The authors thank the staff of the Center for Comparative Medicine and Research (CCMR) at Dartmouth for their expert care of the mice used for these studies. The authors also acknowledge Emily Clough for her excellent administrative support.

Todo el trabajo con animales utiliza protocolos revisados ​​y aprobados por el Comité de Cuidado y Uso de Animales Institucional (IACUC) de la Escuela Geisel de Medicina de Dartmouth. 1. El modelo de ratón <ol><li> La inducción de la enfermedad desmielinizante inducida por TMEV <ol><li> Mover las jaulas que contienen de 4 a 6 semanas de edad, hembra SJL / JHAN ratones del bastidor a un espacio de trabajo confortable. Marcar los ratones (por ejemplo, con una marca auricular o un punzón oído) para permitir una evaluación individual de la enfermedad clínica e histológica. </li><li> Dibujar 30 l de TMEV Stock infectar (unidades formadoras de 2 x 10 6 placa; UFP) en PBS en una jeringa de insulina de calibre 29 y una aguja. </li><li> Preparar la máquina de anestesia de gas: compruebe el sistema para asegurar la presencia de cantidades adecuadas de oxígeno e isoflurano durante la duración del procedimiento. </li><li> Encienda el medidor de flujo de 1 L / min. Colocar el animal en la cámara de inducción y sellar la parte superior. Encienda el VAporizer a 3,5% y controlar al animal hasta reclinada. </li><li> Retire el animal de la cámara y poner a prueba el ratón pellizcando la almohadilla para asegurar una anestesia adecuada. La falta de respuesta a una pizca fuerte indica una anestesia adecuada. </li><li> Limpiar el sitio de la inyección con alcohol isopropílico al 70%. </li><li> Inyectar los 30 l de TMEV Stock infectando en el hemisferio cerebral derecho por inyección a mano alzada (Figura 1). El sitio de la inyección es aproximadamente a medio camino entre la línea de vista y el oído, y justo al lado de la línea media. </li><li> Vuelva a colocar el puntero del ratón a su jaula de retención una vez completamente alerta y móvil (generalmente de 3 - 5 min). </li><li> La eutanasia a los ratones por exanguinación o perfusión cardíaca 3 a 6 meses después de la infección TMEV, en función de la rapidez del desarrollo de la enfermedad. </li></ol></li></ol> 2. Análisis de Rotarod <ol><li> El Aparato de Rotarod <ol><li> ratones de prueba antes de la infección por TMEV que se familiaricen con elmáquina y para evaluar su coordinación balance de línea de base y el motor de control normal. </li><li> Iniciar el protocolo de adaptación en los días -5 después de la infección (dpi; es decir, 5 días antes de la infección por TMEV). </li><li> Permitir que los ratones se aclimaten a la cámara de ensayo durante al menos 30 minutos antes de la prueba de Rotarod, con el fin de permitir que se ajustan al medio ambiente. </li><li> Asegúrese de que tanto la unidad de Rotarod y el ordenador están conectados y conectados entre sí (Figura 2). </li><li> Programado en el rotarod con los parámetros del protocolo de entrenamiento dpi -5, tal como se describe en la Tabla 1. </li><li> Guarde el archivo de trabajo con la información de la fecha y la identificación. </li><li> Mover la jaula que contiene el equipo a ensayar desde el bastidor a una mesa junto a la rotarod. Los ratones se prueban normalmente en escuadrones de 4. </li><li> Recoger a un ratón por la cola y lo coloca en la barra, de espaldas a la operadora. Repita para el segundo hasta el cuarto ratón. Si un ratón falls o saltos, colocarlo de nuevo en su carril de la rotarod hasta que todos los ratones están en posición. No haga caso de cualquier ratones dan la vuelta para hacer frente al operador. </li><li> Después de cargar todos los ratones, presione el botón &quot;Enter&quot; para iniciar el experimento. Observar los temporizadores se inician automáticamente y las revoluciones por minuto (rpm) en la pantalla para cada carril. <ol><li> Como cada animal se cae de la barra, registrar la velocidad de la varilla en el momento de la caída, así como la duración de tiempo que el animal permanecía en la varilla. La varilla seguirá girando hasta que el último animal ha caído del conjunto de la varilla. </li></ol></li><li> Después de que todos los ratones han caído, utilice un pañuelo de papel para eliminar cualquier bolos fecales y la orina de la varilla. La presencia de orina y la materia fecal puede afectar a la capacidad de los ratones para agarrar la barra. <ol><li> Después de un descanso de 3 minutos, dar a los ratones una segunda y una tercera prueba. El tiempo máximo por solo ensayo es de 240 seg. Administrar un total de 3 pruebas durante cada día de la prueba. </li></ol></li><li> Devolver los ratones a sus jaulas y devolverlos de nuevo al bastidor. Al final de la sesión experimental, limpiar el rotarod con agua y jabón para eliminar toda la materia fecal de la máquina. </li><li> Limpiar la placa base con etanol al 70%. Rocíe toda la máquina con dióxido de cloro para desinfectar. </li><li> En días - 4, - 3, - 2, y - 1 pi, pre-establecer el rotarod con los parámetros del protocolo de entrenamiento apropiadas, tal como se describe en la Tabla 1, y repita los pasos 2.1.2 a 2.1.12. </li><li> Después de obtener las medidas de referencia, infectar a los ratones con TMEV. Permitir un período de recuperación pi a 6 días. </li></ol></li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td rowspan="2"> Protocolo </td><td rowspan="2"> Día de la prueba </td><td rowspan="2"> Frecuencia </td><td rowspan="2"> Velocidad de arranque (rpm) </td><td rowspan="2"> MaxVelocidad (rpm) </td><td> Aceleración </td><td> ensayos </td><td> ITI </td></tr><tr><td> (rpm / sec) </td><td> (N x seg) </td><td> (min) </td></tr><tr><td rowspan="4"> Formación </td><td> - 5 dpi </td><td> 1 día </td><td> 1 </td><td> 12 </td><td> 01/03 </td><td> 3x240 seg </td><td> 3 </td></tr><tr><td> - 4 ppp </td><td> 1 día </td><td> 1 </td><td> 13 </td><td> 01/03 </td><td> 3x240 seg </td><td> 3 </td></tr><tr><td> - 3 dpi </td><td> 1 día </td><td> 1 </td><td> 14 </td><td> 01/03 </td><td> 3x240 seg </td><td> 3 </td></tr><tr><td> - 2; - 1 ppp </td><td> 1 día </td><td> 5 </td><td> 40 </td><td> 01/03 </td><td> 3x240 seg </td><td> 3 </td></tr><tr><td rowspan="2"> Experimental </td><td> Entre +7 +50 dpi </td><td> 2 semanas </td><td>5 </td><td> 40 </td><td> 05/30 </td><td> 3x240 seg </td><td> 3 </td></tr><tr><td> De 51 a 150 dpi </td><td> 1 semana </td><td> 5 </td><td> 40 </td><td> 05/30 </td><td> 3x240 seg </td><td> 3 </td></tr></tbody></table> Tabla 1: Parámetros rodillo giratorio en la formación y protocolos experimentales. <ol start="2"><li> El protocolo experimental de Rotarod <ol><li> En 7 dpi, pre-establecer el rotarod con los parámetros de protocolo experimentales apropiados, como se describe en la Tabla 1. Repetir los pasos 2.1.2 a 2.1.10. </li><li> Al final de la prueba # 3, se pesa cada ratón y hacer una nota del peso corporal en la hoja de datos. Limpiar y desinfectar el rotarod según los pasos 2.1.11 y 2.1.12. </li><li> Prueba de los ratones dos veces a la semana para las 6 semanas siguientes, tal como se describe anteriormente. Después de 6 semanas (en el que los ratones propensos han llegado a una fase de meseta) 8,9, prueba los ratones una vez a la semana con el mismo Experimental protocolo. El protocolo de prueba real tiene una duración media de 150 días, dependiendo de la evolución de la enfermedad específica. </li></ol></li><li> Índice funcional neurológica <ol><li> Exportar los datos en bruto en un archivo de hoja de cálculo y analizar los resultados. </li><li> Data Express como correr del tiempo (Figura 3A): este es el tiempo de funcionamiento normal más el tiempo de rotación pasiva menos el tiempo de retardo de rotación (tabla 2) 10. Calcular el tiempo de funcionamiento media de los tres ensayos por día. </li><li> Expresar los datos como un índice funcional neurológica (NFI; Figura 3B). <ol><li> Calcular el umbral de rendimiento de línea de base de cada ratón individual. El umbral de rendimiento de referencia se determina como la media de todos los tiempos de funcionamiento de día + 15 a + 45 pi 6,7. </li><li> Calcular la NFI como la media de los tres tiempos de funcionamiento promedio más recientes dividida por el umbral de rendimiento de línea de base de que 6,7 de ratón específico &lt;/ Sup&gt;. NOTA: Si los tiempos de funcionamiento probado para un ratón en el día +72, +76 y + 79 pi son 55 seg, 45 seg, y 50 segundos, y el tiempo de la línea de base para el mismo ratón fue de 135 segundos, el NFI para ese ratón en 79 dpi será [(45 + 50 + 55) / 3] / 135 o 0,37. </li></ol></li><li> Expresar los datos como un NFI ajustado (adjNFI; Figura 3C): ajustar los datos de artículos no alimentarios por un valor de la población para el único experimento. <ol><li> Calcular la adjNFI dividiendo el valor NFI por el promedio NFI obtenido por el grupo tratado de referencia en ese día específico. </li></ol></li></ol></li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> Término </td><td> Definición </td></tr><tr><td> tiempo de funcionamiento normal </td><td> El tiempo total que el ratón gasta en ejecución activa en la barra giratoria, es decir, la latencia se caiga. </td></tr><tr><td> tiempo de rotación pasiva </td><td> La Amontaje de tiempo que el ratón se ha mantenido en la varilla en el modo de rotación pasiva. </td></tr><tr><td> tiempo de retardo de rotación </td><td> La cantidad de tiempo que el ratón permanece en la varilla durante el modo de rotación pasiva </td></tr><tr><td> modo de rotación pasiva </td><td> Cuando el ratón agarra la varilla y gira sin tener que deambular. </td></tr><tr><td> El tiempo total de la sesión </td><td> La cantidad total de tiempo que el ratón permanece en la barra giratoria durante la sesión. </td></tr><tr><td> el rendimiento de referencia </td><td> Pre-daños rendimiento del motor evaluado para determinar el umbral mínimo de rendimiento. </td></tr><tr><td> índice de la función neurológica (NFI) </td><td> Índice clínico, que compara cada ratón el rendimiento del motor, es decir, el tiempo de funcionamiento, en cualquier momento para su máximo rendimiento. </td></tr><tr><td> índice de la función neurológica ajustado (adjNFI) </td><td> Cuando se aplica un proceso de normalización para ajustar los datos NFI por un valor de la población parael experimento sencillo. </td></tr><tr><td> valor de la población </td><td> NFI valor medio obtenido por el grupo de tratamiento simulado-tratado en un día específico. </td></tr></tbody></table> Tabla 2: Definición de los parámetros adoptados para cuantificar el cilindro giratorio Neurológica deterioro.

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The authors have nothing to disclose.

A pesar de algunas limitaciones, la prueba de rendimiento de Rotarod representa una herramienta importante para la evaluación de la función motora y disfunción en TMEV-IDD, así como el efecto de las intervenciones farmacológicas en la progresión de la discapacidad en los ratones. La prueba de Rotarod fue descrita por primera vez en 1957 como una herramienta para medir los déficits neurológicos en los roedores 11. Los roedores tienen que caminar sobre una barra giratoria, a...

Virus Murino encefalomielitis de Theiler (TMEV) es un virus de ARN de cadena simple neurotrópico que persistentemente infecta el sistema nervioso central murino (CNS). En ratones susceptibles, la infección con TMEV causa una, enfermedad desmielinizante crónica progresiva inmunomediada, conocida como enfermedad desmielinizante inducida por TMEV (TMEV-IDD). La infección experimental de ratones toma un curso de la enfermedad se asemeja a la observada en las formas progresivas de la esclerosis múltiple (MS). TMEV-IDD se caracteriza por dos fases distintas: la fase aguda y la fase crónica. La fase aguda es una, 1,2 encefalitis generalmente subclínica leve. La segunda fase, crónica, que comienza alrededor de un mes después de la infección, se compone de una discapacidad aumento que se caracteriza por la desmielinización, la inflamación y el daño axonal 1,2. La debilidad observada en ratones se asocia con la espasticidad y, en ocasiones, graves espasmos tónicos. Debido a que hay actualmente ningún medicamenns para mejorar la discapacidad progresiva en los pacientes, los investigadores se sienten especialmente atraídos por TMEV-DDI, lo que representa un modelo animal óptimo para el seguimiento del impacto de los fármacos modificadores de la enfermedad en la progresión de la enfermedad. Sin embargo, en ratones, así como en pacientes con EM, la monitorización de la progresión de la discapacidad requiere una observación clínica continua durante largos períodos de tiempo. En ratones, el seguimiento a largo plazo para la progresión de la discapacidad se puede lograr con la prueba de rendimiento de Rotarod. La prueba de rendimiento de Rotarod es una prueba de comportamiento estándar que evalúa las funciones motoras asociadas a tales como la coordinación, el equilibrio y la fatiga en los roedores. Los ratones tienen que mantener el equilibrio sobre una varilla giratoria, que está girando en aceleración continua; la latencia de tiempo para caer de esta barra se registra. Los animales con disfunción neurológica no pueden permanecer en la barra giratoria, siempre que los controles, y que normalmente entrega cuando la velocidad de rotación es superior a sula capacidad del motor. El deterioro neurológico más los animales tienen, más pronto se caiga la varilla, y el más corto es el tiempo de latencia es. La ventaja de la prueba de rotarod en los sistemas tradicionales de puntuación visual es que genera un objetivo, medible variable el tiempo de latencia-que en última instancia puede ser utilizado para los análisis estadísticos para cuantificar los efectos de las terapias y procedimientos experimentales 3. En el Laboratorio de Neuroinmunología (LONI) en Dartmouth, los ratones son sometidos a un protocolo de adaptación, en el que se prueban antes de la infección por TMEV con el fin de que se familiaricen con la máquina y para evaluar su normal &quot;línea de base&quot; equilibrio coordinación y el control motor 4, 5. Una vez que se estableció la línea de base y los ratones están infectados con TMEV, que se ponen a prueba una vez o dos veces a la semana durante un período de varios meses. El protocolo de prueba real tiene una duración media de 150 días, lo que permite una evaluación dela disminución de equilibrio, coordinación, y control del motor durante todo el curso de la enfermedad desmielinizante. Varios cientos de TMEV-IDD y tratada simulacro que los ratones se han probado hasta el momento para la disfunción neurológica en Dartmouth. Estos ratones habían recibido varios tratamientos inmunomoduladores, pero ningún agente farmacológico ha demostrado ser eficaz en la mejora de progresión de la discapacidad 6,7. El presente artículo y el protocolo relacionado describen cómo caracterizar el deterioro neurológico progresivo mostrada por los ratones TMEV-IDD. En particular, el protocolo ofrece recomendaciones de parámetros de prueba específicos que se consideran generalmente adecuados para el estudio de la discapacidad neurológica en ratones TMEV-IDD utilizando la prueba de Rotarod. Este procedimiento proporciona una línea de base que sirve para evaluar (1) la relevancia de este modelo de ratón para la EM progresiva y (2) su utilidad para probar terapias destinadas al tratamiento de enfermedades neurológicas progresivas como la esclerosis múltiple. Claramente, laprueba de rendimiento de Rotarod y el protocolo de parámetros de prueba optimizados y actual no sólo son útiles en la detección de la discapacidad neurológica progresiva en el modelo de ratón TMEV-IDD, pero también son útiles en el descubrimiento de las deficiencias en otros modelos de virus inducida y / o de ratón genético de las enfermedades neurodegenerativas.

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			<td height="42" style="height:42px;width:216px;">Mice SJL/JCrHsd 4 to 6 week old</td>
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			<td height="21" style="height:21px;width:216px;">TMEV virus stock</td>
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			<td height="42" style="height:42px;width:216px;">Isoflurane vaporizer</td>
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			<td height="42" style="height:42px;width:216px;">Insulin Syringes U- 100 29g x 0.5cc</td>
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			<td height="63" style="height:63px;width:216px;">Rotamex-5 4 Lane Rota-Rod for Mice with RS-232 and Software</td>
			<td style="width:83px;">Columbus Instruments</td>
			<td style="width:119px;">#0890M</td>
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measuring progressive neurological disability in a mouse model of multiple sclerosis

After intracerebral infection with the Theiler's Murine Encephalomyelitis Virus (TMEV), susceptible SJL mice develop a chronic-progressive demyelinating disease, with clinical features similar to the progressive forms of multiple sclerosis (MS). The mice show progressive disability with loss of motor and sensory functions, which can be assessed with multiple apparatuses and protocols. Among them, the Rotarod performance test is a very common behavioral test, its advantage being that it provides objective measurements, but it is often used assuming that it is straightforward and simple. In contrast to visual scoring systems used in some models of MS, which are highly subjective, the Rotarod test generates an objective, measurable, continuous variable (i.e., length of time), allowing almost perfect inter-rater concordances. However, inter-laboratory reliability is only achieved if the various testing parameters are replicated. In this manuscript, recommendations of specific testing parameters, such as size, speed, and acceleration of the rod; amount of training given to the animals; and data processing, are presented for the Rotarod test.

El objetivo de este experimento representativo fue comparar la discapacidad neurológica inducida por la cepa Daniels (DA) y la tensión de la haba de TMEV. Para los propósitos del presente estudio, un grupo de 32 ratones SJL hembra fueron infectados por vía intracraneal con TMEV, ya sea la cepa DA (n = 16) o de la cepa de frijol (n = 16), y sus signos clínicos se monitorizaron con el tiempo. Un grupo adicional de 20 ratones se aplicó el tratamiento simulado (es decir, soluc...

Watch this Scientific Journal Video about Measuring Progressive Neurological Disability in a Mouse Model of Multiple Sclerosis at JoVE.com

Measuring Progressive Neurological Disability in a Mouse Model of Multiple Sclerosis

An optimized testing protocol is presented in this paper for the Rotarod performance test, used for measuring progressive neurological disability in TMEV-infected mice.

La medición de la discapacidad neurológica progresiva en un modelo de ratón de la esclerosis múltiple

After intracerebral infection with the Theiler's Murine Encephalomyelitis Virus (TMEV), susceptible SJL mice develop a chronic-progressive demyelinating ...

measuring-progressive-neurological-disability-mouse-model-multiple

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

10.9K vistas.  Geisel School of Medicine at Dartmouth. An optimized testing protocol is presented in this paper for the Rotarod performance test, used for measuring progressive neurological disability in TMEV-infected mice. 

Video: Measuring Progressive Neurological Disability in a Mouse Model of Multiple Sclerosis

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

Múltiples imágenes del ratón neuroanatómicos de Resonancia Magnética

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

Investigación

Neurociencias

Quantitative Analysis of Climbing Defects in a Drosophila Model of Neurodegenerative Disorders

Locomotive defects resulting from neurodegenerative disorders can be a late onset symptom of disease, following years of subclinical degeneration, and thus current therapeutic treatment strategies are not curative. Through the use of whole exome sequencing, an increasing number of genes have been identified to play a role in human locomotion. Despite identifying these genes, it is not known how these genes are crucial to normal locomotive functioning. Therefore, a reliable assay, which utilizes model organisms to elucidate the role of these genes in order to identify novel targets of therapeutic interest, is needed more than ever. We have designed a sensitized version of the negative geotaxis assay that allows for the detection of milder defects earlier and has the ability to evaluate these defects over time. The assay is performed in a glass graduated cylinder, which is sealed with a wax barrier film. By increasing the threshold distance to be climbed to 17.5 cm and increasing the experiment duration to 2 min&#160;we have observed a greater sensitivity in detecting mild mobility dysfunctions. The assay is cost effective and does not require extensive training to obtain highly reproducible results. This makes it an excellent technique for screening candidate drugs in Drosophila mutants with locomotion defects.

We present an optimized inexpensive and reliable negative geotaxis assay in Drosophila melanogaster as a model for neurodegenerative disorders. Being more sensitive to mild locomotor defects, this assay will help screen for potential genetic interactions and drug targets.

Análisis cuantitativo de los Defectos de escalada en un modelo de Drosophila de enfermedades neurodegenerativas

Locomotive defects resulting from neurodegenerative disorders can be a late onset symptom of disease, following years of subclinical degeneration, and ...

Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic transmission during repetitive nerve firing. Impaired endocytosis in pathological conditions leads to decreases in synaptic strength and brain functions. Here, we describe methods used to measure synaptic vesicle endocytosis at the mammalian hippocampal synapse in neuronal culture. We monitored synaptic vesicle protein endocytosis by fusing a synaptic vesicular membrane protein, including synaptophysin and VAMP2/synaptobrevin, at the vesicular lumenal side, with pHluorin, a pH-sensitive green fluorescent protein that increases its fluorescence intensity as the pH increases. During exocytosis, vesicular lumen pH increases, whereas during endocytosis vesicular lumen pH is re-acidified. Thus, an increase of pHluorin fluorescence intensity indicates fusion, whereas a decrease indicates endocytosis of the labelled synaptic vesicle protein. In addition to using the pHluorin imaging method to record endocytosis, we monitored vesicular membrane endocytosis by electron microscopy (EM) measurements of Horseradish peroxidase (HRP) uptake by vesicles. Finally, we monitored the formation of nerve terminal membrane pits at various times after high potassium-induced depolarization. The time course of HRP uptake and membrane pit formation indicates the time course of endocytosis.

Synaptic vesicle endocytosis is detected by light microscopy of pHluorin fused with synaptic vesicle protein and by electron microscopy of vesicle uptake.

Medición de endocitosis vesícula sináptica en neuronas Hippocampal cultivadas

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic ...

Establishing Mouse Models for Zika Virus-induced Neurological Disorders Using Intracerebral Injection Strategies: Embryonic, Neonatal, and Adult

The Zika virus (ZIKV) is a flavivirus currently endemic in North, Central, and South America. It is now established that the ZIKV can cause microcephaly and additional brain abnormalities. However, the mechanism underlying the pathogenesis of ZIKV in the developing brain remains unclear. Intracerebral surgical methods are frequently used in neuroscience research to address questions about both normal and abnormal brain development and brain function. This protocol utilizes classical surgical techniques and describes methods that allow one to model ZIKV-associated human neurological disease in the mouse nervous system. While direct brain inoculation does not model the normal mode of virus transmission, the method allows investigators to ask targeted questions concerning the consequence after ZIKV infection of the developing brain. This protocol describes embryonic, neonatal, and adult stages of intraventricular inoculation of ZIKV. Once mastered, this method can become a straightforward and reproducible technique that only takes a few hours to perform.

Here we describe a method for establishing a model of Zika virus-induced microcephaly in mouse. This protocol includes methods for embryonic, neonatal, and adult-stage intracerebral inoculation of the Zika virus.

Establecimiento de modelos de ratón para trastornos neurológicos inducidos por el Virus Zika utilizando estrategias de inyección Intracerebral: embrionario, Neonatal y adulto

The Zika virus (ZIKV) is a flavivirus currently endemic in North, Central, and South America. It is now established that the ZIKV can cause microcephaly ...

Simultaneous Cryosectioning of Multiple Rodent Brains

Histology and immunohistochemistry are routine methods of analysis to visualize microscopic anatomy and localize proteins within biological tissue. In neuroscience, as well as a plethora of other scientific fields, these techniques are used. Immunohistochemistry can be done on slide mounted tissue or free-floating sections. Preparing slide-mounted samples is a time intensive process. The following protocol for a technique, called the Megabrain, reduced the time taken to cryosection and mount brain tissue by up to 90% by combining multiple brains into a single frozen block. Furthermore, this technique reduced variability seen between staining rounds, in a large histochemical study. The current technique has been optimized for using rodent brain tissue in downstream immunohistochemical analyses; however, it can be applied to different scientific fields that use cryosectioning.

Here, we present a protocol to freeze and section brain tissue from multiple animals as a timesaving alternative to processing single brains. This reduces staining variability during immunohistochemistry and reduces time cryosectioning and imaging.

Cryosectioning simultánea de varios cerebros de roedores

Histology and immunohistochemistry are routine methods of analysis to visualize microscopic anatomy and localize proteins within biological tissue. In ...

Lipidomics and Transcriptomics in Neurological Diseases

Lipids serve as the primary interface to brain insults or stimuli conducive to neurological diseases and are a reservoir for the synthesis of lipids with various signaling or ligand function that can underscore the onset and progression of diseases. Often changing at the presymptomatic level, lipids are an emerging source of drug targets and biomarkers. Many neurological diseases exhibit neuroinflammation, neurodegeneration, and neuronal excitability as common hallmarks, partly modulated by specific lipid signaling systems. The interdependence and interrelation of synthesis of various lipids prompts a multilipid, multienzyme, and multireceptor analysis in order to derive the commonalities and specificities of neurological contexts and to expedite the unravelling of mechanistic aspects of disease onset and progression. Ascribing lipid roles to distinct brain regions advances the determination of lipid molecular phenotype and morphology associated with a neurological disease.
Presented here is a modular protocol suitable for the analysis of membrane lipids and downstream lipid signals along with mRNA of enzymes and mediators underlying their functionality, extracted from discrete brain regions that are relevant for a particular neurological disease and/or condition. To ensure accurate comparative lipidomic profiling, the workflows and operating criteria were optimized and standardized for: i) brain sampling and dissection of regions of interest, ii) co-extraction of multiple lipid signals and membrane lipids, iii) dual lipid/mRNA extraction, iv) quantification by liquid chromatography multiple reaction monitoring (LC/MRM), and v) standard mRNA profiling. This workflow is amenable for the low tissue amounts obtained by sampling of the functionally discrete brain subregions (i.e. by brain punching), thus preventing bias in multimolecular analysis due to tissue heterogeneity and/or animal variability. To reveal peripheral consequences of neurological diseases and establish translational molecular readouts of neurological disease states, peripheral organ sampling, processing, and their subsequent lipidomic analysis, as well as plasma lipidomics, are also pursued and described. The protocol is demonstrated on an acute epilepsy mouse model.

This article presents a modular protocol for tissue lipidomics and transcriptomics, and plasma lipidomics in neurological disease mouse models targeting lipids underlying inflammation and neuronal activity, membrane lipids, downstream messengers, and mRNA-encoding enzymes/receptors underlying lipid function. Sampling, sample processing, extraction, and quantification procedures are outlined.

Lipidómica y Transcriptómica en Enfermedades Neurológicas

Lipids serve as the primary interface to brain insults or stimuli conducive to neurological diseases and are a reservoir for the synthesis of lipids with ...

Comprehensive Autopsy Program for Individuals with Multiple Sclerosis

We describe a rapid tissue donation program for individuals with multiple sclerosis (MS) that requires scientists and technicians to be on-call 24/7, 365 days a year. Participants consent to donate their brain and spinal cord. Most patients were followed by neurologists at the Cleveland Clinic Mellen Center for MS Treatment and Research. Their clinical courses and neurological disabilities are well-characterized. Soon after death, the body is transported to the MS Imaging Center, where the brain is scanned in situ by 3 T magnetic resonance imaging (MRI). The body is then transferred to the autopsy room, where the brain and spinal cord are removed. The brain is divided into two hemispheres. One hemisphere is immediately placed in a slicing box and alternate 1 cm-thick slices are either fixed in 4% paraformaldehyde for two days or rapidly frozen in dry ice and 2-methylbutane. The short-fixed brain slices are stored in a cryopreservation solution and used for histological analyses and immunocytochemical detection of sensitive antigens. Frozen slices are stored at -80 &#176;C and used for molecular, immunocytochemical, and in situ hybridization/RNA scope studies. The other hemisphere is placed in 4% paraformaldehyde for several months, placed in the slicing box, re-scanned in the 3 T magnetic resonance (MR) scanner and sliced into centimeter-thick slices. Postmortem in situ MR images (MRIs) are co-registered with 1 cm-thick brain slices to facilitate MRI-pathology correlations. All brain slices are photographed and brain white-matter lesions are identified. The spinal cord is cut into 2 cm segments. Alternate segments are fixed in 4% paraformaldehyde or rapidly frozen. The rapid procurement of postmortem MS tissues allows pathological and molecular analyses of MS brains and spinal cords and pathological correlations of brain MRI abnormalities. The quality of these rapidly-processed postmortem tissues (usually within 6 h of death) is of great value to MS research and has resulted in many high-impact discoveries.

Multiple sclerosis is an inflammatory demyelinating disease with no cure. Analysis of brain tissue provides important clues to understanding the pathogenesis of the disease. Here we discuss the methodology and downstream analysis of MS brain tissue collected through a unique rapid autopsy program in operation at the Cleveland Clinic.

Programa integral de autopsias para individuos con esclerosis múltiple

We describe a rapid tissue donation program for individuals with multiple sclerosis (MS) that requires scientists and technicians to be on-call 24/7, 365 ...

Applying the RatWalker System for Gait Analysis in a Genetic Rat Model of Parkinson's Disease

Parkinson's disease (PD) is a progressive neurodegenerative disorder caused by the loss of dopaminergic (DA) neurons in the substantia nigra pars compacta. Gait abnormalities, including decreased arm swing, slower walking speed, and shorter steps are common in PD patients and appear early in the course of disease. Thus, the quantification of motor patterns in animal models of PD will be important for phenotypic characterization during disease course and upon therapeutic treatment. Most cases of PD are idiopathic; however, the identification of hereditary forms of PD uncovered gene mutations and variants, such as loss-of-function mutations in Pink1 and Parkin, two proteins involved in mitochondrial quality control that could be harnessed to create animal models. While mice are resistant to neurodegeneration upon loss of Pink1 and Parkin (single and combined deletion), in rats, Pink1 but not Parkin deficiency leads to nigral DA neuron loss and motor impairment. Here, we report the utility of FTIR imaging to uncover gait changes in freely walking young (2 months of age) male rats with combined loss of Pink1 and Parkin prior to the development of gross visually apparent motor abnormality as these rats age (observed at 4-6 months), characterized by hindlimb dragging as previously reported in Pink1 knockout (KO) rats.

Here we describe the RatWalker system, built by redesigning the MouseWalker apparatus to accommodate for the increased size and weight of rats. This system uses frustrated total internal reflection (FTIR), high-speed video capture, and open-access analysis software to track and quantify gait parameters.

Aplicación del sistema RatWalker para el análisis de la marcha en un modelo genético de ratas de la enfermedad de Parkinson

Parkinson's disease (PD) is a progressive neurodegenerative disorder caused by the loss of dopaminergic (DA) neurons in the substantia nigra pars ...

Positron Emission Tomography Imaging for In Vivo Measuring of Myelin Content in the Lysolecithin Rat Model of Multiple Sclerosis

Multiple sclerosis (MS) is a neuroinflammatory disease with expanding axonal and neuronal degeneration and demyelination in the central nervous system, leading to motor dysfunctions, psychical disability, and cognitive impairment during MS progression. Positron emission tomography (PET) is an imaging technique able to quantify in vivo cellular and molecular alterations.
Radiotracers with affinity to intact myelin can be used for in vivo imaging of myelin content changes over time. It is possible to detect either an increase or decrease in myelin content, what means this imaging technique can detect demyelination and remyelination processes of the central nervous system. In this protocol we demonstrate how to use PET imaging to detect myelin changes in the lysolecithin rat model, which is a model of focal demyelination lesion (induced by stereotactic injection) (i.e., a model of multiple sclerosis disease). 11C-PIB PET imaging was performed at baseline, and 1 week and 4 weeks after stereotaxic injection of lysolecithin 1% in the right striatum (4 &#181;L) and corpus callosum (3 &#181;L) of the rat brain, allowing quantification of focal demyelination (injection site after 1 week) and the remyelination process (injection site at 4 weeks).
Myelin PET imaging is an interesting tool for monitoring in vivo changes in myelin content which could be useful for monitoring demyelinating disease progression and therapeutic response.

Positron Emission Tomography Imaging for In Vivo Measuring of Myelin Content in the Lysolecithin Rat Model of Multiple Sclerosis

This protocol has the aim of monitoring in vivo myelin changes (demyelination and remyelination) by positron emission tomography (PET) imaging in an animal model of multiple sclerosis.

Imágenes de tomografía por emisión de Positron para la medición in vivo del contenido de mielina en el modelo de rata de lysolecitina de esclerosis múltiple

Multiple sclerosis (MS) is a neuroinflammatory disease with expanding axonal and neuronal degeneration and demyelination in the central nervous system, ...

Flow Cytometric Analysis of Multiple Mitochondrial Parameters in Human Induced Pluripotent Stem Cells and Their Neural and Glial Derivatives

Mitochondria are important in the pathophysiology of many neurodegenerative diseases. Changes in mitochondrial volume, mitochondrial membrane potential (MMP), mitochondrial production of reactive oxygen species (ROS), and mitochondrial DNA (mtDNA) copy number are often features of these processes. This report details a novel flow cytometry-based approach to measure multiple mitochondrial parameters in different cell types, including human induced pluripotent stem cells (iPSCs) and iPSC-derived neural and glial cells. This flow-based strategy uses live cells to measure mitochondrial volume, MMP, and ROS levels, as well as fixed cells to estimate components of the mitochondrial respiratory chain (MRC) and mtDNA-associated proteins such as mitochondrial transcription factor A (TFAM). 
By co-staining with fluorescent reporters, including MitoTracker Green (MTG), tetramethylrhodamine ethyl ester (TMRE), and MitoSox Red, changes in mitochondrial volume, MMP, and mitochondrial ROS can be quantified and related to mitochondrial content. Double staining with antibodies against MRC complex subunits and translocase of outer mitochondrial membrane 20 (TOMM20) permits the assessment of MRC subunit expression. As the amount of TFAM is proportional to mtDNA copy number, the measurement of TFAM per TOMM20 gives an indirect measurement of mtDNA per mitochondrial volume. The entire protocol can be carried out within 2-3 h. Importantly, these protocols allow the measurement of mitochondrial parameters, both at the total level and the specific level per mitochondrial volume, using flow cytometry.

This study reports a novel approach to measure multiple mitochondrial functional parameters based on flow cytometry and double staining with two fluorescent reporters or antibodies to detect changes in mitochondrial volume, mitochondrial membrane potential, reactive oxygen species level, mitochondrial respiratory chain composition, and mitochondrial DNA.

Análisis citométrico de flujo de múltiples parámetros mitocondriales en células madre pluripotentes inducidas por humanos y sus derivados neuronales y gliales

Mitochondria are important in the pathophysiology of many neurodegenerative diseases. Changes in mitochondrial volume, mitochondrial membrane potential ...