,23,24 ,25. Em experimentos EAE, controle de ratos são injetados com completa adjutor de Freund (CFA) sem o antígeno de mielina. EAE é caracterizada pela crescente paralisia que começa com fraqueza de cauda e potencialmente pode envolver os membros dianteiros, resultando em ataxia e paralisia20. Nós recentemente têm caracterizado as mudanças de marcha em camundongos C57Bl/6 sujeitados a glicoproteína de mielina oligodendrocyte 35-55 (MOG35-55)-induzido EAE. Estes estudos mostraram-se análise de marcha ser superior do que a clássica análise comportamental porque desvios do movimento normal do tornozelo são altamente correlacionados com o grau de perda de substância branca na medula espinhal lombar de EAE ratos26. Por outro lado, a força da correlação entre perda de substância branca e duas outras medidas comportamentais tradicionais (marcando clínicos e rotarod) foi muito mais fraco26.
Descrevemos aqui a utilização da análise cinemática da marcha para detectar déficits de movimento no plano sagital de ratos EAE andando em uma esteira. Cinco marcadores reflexivos foram colocados em um membro posterior para identificar o movimento de quadril, joelho e articulações do tornozelo em gravações de vídeo de alta velocidade. Software de análise de movimento foi usado para extrair dados cinemáticos sobre excursões conjuntas. A utilidade destas técnicas para quantificar os défices de movimento para o modelo de35-55 MOG da EAE são discutidos. Essas técnicas também são aplicáveis para o estudo de déficits da marcha em outros modelos de rato de doenças neurodegenerativas.

<table border="0" cellpadding="0" cellspacing="0" width="893">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Camera</td>
			<td style="width:167px;">Nikon</td>
			<td style="width:344px;">Nikon D750</td>
			<td style="width:167px;">Used to film the video</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Reflective tape</td>
			<td>B&#38;L Engineering</td>
			<td>MKR-Tape-2</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Fine scissors</td>
			<td>Fine Science Tools</td>
			<td>15023-10</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Forceps</td>
			<td>Fine Science Tools</td>
			<td>11252-20</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Glue gun</td>
			<td>Craftsmart</td>
			<td>E231647</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">scalpel handle #4</td>
			<td>Roboz</td>
			<td>R5-9884</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Scalpel Blade No.10</td>
			<td>Feather</td>
			<td>2020-12</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">C57BL/6 mice</td>
			<td>Charles River Laboratories</td>
			<td style="width:344px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Anesthetic machine</td>
			<td>EZ Anesthesia</td>
			<td>EZ-AF9000 Auto Flow System</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Recirculating water heating blanket</td>
			<td>Androit</td>
			<td>HTP-1500</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">topical eye lubricant</td>
			<td>Refresh</td>
			<td>DIN00210889</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Shaver</td>
			<td>Oster</td>
			<td>78997-010</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">High speed camera</td>
			<td>Fastec</td>
			<td>Fastec IL3-100</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">High power light</td>
			<td>Smith Victor Corporation</td>
			<td>Model 700 SG (600 Watt quartz light, 120 Volts)</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Light Stand</td>
			<td>Promaster</td>
			<td>LS1</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Treadmill</td>
			<td colspan="2">Custom built at the Zoological Institute, University of Cologne</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Microsoft Excel 2016</td>
			<td>Microsoft</td>
			<td>Version 2016</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">KinemaJ</td>
			<td>Nicolas Stifani</td>
			<td></td>
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sagittal plane kinematic gait analysis in c57bl/6 mice subjected to mog35-55 induced experimental autoimmune encephalomyelitis

Análise cinemática da marcha no plano sagital frequentemente tem sido usado para caracterizar o déficit motor em esclerose múltipla (MS). Descrevemos a aplicação destas técnicas para identificar déficits da marcha em um modelo do rato do MS, conhecido como encefalomielite auto-imune experimental (EAE). Défices de paralisia e motor em ratos submetidos a EAE normalmente são avaliados usando uma escala de Pontuação clínica. No entanto, esta escala produz somente dados ordinais que fornece pouca informação acerca da natureza exacta dos défices motor. Severidade da doença EAE também tenha sido avaliada por desempenho rotarod, que fornece uma medida de coordenação motora geral. Por outro lado, análise cinemática da marcha do membro posterior no plano sagital gera informações altamente precisas sobre como o movimento é prejudicado. Para executar este procedimento, marcadores reflexivos são colocados em um membro posterior para detectar movimento comum, enquanto que um rato está caminhando em uma esteira. Software de análise de movimento é usado para medir o movimento dos marcadores durante a caminhada. Parâmetros cinemáticos da marcha em seguida são derivados de dados resultantes. Mostramos como esses parâmetros da marcha podem ser usados para quantificar movimentos prejudicados das articulações do tornozelo, joelho e quadril na EAE. Estas técnicas podem ser usadas para melhor compreender os mecanismos da doença e identificar potenciais tratamentos para MS e outras doenças neurodegenerativas que afectar a mobilidade.

A Figura 1 é uma representação esquemática do procedimento utilizado para a análise cinemática da marcha. Primeiros, reflexivos marcadores são feitos e colocados em um mouse em 5 pontos anatômicos. Marcha é então gravada enquanto o mouse está caminhando em uma esteira. Software de análise de movimento é usado para extrair dados cinemáticos para análise posterior.
Figura 2A C representar o ciclo do passo...

Watch this Scientific Journal Video about Sagittal Plane Kinematic Gait Analysis in C57BL/6 Mice Subjected to MOG35-55 Induced Experimental Autoimmune Encephalomyelitis at JoVE.com

Sagittal Plane Kinematic Gait Analysis in C57BL/6 Mice Subjected to MOG35-55 Induced Experimental Autoimmune Encephalomyelitis

Análise cinemática da marcha de sagital em camundongos C57BL/6 sujeitado a MOG35-55 induzido encefalomielite auto-imune Experimental

Análise cinemática da marcha no plano sagital produz informações altamente precisas sobre como o movimento é executado. Descrevemos a aplicação destas técnicas para identificar déficits da marcha de ratos submetidos a desmielinização mediada por doença auto-imune. Esses métodos também podem ser usados para caracterizar os déficits da marcha para outros modelos de rato com locomoção prejudicada.

Kinematic gait analysis in the sagittal plane has frequently been used to characterize motor deficits in multiple sclerosis (MS). We describe the ...

The overall goal of this procedure is to measure functional motor impairments in the experimental autoimmune encephalomyelitis or EAE, mouse model multiple sclerosis using kinematic gait analysis. The application of kinematic gait analysis to mouse-walking behavior has been previously established and described by others. Neurological deficits in an EAE result from neural inflammation and sporadic white matter loss throughout the spinal cord and the cerebellum.Traditionally, motor stability in EAE mice has been assessed using clinical scoring systems, in which mice are assigned a clinical score based on the observer's impression of the severity of motor deficits. Data from clinical scores are ordinal and do not correlate well with the spinal cord histopathology. Kinematic gait analysis has recently been shown to be a better behavioral correlate of white matter loss than clinical scores, in addition to providing an objective description of walking deficits in the EAE mice.This technique involves placing reflective markers on the high limbs of mice and allowing them to walk on a treadmill while being recorded with a high-speed camera. Kinematic parameters are then extracted from the video using motion analysis software. The first step is to make the markers that will be placed on the animal's hind leg.The reflection of light off of these markers allows the coordinates of anatomical points on the leg to be extracted from the videos. To begin, punch the desired number of circles from the sheet of reflective paper. Each mouse requires five markers per recording, two large and three small.Using fine scissors, make a cut extending from the perimeter to the center of the circle. Peel off the paper backing to reveal the adhesive surface using fine forceps. Grip the marker with forceps and using your finger, roll the marker in on itself to form a cone.To make a small marker, make a longer cut and curl the cone more tightly. To make a large marker, make a shorter cut and curl the cone more loosely. Using a hot glue gun, fill the inside of the cone with glue and adhere it to a piece of cardboard.It is important to fully fill the marker with glue to prevent the marker from collapsing when handling during recording. Once the glue is dry, cut the marker off using a scalpel. Ensure that you cut away from your body.The next step is to prepare the animal for recording. To do this, the reflective markers must be adhered to the hind limb of the mouse at the proper anatomical locations. This is conducted under light anesthesia.Place the mouse in an induction chamber and anesthetize with 2.5%isoflurane. Once unconscious, transfer the mouse from the induction chamber to a nose cone or a recirculating water heating pad. Apply a topical lubricant to both eyes.Shave the desired hind limb extending from the ankle to the spine and the bottom of the ribs. In this demonstration, we are recoding movements of the right hind limb, but either or both limbs may be used. Locate the iliac crest by bringing both knees together with your thumb and forefinger and palpating just below the ribs.Mark this location using a permanent marker. Locate the hip by extending the leg and moving it back and forth. Place a marker over the hip joint, which is the point of articulation between the head of the femur and the pelvis.Using a flexible ruler, measure and record the length of the tibia or shank of the mouse. Measure and record the length of the femur or thigh as well. To isolate the fourth toe for marker placement, tape down the rest of the foot.Grasp a small marker with forceps and dip the flat end in glue. Place the small marker on the tip of the fourth toe. Place another small marker on the metatarsophalangeal joint.Place the last small marker on the ankle. Place a large marker on the hip joint directly over the mark on the skin. Place the second large marker on the mark over the iliac crest.Remove the tape from the foot. Place the mouse in a recovery cage and transport immediately to the gait recording room. The next step is recording the mouse walking on a treadmill.This picture is of our treadmill recording setup, showing the light, high-speed camera, and treadmill. Prior to recording the mouse's gait, take a picture of a calibration block with known dimensions on the treadmill. This will allow the pixels in the video to be converted to real measurements.It is critical that the camera angle and position at which the calibration picture is taken remains the same during the recording of walking behavior. Place the mouse in the treadmill. Increase the treadmill's speed gradually to orient the mouse in the correct direction.Slowly accelerate to 20 centimeters per second, which is the ideal running speed for obtaining recordings of steady gait. For accurate analysis, it's best to record eight to 12 step cycles of steady walking behavior. This is an example of consistent walking.The following is the same video shown at half speed. Because mice walk so quickly, it can be useful to view the videos at slower speeds to count step cycles and to better appreciate the gait pattern. This is one example of an EAE mouse walking on the treadmill.This mouse is unable to support its body weight as its pelvis is very low to the ground. Additionally, it is having difficulty lifting its foot off the ground during the swing phase. This is an example of another EAE mouse that has less severe motor deficits.The EAE mouse is walking on its toes with reduced movement at the ankle joint. This gives the mouse an uneven gait. The following are examples of behaviors that will compound analysis.Recordings, including these behaviors, should not be used. Lagging occurs when the mouse stops walking and travels to the back of the treadmill, but then resumes walking. This may occur in any mouse, but will occur more frequently at lower speeds.Rearing occurs when the mouse shifts its weight on its back hind limbs and raises its head and upper body. This behavior is common in anxious mice. The following are examples of poor lighting that will lead to issues with analysis of the markers'position in time and space.If the lighting is too dim, markers may not reflect enough light to be recognized by the computer software. In this video, the toe markers are not easily visible due to insufficient lighting. If the lighting is too bright, objects other than the markers may reflect too much light and be recognized as a marker.In this video, the toe markers appear to be merging with the reflective parts of the treadmill. Data from the reflective markers on mice walking can be used to create stick models of the leg from which kinematic parameters can be extracted. This is a recording of a mouse walking with a stick model of the leg superimposed.Note that in this video, there is a sixth marker on the knee, which is not necessary because the location of the knee can be triangulated from the positions of the hip and ankle joints, and the measured lengths of the femur and tibia. Because markers over the knee joint are often inaccurate due to skin slippage, triangulation is the preferred method. A mouse's step cycle can be divided into two main phases.The stance phase and the swing phase. This stick diagram can be used to qualitatively illustrate the movement of the hind limb in time and space. An example of this could be to assess the movement of the hind limb at different times points throughout the study.This example shows that the hind leg is compressed during the stance phase, indicating that the mouse has difficulty supporting its body weight. This is reflected by increased flexion of the knee and ankle joints. At the later time point, this has partially recovered.This video illustrates the relationship between the walking behavior and joint angles over time. The hip, knee, and ankle angle waveforms can be extracted from each recording. The joint angles can then be averaged over eight to 12 consecutive step cycles, resulting in an average step cycle that can be used for further analysis.This graph represents a knee joint waveform averaging from 10 consecutive step cycles. The data has been normalized, so that the length of the stance and swing phases are 100 frames respectively. The clear background represents the swing phase and the green background represents the stance phase.For this mouse, the walking behavior from week to week was very consistent and the step-cycle waveforms from each week overlapped considerably. However, there can also be substantial variability in gait in healthy mice as seen in this graph. The degree of variability shown here is acceptable and within the range of what might be expected from a mouse.This graph represents the knee step cycle from the EAE mouse recorded on three consecutive weeks. There is a slight change in the shape of the step cycle at week two and a substantial deviation by week three, which the mouse's knee is much more flexed and does not extend during walking. There are numerous parameters that can be measured using this technique.We'll briefly describe three. The average angle is obtained by averaging all angles throughout the normalized step cycle. In this instance, the average angle decreases throughout the study, suggesting that the mice are not extending their knees as much as normal.The range of motion is obtained by subtracting the smallest angle from the largest angle in the normalized step cycle. This parameter can give you insight into joint flexibility, rigidity, or weakness. In this example, the knee range of motion decreases throughout the study indicating that the mice are not able to move their knee normally, possibly due to muscle weakness.Root-Mean-Square difference is a method used to measure deviation of step-cycle waveforms from the baseline recording. This parameter tells you how much deviation there is from the initial recording. Kinematic gait analysis is a valuable technique that can be used to sensitively detect and describe changes in gait.The application of kinematic gait analysis to EAE studies may be a valuable tool in understanding the functional consequences of spinal cord pathology in this model. It may facilitate discovery of novel treatments for multiple sclerosis. Additionally, kinematic gait analysis is not limited to the context of EAE.This technique has previously been used in mouse models with spinal cord injury, amyotrophic lateral sclerosis, Huntington's disease and stroke, and can be applied to other mouse models with neurological disorders, including Parkinson's disease.

Research

JoVE Journal

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