Esta investigación fue financiada por becas de la Consejo de Investigación Médica, las Ciencias Naturales e Ingeniería de Investigación de Canadá, y la Red Canadiense de accidente cerebrovascular. GAM también fue apoyada por la Fundación del Patrimonio de Alberta para la Investigación Médica.

Escalera de mano a pie aparato de prueba El peldaño de la escalera horizontal caminar aparato de prueba consistía en las paredes laterales de plexiglás transparente y los peldaños de metal (3 mm de diámetro), que puede ser insertado para crear un piso con una distancia mínima de 1 cm entre los peldaños (ver Fig. 1;. Metz y Whishaw , 2003). Las paredes laterales eran de 1 m de largo y 19 cm de alto medido desde la altura de los peldaños. La escalera fue elevada a 30 cm del suelo, con una jaula de arranque en punto muerto y un refugio (jaula) al final. Porque los animales se habituaron durante el entrenamiento, la elevación de los aparatos era poco probable que cause ansiedad. La anchura del callejón se ajusta al tamaño del animal, por lo que era de aproximadamente 1 cm más ancho que un animal para evitar que el animal darse vuelta. La dificultad de la tarea fue modificada mediante la variación de la posición de los peldaños de metal. Un patrón regular de los peldaños permitió a los animales para aprender el patrón a lo largo de varios talleres, y para anticipar la posición de los peldaños (Fig. 1, un patrón). Un patrón irregular que se ha cambiado de un ensayo a evitar que el animal aprenda el modelo (Fig. 1, el patrón B). Para la disposición regular, los peldaños estaban espaciados a intervalos de 2 cm. Para el patrón irregular, la distancia de los peldaños variado sistemáticamente de 1 a 5 cm. Cinco plantillas de los patrones de escalón irregular se utilizaron, de manera que las mismas pautas se aplican a todos los animales para estandarizar la dificultad de la prueba y mejorar la comparabilidad de los resultados (ver resultados). Grabación de video Una cámara (Canovision, Canon Inc.) se colocó en un ángulo ventral leve, por lo que las posiciones de los cuatro miembros podría ser grabado simultáneamente. La velocidad de obturación se fija en 500 - 2000 s. El videograbaciones fueron analizados mediante el cuadro-por-marco de análisis a 30 f / seg. La formación del comportamiento y análisis de pruebas Los animales fueron entrenados para cruzar la escalera de una jaula neutral para llegar a su jaula, por lo que la jaula con los compañeros de camada siempre que el refuerzo positivo para caminar. Todos los animales cruzaron la escalera en la misma dirección. No se le dio mayor refuerzo para motivar a los animales para cruzar la escalera. Todos los animales fueron entrenados y evaluados cinco veces por sesión. Puntuación falta de pie La evaluación cualitativa de la colocación de patas delanteras y la extremidad posterior se realizó con un sistema de fallas de puntuación pie como se describió anteriormente (Metz y Whishaw, 2003). Análisis se realizó mediante la inspección de las grabaciones de video cuadro por cuadro. A unos pasos consecutivos de cada miembro se analizaron. Por lo tanto, el último paso antes de una interrupción de la marcha, tales como una parada o una falta de pie, y el primer paso después de una interrupción no se anotó. El ciclo de intensificación actuó por última vez al final de la escalera también se excluyó de la puntuación. La colocación de la extremidad fue evaluado en términos de la colocación de las extremidades en un peldaño y el miembro saliente entre peldaños cuando se produjo un fallo. Los tipos de colocación de los pies o la pata en los peldaños se calificaron en una escala de 7 categorías (ver fig. 2). Colocación de los pies o la pata en el último peldaño se clasifican de acuerdo a su posición y los errores producidos en la precisión de la colocación. (0) Total de menos. 0 puntos se da cuando el miembro perdido completamente un peldaño, es decir, no lo he tocado, y se produjo una caída. Una caída se define como una rama profunda caída en entre los peldaños y la postura corporal y el equilibrio fueron molestados. (1) de deslizamiento profundo. La extremidad fue colocado inicialmente en un peldaño, luego se deslizó al soporte de peso y causó una caída. (2) deslizamiento leve. La extremidad se colocaba en un escalón, resbaló cuando se soporta peso, pero no se tradujo en una caída, ni interrumpir el ciclo de la marcha. En este caso, el animal era capaz de mantener el equilibrio y continuar con una marcha coordinada. (3) de repuesto. El miembro fue colocado en un peldaño, pero antes de que se carga de peso se levantó rápidamente y se coloca en otro peldaño. (4) Corrección. El miembro dirigido por un peldaño, pero fue colocado en otro peldaño, sin tocar la primera. Por otra parte, una puntuación de 4 se registró cuando un miembro se coloca en un peldaño y se reposicionó rápidamente sin dejar de ser en el mismo peldaño. (5) la colocación parcial. La extremidad se colocaba en un renglón, ya sea con la muñeca o dedos de las patas delanteras o en el talón o los dedos de las extremidades inferiores. (6) La correcta colocación. La parte media de la palma de una extremidad se colocó en el peldaño con el apoyo de peso. Cuando los errores se produjeron diferentes al mismo tiempo, la más baja de las puntuaciones se registró. Por ejemplo, si un pie se colocó por primera vez en un peldaño y se coloca en otro en el mismo paso (Puntuación 3), y luego se resbaló y cayó en entre los peldaños (Puntuación 1), una puntuación de 1 se registró. Blancoen una caída se produjo, sólo el miembro de iniciar el error fue calificado y ninguno de los otros miembros se anotó hasta que el animal había reposicionado todos los miembros. Número de errores de los cinco ensayos fueron en promedio para el análisis. Colocación de los pies análisis de precisión (número de errores) El número de errores en cada cruce se había contado. Los errores se determinaron con base en el sistema de fallas de puntuación pie. Un error se define como la colocación de cada miembro que recibió una puntuación de 0 puntos, 1 ó 2, es decir, representa un error de cualquier tipo de deslizamiento del pie o se pierda total. El número de errores y el número de pasos que se registró para cada miembro por separado. A partir de estos datos, el número medio de errores por paso y se calculó un promedio de cinco ensayos. El segundo parámetro cuantitativo se analizó el tiempo medio necesario para atravesar toda la longitud de la tarea de la escalera. La medición del tiempo se inició después de que el animal fue colocado en la escalera y comenzó a caminar a todo lo largo de la misma. El tiempo que el animal pasó en una parada no fue incluido en la medición. Pata dígitos puntuación La puntuación pata dígitos fue grabado cuando la pata se coloca correctamente con su parte media en un peldaño (puntuación es decir, 6 en el sistema de fallas de puntuación pie). El grado en que los dígitos de la pata puede ser flexionada en torno a un sonado fue clasificado en una escala de tres puntos (Fig. 2). La puntuación se aplica a la posición con el apoyo de peso máximo, es decir, la extremidad anterior se coloca verticalmente en un peldaño. Los resultados se dieron de la siguiente manera: (0) dígitos se cerró en un aproximado de 90 grados de ángulo. (1) Los dígitos se cerró en un ángulo aproximado de 45. (2) dígitos completamente flexionadas alrededor de la campana. Decenas de cinco pasos fueron en promedio y para el análisis. Leyendas de las figuras <img src="/files/ftp_upload/old/1204_2009_1_20_6/Fig 1 Grid.png" alt="la figura 1" /> Fig. 1. El peldaño de la escalera calificados caminar aparato de prueba en la vista frontolateral, con las medidas específicas del aparato indicado. Dos patrones diferentes peldaño se utilizaron en el presente estudio: un patrón, arreglo sonado regular, B patrón, arreglo escalón irregular. La disposición irregular peldaño variaron entre los ensayos consecutivos. <img src="/files/ftp_upload/old/1204_2009_1_20_7/Fig 2 (angle lines).png" alt="la figura 2" /> Fig. 2. Fotografías que ilustran Representante de las tres categorías de la puntuación dígitos. Tenga en cuenta el ángulo de las cifras en relación con la extremidad anterior en apoyo al peso.

<ol>
	<li>Chu, C. J., Jones, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experience-dependent+structural+plasticity+in+cortex+heterotopic+to+focal+sensorimotor+cortical+damage.">Experience-dependent structural plasticity in cortex heterotopic to focal sensorimotor cortical damage.</a> Exp Neurol. 166, 403-414 (2000).</li><li>Emerick, A. J., Kartje, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+recovery+and+anatomical+plasticity+in+adult+rats+after+cortical+lesion+and+treatment+with+monoclonal+antibody+IN-1.">Behavioral recovery and anatomical plasticity in adult rats after cortical lesion and treatment with monoclonal antibody IN-1.</a> Behav Brain Res. 152, 315-325 (2004).</li><li>Faraji, J., Metz, 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=Sequential+bilateral+striatal+lesions+have+additive+effects+on+single+skilled+limb+use+in+rats.">Sequential bilateral striatal lesions have additive effects on single skilled limb use in rats.</a> Behav Brain Res. 177, 195-204 (2007).</li><li>Farr, T. D., Liu, L., Colwell, K. L., Whishaw, I. Q., Metz, 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=Bilateral+alteration+in+stepping+pattern+after+unilateral+motor+cortex+injury:+a+new+test+strategy+for+analysis+of+skilled+limb+movements+in+neurological+mouse+models.">Bilateral alteration in stepping pattern after unilateral motor cortex injury: a new test strategy for analysis of skilled limb movements in neurological mouse models.</a> J Neurosci Methods. 153, 104-113 (2006).</li><li>Kleim, J. A., Barbay, S., Nudo, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+reorganization+of+the+rat+motor+cortex+following+motor+skill+learning.">Functional reorganization of the rat motor cortex following motor skill learning.</a> J Neurophysiol. 80, 3321-3325 (1998).</li><li>Merkler, D., Metz, G. A., Raineteau, O., Dietz, V., Schwab, M. E., Fouad, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Locomotor+recovery+in+spinal+cord-injured+rats+treated+with+an+antibody+neutralizing+the+myelin-associated+neurite+growth+inhibitor+Nogo-A.">Locomotor recovery in spinal cord-injured rats treated with an antibody neutralizing the myelin-associated neurite growth inhibitor Nogo-A.</a> J Neurosci. 21, 3665-3673 (2001).</li><li>McVea, D. A., Pearson, 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=Stepping+of+the+forelegs+over+obstacles+establishes+long-lasting+memories+in+cats.">Stepping of the forelegs over obstacles establishes long-lasting memories in cats.</a> Curr Biol. 17, R621-R623 (2007).</li><li>Metz, G. A. S., Dietz, V., Schwab, M. E., Meent, H. v. a. n. d. 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+effects+of+unilateral+pyramidal+tract+section+on+hindlimb+motor+performance+in+the+rat.">The effects of unilateral pyramidal tract section on hindlimb motor performance in the rat.</a> Behav Brain Res. 95, 37-46 (1998).</li><li>Metz, G. A. S., Dietz, V., Schwab, M. E., Fouad, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+testing+of+motor+function+in+spinal+cord+injured+rats.">Efficient testing of motor function in spinal cord injured rats.</a> Brain Res. 883, 165-177 (2000).</li><li>Metz, G. A., Whishaw, I. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug-induced+rotation+intensity+in+unilateral+dopamine-depleted+rats+is+not+correlated+with+end+point+or+qualitative+measures+of+forelimb+or+hindlimb+motor+performance.">Drug-induced rotation intensity in unilateral dopamine-depleted rats is not correlated with end point or qualitative measures of forelimb or hindlimb motor performance.</a> Neuroscience. 111, 325-336 (2002).</li><li>Miklyaeva, E. I., Castaneda, E., Whishaw, I. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skilled+reaching+deficits+in+unilateral+dopamine-depleted+rats:+impairments+in+movement+and+posture+and+compensatory+adjustments.">Skilled reaching deficits in unilateral dopamine-depleted rats: impairments in movement and posture and compensatory adjustments.</a> J Neurosci. 14, 7148-7158 (1994).</li><li>Muir, G. D., Whishaw, I. Q., Kolb, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Locomotion in The Behavior of the laboratory rat. , 150-161 (2005).</li><li>Muir, G. D., Whishaw, I. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ground+reaction+forces+in+locomoting+hemi-parkinsonian+rats:+a+definitive+test+for+impairments+and+compensations.">Ground reaction forces in locomoting hemi-parkinsonian rats: a definitive test for impairments and compensations.</a> Exp Brain Res. 126, 307-314 (1999).</li><li>Muir, G. D., Whishaw, I. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Red+nucleus+lesions+impair+overground+locomotion+in+rats:+a+kinetic+analysis.">Red nucleus lesions impair overground locomotion in rats: a kinetic analysis.</a> Eur J Neurosci. 12, 1113-1122 (2000).</li><li>Riek-Burchardt, M., Henrich-Noack, P., Metz, G. A., Reymann, 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=Detection+of+chronic+sensorimotor+impairments+in+the+ladder+rung+walking+task+in+rats+with+endothelin-1-induced+mild+focal+ischemia.">Detection of chronic sensorimotor impairments in the ladder rung walking task in rats with endothelin-1-induced mild focal ischemia.</a> J Neurosci Methods. 137, 227-233 (2004).</li><li>Ploughman, M., Attwood, Z., White, N., Dor&#233;, J. J., Corbett, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endurance+exercise+facilitates+relearning+of+forelimb+motor+skill+after+focal+ischemia.">Endurance exercise facilitates relearning of forelimb motor skill after focal ischemia.</a> Eur J Neurosci. 25, 3453-3460 (2007).</li><li>Thallmair, M., Metz, G. A. S., Z'Graggen, W. J., Kartje, G. L., Schwab, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+recovery+parallels+enhanced+plasticity+of+the+lesioned+adult+CNS+by+antibodies+to+neurite+growth+inhibitors.">Functional recovery parallels enhanced plasticity of the lesioned adult CNS by antibodies to neurite growth inhibitors.</a> Nature Neurosci. 1, 124-131 (1998).</li><li>Meyenburg, J. v. o. n., Brosamle, C., Metz, G. A., Schwab, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+and+sprouting+of+chronically+injured+corticospinal+tract+fibers+in+adult+rats+promoted+by+NT-3+and+the+mAb+IN-1,+which+neutralizes+myelin-associated+neurite+growth+inhibitors.">Regeneration and sprouting of chronically injured corticospinal tract fibers in adult rats promoted by NT-3 and the mAb IN-1, which neutralizes myelin-associated neurite growth inhibitors.</a> Exp Neurol. 154, 583-594 (1998).</li><li>Whishaw, I. Q., Coles, B. L., Pellis, S. M., Miklyaeva, E. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impairments+and+compensation+in+mouth+and+limb+use+in+free+feeding+after+unilateral+dopamine+depletions+in+a+rat+analog+of+human+Parkinson's+disease.">Impairments and compensation in mouth and limb use in free feeding after unilateral dopamine depletions in a rat analog of human Parkinson's disease.</a> Behav Brain Res. 84, 167-177 (1997a).</li><li>Whishaw, I. Q., Woodward, N. C., Miklyaeva, E., Pellis, 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=Analysis+of+limb+use+by+control+rats+and+unilateral+DA-depleted+rats+in+the+Montoya+staircase+test:+movements,+impairments+and+compensatory+strategies.">Analysis of limb use by control rats and unilateral DA-depleted rats in the Montoya staircase test: movements, impairments and compensatory strategies.</a> Behav Brain Res. 89, 167-177 (1997b).</li><li>Whishaw, I. Q., Gorny, B., Sarna, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paw+and+limb+use+in+skilled+and+spontaneous+reaching+after+pyramidal+tract,+red+nucleus+and+combined+lesions+in+the+rat:+behavioral+and+anatomical+dissociations.">Paw and limb use in skilled and spontaneous reaching after pyramidal tract, red nucleus and combined lesions in the rat: behavioral and anatomical dissociations.</a> Behav Brain Res. 93, 167-183 (1998).</li><li>Z'Graggen, W. J., Metz, G. A. S., Kartje, G. L., Schwab, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+recovery+and+enhanced+ccrtico-fugal+plasticity+in+the+adult+rat+after+unilateral+pyramidal+tract+section+and+blockade+of+myelin-associated+neurite+growth+inhibitors.">Functional recovery and enhanced ccrtico-fugal plasticity in the adult rat after unilateral pyramidal tract section and blockade of myelin-associated neurite growth inhibitors.</a> J Neurosci. 18, 4744-4757 (1998).</li></ol>

El presente estudio presenta el peldaño de la escalera caminando tarea como una nueva prueba para evaluar el caminar expertos, la colocación de las extremidades y la integridad física de coordinación. El peldaño de la escalera caminando tarea permite la discriminación entre las alteraciones sutiles de la función motora mediante la combinación de análisis cualitativo y cuantitativo de caminar calificado. El procedimiento de análisis de vídeo se puede utilizar para obtener una cuenta más descriptivo de las def...

the ladder rung walking task: a scoring system and its practical application.

El progreso en el desarrollo de modelos animales para / carrera, lesión de la médula espinal, enfermedades neurodegenerativas y otras requieren pruebas de alta sensibilidad para elaborar distintos aspectos de la función motora y para determinar las pérdidas más leves de la capacidad de movimiento. Para mejorar la eficacia y la resolución de las pruebas, las pruebas deben permitir mediciones cualitativas y cuantitativas de la función motora y ser sensible a cambios en el rendimiento durante los períodos de recuperación. El presente estudio describe una nueva tarea para evaluar caminar un experto en la rata para medir tanto la pata delantera y patas traseras función al mismo tiempo. Los animales son obligados a caminar por una escalera horizontal en la que el espaciamiento de los escalones es variable y se cambia periódicamente. Los cambios en el espaciamiento sonado evitar que los animales aprenden la ubicación absoluta y relativa de los peldaños y así minimizar la capacidad de los animales para compensar el deterioro a través del aprendizaje. Además, cambiar el espacio entre los escalones permite que la prueba se usa repetidamente en estudios a largo plazo. Se describen los métodos para la descripción cuantitativa y cualitativa de los resultados anteriores y extremidades inferiores, incluyendo la colocación de las extremidades, paso a paso, la coordinación. Además, el uso de estrategias compensatorias se indica por errores o medidas compensatorias en respuesta a la mala colocación de otro miembro.

Watch this Scientific Journal Video about The Ladder Rung Walking Task: A Scoring System and its Practical Application. at JoVE.com

The Ladder Rung Walking Task: A Scoring System and its Practical Application.

El peldaño de la escalera caminando tarea es una nueva prueba para evaluar el caminar calificados y medir patas delanteras y la extremidad posterior colocación, paso a paso, y entre los miembros de coordinación.

El peldaño de la escalera a pie de la tarea: un sistema de puntuación y su aplicación práctica.

Progress in the development of animal models for/stroke, spinal cord injury, and other neurodegenerative disease requires tests of high sensitivity to ...

the-ladder-rung-walking-task-scoring-system-its-practical

Research

JoVE Journal

Biology

25.9K vistas.  University of Lethbridge. El peldaño de la escalera caminando tarea es una nueva prueba para evaluar el caminar calificados y medir patas delanteras y la extremidad posterior colocación, paso a paso, y entre los miembros de coordinación.

Video: The Ladder Rung Walking Task: A Scoring System and its Practical Application.

Induction and Clinical Scoring of Chronic-Relapsing Experimental Autoimmune Encephalomyelitis

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) that commonly affects young adults. It is characterized by demyelination and glial scaring in areas disseminated in the brain and spinal cord. These lesions alter nerve conduction and induce the disabling neurological deficits that vary with the location of the demyelinated plaques in the CNS (e.g. paraparesis, paralysis, blindness, incontinence).
Experimental autoimmune encephalomyelitis (EAE) is a model for MS. EAE was first induced accidentally in humans during vaccination against rabies, using viruses grown on rabbit spinal cords. Residues of spinal injected with the inactivated virus induced the CNS disease. Following these observations, a first model of EAE was described in non-human primates immunized with a CNS homogenate by Rivers and Schwenther in 1935. EAE has since been generated in a variety of species and can follow different courses depending on the species/strain and immunizing antigen used. For example, immunizing Lewis rats with myelin basic protein in emulsion with adjuvant induces an acute model of EAE, while the same antigen induces a chronic disease in guinea pigs.
The EAE model described here is induced by immunizing DA rats against DA rat spinal cord in emulsion in complete Freund's adjuvant. Rats develop an ascending flaccid paralysis within 7-14 days post-immunization. Clinical signs follow a relapsing-remitting course over several weeks. Pathology shows large immune infiltrates in the CNS and demyelination plaques. Special considerations for taking care for animals with EAE are described at the end of the video.

This video demonstrates the induction and clinical scoring of an animal model of multiple sclerosis: chronic-relapsing experimental autoimmune encephalomyelitis in DA rats. The disease, induced by immunizing rats with an emulsion containing whole rat spinal cord and complete Freund's adjuvant, presents clinical signs resembling the human disease.

La inducción y la puntuación clínica de enfermedades crónico-recurrente encefalomielitis autoinmune experimental

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) that commonly affects young adults. It is characterized by ...

Investigación

Biología

Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat

High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a rotorod, animals remain in approximately the same place making repetitive movements of stepping. Thus the task provides a rich source of information on the details of foot stepping movements. Subjects were hemi-Parkinson analogue rats, produced by injection of 6-hydroxydopamine (6-OHDA) into the right nigrostriatal bundle to deplete nigrostriatal dopamine (DA). The present report provides a video analysis illustration of animals previously were filmed from frontal, lateral, and posterior views as they walked (15). Rating scales and frame-by-frame replay of the video records of stepping behavior indicated that the hemi-Parkinson rats were chronically impaired in posture and limb use contralateral to the DA-depletion. The contralateral limbs participated less in initiating and sustaining propulsion than the ipsilateral limbs. These deficits secondary to unilateral DA-depletion show that the rotorod provides a use task for the analysis of stepping movements.

The rotorod test is used to assess motor status in the walking movement of hemi-Parkinson analogue rats.

El uso de Rotorod como un método para el análisis cualitativo de la Ruta de la rata

High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a ...

Lens Transplantation in Zebrafish and its Application in the Analysis of Eye Mutants

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development can be addressed. The zebrafish is useful for genetic studies due to several advantageous characteristics, including small size, high fecundity, short lifecycle, and ease of care. Lens development occurs rapidly in zebrafish. By 72 hpf, the zebrafish lens is functionally mature [3]. Abundant genetic and molecular resources are available to support research in zebrafish. In addition, the similarity of the zebrafish eye to those of other vertebrates provides basis for its use as an excellent animal model of human defects[4-7]. Several zebrafish mutants exhibit lens abnormalities, including high levels of cell death, which in some cases leads to a complete degeneration of lens tissues [8]. To determine whether lens abnormalities are due to intrinsic causes or to defective interactions with the surrounding tissues, transplantation of a mutant lens into a wild-type eye is performed. Using fire-polished metal needles, mutant or wild-type lenses are carefully dissected from the donor animal, and transferred into the host. To distinguish wild-type and mutant tissues, a transgenic line is used as the donor. This line expresses membrane-bound GFP in all tissues, including the lens. This transplantation technique is an essential tool in the studies of zebrafish lens mutants.

Lens development involves interactions with other tissues. Several zebrafish eye mutants are characterized by an abnormally small lens size. Here we demonstrate a lens transplantation experiment to determine whether this phenotype is due to intrinsic causes or defective interactions with tissues that surround the lens.

Trasplante de lentes en el pez cebra y su aplicación en el análisis de mutantes de ojos

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development ...

Tomato Analyzer: A Useful Software Application to Collect Accurate and Detailed Morphological and Colorimetric Data from Two-dimensional Objects

Measuring fruit morphology and color traits of vegetable and fruit crops in an objective and reproducible way is important for detailed phenotypic analyses of these traits. Tomato Analyzer (TA) is a software program that measures 37 attributes related to two-dimensional shape in a semi-automatic and reproducible manner1,2. Many of these attributes, such as angles at the distal and proximal ends of the fruit and areas of indentation, are difficult to quantify manually. The attributes are organized in ten categories within the software: Basic Measurement, Fruit Shape Index, Blockiness, Homogeneity, Proximal Fruit End Shape, Distal Fruit End Shape, Asymmetry, Internal Eccentricity, Latitudinal Section and Morphometrics. The last category requires neither prior knowledge nor predetermined notions of the shape attributes, so morphometric analysis offers an unbiased option that may be better adapted to high-throughput analyses than attribute analysis. TA also offers the Color Test application that was designed to collect color measurements from scanned images and allow scanning devices to be calibrated using color standards3. 
TA provides several options to export and analyze shape attribute, morphometric, and color data. The data may be exported to an excel file in batch mode (more than 100 images at one time) or exported as individual images. The user can choose between output that displays the average for each attribute for the objects in each image (including standard deviation), or an output that displays the attribute values for each object on the image. TA has been a valuable and effective tool for indentifying and confirming tomato fruit shape Quantitative Trait Loci (QTL), as well as performing in-depth analyses of the effect of key fruit shape genes on plant morphology. Also, TA can be used to objectively classify fruit into various shape categories. Lastly, fruit shape and color traits in other plant species as well as other plant organs such as leaves and seeds can be evaluated with TA.

Tomato Analyzer (TA) quantifies attributes of two dimensional shapes and color in a reproducible and accurate manner. A step-by-step procedure for obtaining high quality digitalized images of tomato fruit, morphological and color analyses of these images and several applications using the data generated through this software are described.

Analizador de tomate: una aplicación de software útiles para recopilar los datos morfológicos y colorimétrico precisa y detallada de objetos en dos dimensiones

Measuring fruit morphology and color traits of vegetable and fruit crops in an objective and reproducible way is important for detailed phenotypic ...

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the environment, but also determines body shape and plays a role in motility4-6. Several layers secreted by epidermal cells comprise the cuticle, including an outermost lipid layer7.
Circumferential ridges in the cuticle called annuli pattern the length of the animal and are present during all stages of development8. Alae are longitudinal ridges that are present during specific stages of development, including L1, dauer, and adult stages2,9. Mutations in genes that affect cuticular collagen organization can alter cuticular structure and animal body morphology5,6,10,11. While cuticular imaging using compound microscopy with DIC optics is possible, current methods that highlight cuticular structures include fluorescent transgene expression12, antibody staining13, and electron microscopy1. Labeled wheat germ agglutinin (WGA) has also been used to visualize cuticular glycoproteins, but is limited in resolving finer cuticular structures14. Staining of cuticular surface using fluorescent dye has been observed, but never characterized in detail15. We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. This optimized protocol for DiI staining is a simple, robust method for high resolution fluorescent visualization of annuli, alae, vulva, male tail, and hermaphrodite tail spike in C. elegans.

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. With this optimized protocol, alae and annular cuticular structures are stained by DiI and observed using compound microscopy.

La visualización de Caenorhabditis Elegans Estructuras de la cutícula uso del colorante lipofílico Vital, DII

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

RNAi de detección para identificar fenotipos postembrionarios en C. elegans</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

A Fluorescence-based Exonuclease Assay to Characterize DmWRNexo, Orthologue of Human Progeroid WRN Exonuclease, and Its Application to Other Nucleases

WRN exonuclease is involved in resolving DNA damage that occurs either during DNA replication or following exposure to endogenous or exogenous genotoxins. It is likely to play a role in preventing accumulation of recombinogenic intermediates that would otherwise accumulate at transiently stalled replication forks, consistent with a hyper-recombinant phenotype of cells lacking WRN. In humans, the exonuclease domain comprises an N-terminal portion of a much larger protein that also possesses helicase activity, together with additional sites important for DNA and protein interaction. By contrast, in Drosophila, the exonuclease activity of WRN (DmWRNexo) is encoded by a distinct genetic locus from the presumptive helicase, allowing biochemical (and genetic) dissection of the role of the exonuclease activity in genome stability mechanisms. Here, we demonstrate a fluorescent method to determine WRN exonuclease activity using purified recombinant DmWRNexo and end-labeled fluorescent oligonucleotides. This system allows greater reproducibility than radioactive assays as the substrate oligonucleotides remain stable for months, and provides a safer and relatively rapid method for detailed analysis of nuclease activity, permitting determination of nuclease polarity, processivity, and substrate preferences.

Exonucleases play critical roles in ensuring genome stability. Loss of WRN exonuclease function results in premature aging. Studying substrates and other requirements of the nuclease in vitro can help elucidate its role in vivo. Here we demonstrate a rapid and reproducible fluorescence-based assay to measure its nuclease activity.

WRN exonuclease is involved in resolving DNA damage that occurs either during DNA replication or following exposure to endogenous or exogenous genotoxins. ...

A Practical Guide to Phylogenetics for Nonexperts

Many researchers, across incredibly diverse foci, are applying phylogenetics to their research question(s). However, many researchers are new to this topic and so it presents inherent problems. Here we compile a practical introduction to phylogenetics for nonexperts.&#160;We outline in a step-by-step manner, a pipeline for generating reliable phylogenies from gene sequence datasets. We begin with a user-guide for similarity search tools via online interfaces as well as local executables. Next, we explore programs for generating multiple sequence alignments followed by protocols for using software to determine best-fit models of evolution. We then outline protocols for reconstructing phylogenetic relationships via maximum likelihood and Bayesian criteria&#160;and finally describe tools for visualizing phylogenetic trees. While this is not by any means an exhaustive description of phylogenetic approaches, it does provide the reader with practical starting information on key software applications commonly utilized by phylogeneticists. The vision for this article would be that it could serve as a practical training tool for researchers embarking on phylogenetic studies&#160;and also serve as an educational resource that could be incorporated into a classroom or teaching-lab.

Here we describe a step-by-step pipeline for generating reliable phylogenies from nucleotide or amino acid sequence datasets. This guide aims to serve researchers or students new to phylogenetic analysis.&#160;

Una guía práctica para Phylogenetics para no expertos

Many researchers, across incredibly diverse foci, are applying phylogenetics to their research question(s). However, many researchers are new to this ...

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a novel Drosophila circadian output, called the temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night 3. Surprisingly, the TPR and locomotor activity are controlled through distinct circadian neurons3. Drosophila locomotor activity is a well&#160;known circadian behavioral output and has provided strong contributions to the discovery of many conserved mammalian circadian clock genes and mechanisms4. Therefore, understanding TPR will lead to the identification of hitherto unknown molecular and cellular circadian mechanisms. Here, we describe how to perform and analyze the TPR assay. This technique not only allows for dissecting the molecular and neural mechanisms of TPR, but also provides new insights into the fundamental mechanisms of the brain functions that integrate different environmental signals and regulate animal behaviors. Furthermore, our recently published data suggest that the fly TPR shares features with the mammalian BTR3. Drosophila are ectotherms, in which the body temperature is typically behaviorally regulated. Therefore, TPR is a strategy used to generate a rhythmic body temperature in these flies5-8. We believe that further exploration of Drosophila TPR will facilitate the characterization of the mechanisms underlying body temperature control in animals.

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

We recently identified a novel Drosophila circadian output, temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night. TPR is regulated independently from another circadian output, locomotor activity. Here&#160;we describe the design and analysis of TPR in Drosophila.

Diseño y Análisis Del Comportamiento De Preferencia De Temperatura Y Su Ritmo Circadiano En Drosophila

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a ...

Reconstruction of 3-Dimensional Histology Volume and its Application to Study Mouse Mammary Glands

Histology volume reconstruction facilitates the study of 3D shape and volume change of an organ at the level of macrostructures made up of cells. It can also be used to investigate and validate novel techniques and algorithms in volumetric medical imaging and therapies. Creating 3D high-resolution atlases of different organs1,2,3 is another application of histology volume reconstruction. This provides a resource for investigating tissue structures and the spatial relationship between various cellular features. We present an image registration approach for histology volume reconstruction, which uses a set of optical blockface images. The reconstructed histology volume represents a reliable shape of the processed specimen with no propagated post-processing registration error. The Hematoxylin and Eosin (H&#38;E) stained sections of two mouse mammary glands were registered to their corresponding blockface images using boundary points extracted from the edges of the specimen in histology and blockface images. The accuracy of the registration was visually evaluated. The alignment of the macrostructures of the mammary glands was also visually assessed at high resolution.
This study delineates the different steps of this image registration pipeline, ranging from excision of the mammary gland through to 3D histology volume reconstruction. While 2D histology images reveal the structural differences between pairs of sections, 3D histology volume provides the ability to visualize the differences in shape and volume of the mammary glands.

We present an image registration approach for 3-dimensional (3D) histology volume reconstruction, which facilitates the study of the changes of an organ at the level of macrostructures made up of cells . Using this approach, we studied the 3D changes between wild-type and Igfbp7-null mammary glands.

Reconstrucción de 3-Dimensional Volumen Histología y su aplicación al estudio del ratón glándulas mamarias

Histology volume reconstruction facilitates the study of 3D shape and volume change of an organ at the level of macrostructures made up of cells. It can ...