Con el apoyo de la Fundación de Amparo un Pesquisa do Estado do Rio de Janeiro (FAPERJ) y Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). Estamos muy agradecidos a los árbitros anónimos cuyos comentarios ayudaron a mejorar tanto el manuscrito y video.

Todos los productos químicos almacenados en forma sólida, como la 6-OHDA y apomorfina, se diluye en el agua de inyección estéril y se filtra dentro de una campana de flujo laminar, para evitar la contaminación. Además, las neurotoxinas y sustancias neuroactivos fueron almacenados, preparados, manipulados y eliminados de acuerdo a la reglamentación establecida por las normas internacionales y seis locales de la Comisión de Seguridad de la Biotecnología. 1 - Anestesia y cirugía Ratones machos adultos suizos (25-30g) son premedicados con atropina (0,2 mg / kg, sc) para reducir el tono vagal y anestesiados con una inyección intraperitoneal de ketamina (90-120 mg / kg) y xilazina (10 mg / Kg) . El animal se coloca en una almohadilla de calefacción con control electrónico (Insight) para asegurar la temperatura corporal se mantiene a 37 ° C, como la inducción de la sedación toma unos minutos. El nivel de anestesia se comprueba a través del procedimiento de prueba de la ausencia de reflejo de retirada. Dosis de anestesia debe ser ajustada para cada cepa / colonia. Dependiendo del animal y la duración de la cirugía, las dosis adicionales de ketamina puede ser necesario. Después de eso, la solución salina (NaCl 0,9%) se aplica en ambos ojos para proteger la córnea de la desecación y la prueba de la ausencia del reflejo de parpadeo en este primer momento. La piel en toda la cabeza se afeita con un condensador de ajuste para garantizar el mínimo contacto con zonas estériles. Anestésico local (xilocaína) se aplica a las barras del oído para evitar el malestar. Sólo una vez retirada y reflejo de parpadeo se han desvanecido, lo que indica el nivel de anestesia es lo suficientemente profunda para la cirugía (fase 3), el animal se coloca en el marco estereotáxico con boquilla y bares del oído especialmente diseñado para esta especie (Insight). Una vez positionated, una pomada oftalmológica (MARCA) se aplica para proteger los ojos de los animales a lo largo de toda la cirugía. Sólo después de asegurarse de que los ojos están correctamente protegidas, el procedimiento continuará con la desinfección de la piel de la región quirúrgica mediante la repetición de tres aplicaciones de yodo povidona seguido de 70% de etanol, utilizando hisopos de algodón estériles Después, correcta ubicación de las barras de oído es garantizada por las pruebas de movimientos laterales de la cabeza (la cabeza no debe moverse para que las barras estén bien fijos). Inclinación adecuada dorsoventral de la cabeza se verificará más adelante, cuando se revelan puntos de referencia del cráneo. Se hace una incisión sagital (1,5 cm) se hace con un bisturí estéril (# 15 hojas). El periostio sobre el área de interés se raspa suavemente lejos con una hoja nueva, y el hueso se limpia con la mirada estéril y bastoncillos de algodón, para descubrir puntos de referencia del cráneo: bregma se define como el punto de intersección de las suturas coronal y sagital, mientras que lambda es el punto de intersección de la sutura sagital y la línea de mejor ajuste que pasa por la parte izquierda y derecha de la sutura. La punta de una aguja de referencia alineada verticalmente por primera vez a cero en el bregma, que se examina en el microscopio quirúrgico, y luego se trasladó como tocar el punto lambda. Si la cabeza se coloca correctamente, lambda debe estar en el mismo nivel dorso-ventral como bregma, y ​​ambos lugares, separados por 4,2 mm en el eje rostro-caudal. Si no, las barras de las orejas y el portaobjetos debe aflojar y la cabeza inclinada delicadamente alrededor del eje interaural, a fin de lograr la posición deseada, que define el &quot;cráneo plano&quot; de referencia para las medidas de stereotaxical. La punta de la aguja de referencia se sitúa en las coordenadas deseadas: antero-posterior (AP 0.5 mm) y lateral (L -2,0 mm, izquierda). La ubicación está marcada en el cráneo y un pequeño (1,2 mm de diámetro) el agujero se abrió con una broca de esterilización, el uso intermitente de la acción para evitar el calor de la zona. Fragmentos de hueso se retira cuidadosamente con un raspador dental y lavado con agua estéril tibia NaCl al 0,9%. La esterilización 5 l jeringa Hamilton (26s medidor, 0,47 mm de diámetro exterior) se carga con la solución de 6-OHDA (o el vehículo de los animales de control) y verticalmente en el aparato estereotáxico. La punta de la aguja (tipo 2 o 12 bisel °) se inserta en el agujero abierto, hasta que toque la superficie pial, para determinar el punto de referencia de la coordenada dorso-ventral. Una vez determinada esta coordenada, la aguja es reducida gradualmente para llegar a las coordenadas del cuerpo estriado (AP: 0,5; L: -2,0 y DV: -3,0 mm, debajo de la superficie pial 7). La solución de 6-OHDA (10 mg 6-OHDA en NaCl al 0,9% con ácido ascórbico 0,02%), que se preparó y se filtra dentro de una campana de flujo laminar, y debe ser protegido de la luz con papel de aluminio durante todo el procedimiento, se inyecta en un caudal de 0.1μL/min. Para los animales de control, un volumen igual de vehículo (ácido ascórbico 0,02% y el 0,9% de NaCl en agua estéril) se inyecta. Una vez que la totalidad de l 2.0 ha sido inyectado, la jeringa se mantiene en su lugar por 5 minutos antes de que se retrae muy lentamente desde el cerebro, en no menos que en 5 minutos. El cráneo se limpia y se cierra la incisión con una sutura de nylon # 6-0 (Shalon). Para tener un extraatención de las infecciones, un ungüento antibiótico local, como la neomicina, pueden ser aplicados a la piel suturada. Los animales son retirados de la estructura, dado 0,5 ml de solución salina (sc.) para prevenir la deshidratación, y se mantienen calientes y bajo una estrecha observación hasta la recuperación completa. Luego se encuentra con la comida y agua ad libitum. Analgésicos se añade a la botella de agua potable para los dos primeros días después de la cirugía (dado un consumo medio diario de agua de 5,5 ml, y una dosis recomendada de ibuprofeno 0,03 mg / g de peso corporal / día, añadir 0,82 ml de 20 mg / ml ibuprofeno Abbott suspensión de 100 ml de agua potable). Los ratones se recuperan rápidamente del procedimiento, errante y alcance de los pellets de alimentos el agua con facilidad. En cualquier caso de malestar evidente, o cualquier signo de infección, los animales deben ser sacrificados de acuerdo con las directrices locales. 2 - Ensayo de comportamiento Aunque el deterioro del motor se puede observar después de una semana, un intervalo después de la lesión de un mes se permite antes de la cuantificación fiable con la prueba de rotación. El animal recibe una inyección subcutánea de apomorfina agonista DA (0,5 mg / Kg), y se coloca en un cilindro opaco de 30 cm de diámetro, colocados 45 cm por debajo de la grabación de la cámara. Es importante para eliminar los residuos y los olores de los animales previamente probados antes de cada prueba. Después de un periodo de habituación 5 minutos, los movimientos se registran en un plazo de 5 minutos. Ambas contra-y ipsilateral de todo el cuerpo de rotación se mide y se compara con el grupo control (vehículo de inyección). Si el procedimiento se realiza correctamente, los animales de control no se presenta ninguna rotación, mientras que la 6-OHDA animales lesionados comenzará a girar hacia el lado contralateral, ya que los agonistas predominantemente activa el cuerpo estriado denervado supersensible en el lado lesionado. Animales de puntuación de más de 7 rpm (que muestra por lo menos 7 de cuerpo entero rotaciones contralateral por minuto) son considerados como éxito lesionado. El animal puede ser regresado sano y salvo a su vivienda de 30 minutos después de la prueba, que puede repetirse semanalmente. En nuestras manos, menos del 5% de los ratones lesionados muestran las tasas de rotación óptimo.

<ol>
	<li>Ungerstedt, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=6-hydroxy-dopamine+induced+degeneration+of+central+monoamine+neurons.">6-hydroxy-dopamine induced degeneration of central monoamine neurons.</a> Eur. J. Pharmacol. 5, 107-110 (1968).</li><li>Akerud, P., Canals, J. M., Snyder, E. Y., Arenas, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroprotection+through+delivery+of+glial+cell+line-derived+neurotrophic+factor+by+neural+stem+cells+in+a+mouse+model+of+Parkinson's+Disease.">Neuroprotection through delivery of glial cell line-derived neurotrophic factor by neural stem cells in a mouse model of Parkinson's Disease.</a> J. Neurosci. 21, 8108-8118 (2001).</li><li>Ghosh, A., Roy, A., Liu, X., Kordower, J. H., Mufson, E. J., Hartley, D. M., Ghosh, S., Mosley, R. L., Gendelman, H. E., Pahan, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+inhibition+of+NF-kappaB+activation+prevents+dopaminergic+neuronal+loss+in+a+mouse+model+of+Parkinson's+Disease.">Selective inhibition of NF-kappaB activation prevents dopaminergic neuronal loss in a mouse model of Parkinson's Disease.</a> Proc. Natl. Acad. Sci. USA. 104, 18754-18759 (2007).</li><li>Alvarez-Fischer, D., Henze, C., Strenzke, C., Westrich, J., Ferger, B., H&#246;glinger, G. U., Oertel, W. H., Hartmann, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+striatal+6-OHDA+model+of+Parkinson's+disease+in+wild+type+and+a-synuclein-deleted+mice.">Characterization of the striatal 6-OHDA model of Parkinson's disease in wild type and a-synuclein-deleted mice.</a> Exp. Neurol. 210, 182-193 (2008).</li><li>Ungerstedt, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=6-Hydroxydopamine-induced+degeneration+of+the+nigrostriatal+dopamine+pathway:+The+turning+syndrome.">6-Hydroxydopamine-induced degeneration of the nigrostriatal dopamine pathway: The turning syndrome.</a> Pharmacology and Therapeutics Part B: General and Systematic Pharmacology. 2, 37-40 (1976).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Guidelines for the Care and Use of Mammals in Neuroscience and Behaviour Research. , 209 (2003).</li><li>Franklin, K. B. J., Paxinos, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Mouse Brain in Stereotaxic Coordinates. , (1997).</li><li>Hudson, J. L., Horne, C. G. v. a. n., Str&#246;mberg, I., Brock, S., Clayton, J., Masserano, J., Hoffer, B. J., Gerhardt, 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=Correlation+of+apomorphine-+and+amphetamine-induced+turning+with+nigrostriatal+dopamine+content+in+unilateral+6-hydroxydopamine+lesioned+rats.+Brain+Res.">Correlation of apomorphine- and amphetamine-induced turning with nigrostriatal dopamine content in unilateral 6-hydroxydopamine lesioned rats. Brain Res.</a> , 626-6167 (1993).</li><li>Truong, L., Allbutt, H., Kassiou, M., Henderson, 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=Developing+a+preclinical+model+of+Parkinson's+disease:+A+study+of+behaviour+in+rats+with+graded+6-OHDA+lesions.">Developing a preclinical model of Parkinson's disease: A study of behaviour in rats with graded 6-OHDA lesions.</a> Behav. Brain Res. 169, 1-9 (2006).</li><li>Harvey, B. K., Wang, Y., Hoffer, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+rodent+models+of+Parkinson's+disease.">Transgenic rodent models of Parkinson's disease.</a> Acta Neurochir. (Wien.). 101, 89-92 (2008).</li><li>Isacson, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ole+Isacson:+Development+of+New+Therapies+for+Parkinson's+Disease.">Ole Isacson: Development of New Therapies for Parkinson's Disease.</a> JoVE. 3, (2007).</li></ol>

Los experimentos se realizaron en conformidad con las directrices internacionales para el cuidado y uso de los mamíferos en Neurociencias y de Investigación del Comportamiento<sup&gt; 6</sup&gt;, Tras su aprobación por el Cuidado de Animales institucional y el empleo (Comissão de Avaliação de Uso de Animais em Pesquisa: UFRJ / CAUAP protocolo DAHEICB # 027). Medidas adecuadas fueron tomadas para minimizar el dolor y el malestar de los animales de experimentación.

Extensa lesión unilateral de la vía nigro-estriado se puede lograr de forma fiable en los ratones con una sola inyección estereotáxica de 6-OHDA en el estriado. La extensión de la lesión puede ser verificada post-mortem mediante técnicas de inmunohistoquímica para la tirosina hidroxilasa (que cataliza la etapa limitante de la velocidad de la síntesis de DA, es decir, la conversión de L-tirosina a dihidroxifenilalanina), como se muestra en el video. In vivo, las rotaciones contralat...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Ketamine hydrochloride</td>
<td>&nbsp;</td>
<td>K&ouml;nig</td>
<td>&nbsp;</td>
<td>90-120 mg/Kg</td>
</tr>
<tr>
<td>Xylazine hydrochloride</td>
<td>&nbsp;</td>
<td>K&ouml;nig</td>
<td>&nbsp;</td>
<td>10 mg/Kg</td>
</tr>
<tr>
<td>Atropine Sulfate</td>
<td>&nbsp;</td>
<td>Isofarma</td>
<td>&nbsp;</td>
<td>Isofarma</td>
</tr>
<tr>
<td>6 &#x2013; Hydroxydopamine Hydrochloride</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>&nbsp;</td>
<td>5 &mu;g/&mu;L</td>
</tr>
<tr>
<td>L - Ascorbic Acid</td>
<td>&nbsp;</td>
<td>USB Corporation</td>
<td>&nbsp;</td>
<td>0.2%</td>
</tr>
<tr>
<td>Xylocaine (Lidocaine Chloridrate)</td>
<td>&nbsp;</td>
<td>ASTRA Chemistry</td>
<td>&nbsp;</td>
<td>5% 50 mg</td>
</tr>
<tr>
<td>Nebacetin (Neomycin Sulfate + Bacitracin)</td>
<td>&nbsp;</td>
<td>Altana Pharma</td>
<td>&nbsp;</td>
<td>5 mg/g + 250 Ul/g</td>
</tr>
<tr>
<td>R-(-)-Apomorphine hydrochloride</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>&nbsp;</td>
<td>0.5 mg/Kg</td>
</tr>
<tr>
<td>GenTeal lOphthalmic ointment Hypromellose</td>
<td>&nbsp;</td>
<td>NOVARTIS</td>
<td>&nbsp;</td>
<td> 0.33%</td>
</tr>
<tr>
<td>Ibuprofen</td>
<td>&nbsp;</td>
<td>Abbott</td>
<td>&nbsp;</td>
<td>4 mg/ 100 ml drinking water</td>
</tr>
<tr>
<td>Povidine</td>
<td>&nbsp;</td>
<td>JohnsonDiversey</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>R-(-)-Apomorphine hydrochloride</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>&nbsp;</td>
<td>0.5 mg/Kg</td>
</tr>		</tbody>
		</table>
		</div>

murine model for parkinson's disease: from 6-oh dopamine lesion to behavioral test

La enfermedad de Parkinson (EP) afecta al menos a 6,5 ​​millones de personas en todo el mundo, independientemente de su sexo, las fronteras sociales, étnicas, económicas o geográficas. Los síntomas principales, tales como temblor, rigidez y bradikinesia, se desarrollan cuando aproximadamente 3 / 4 de las células dopaminérgicas se pierden en la sustancia negra, y no pueden proporcionar para la regulación suave y coordinada de los circuitos motores del estriado. Depresión y alucinaciones son comunes, y la demencia con el tiempo ocurre en el 20% de los pacientes. En este momento, no existe un tratamiento para retrasar o detener la progresión de la EP. Por el contrario, los medicamentos disponibles en la actualidad más objetivo hacia el alivio de estos síntomas. Nuevas estrategias quirúrgicas reversible puede cambiar en los circuitos funcionalmente dañadas a través de la estimulación eléctrica de estructuras profundas del cerebro, pero a pesar de la estimulación cerebral profunda es un avance importante, no es adecuado para todos los pacientes. Queda, pues, necesario para probar nuevas estrategias de terapia celular en modelos preclínicos. Interrupción selectiva neurotóxica de las vías dopaminérgicas pueden ser reproducidos por la inyección de 6-hidroxidopamina (6-OHDA) o MPTP (1-metil-4-fenil-1 ,2,3,6-tertahydropyridine), mientras que agotan los medicamentos y los productos químicos oxidantes que dañan puede También se reproducen las características específicas de la EP en los roedores. A diferencia de MPTP, 6-OHDA lesiones causan gran pérdida neuronal irreversible, y puede ser uni o bilateral. El modelo de lesión de 6-OHDA es confiable, da lugar a déficits motores robustos, y es el más utilizado después de 40 años de investigación en rats1. Como las interacciones entre las células injertadas y anfitrión puede ser estudiado más a fondo en los ratones y no en las ratas, el modelo ha sido adaptado a los ratones 2,3, donde recientemente se ha caracterizado 4. En este video, que muestra cómo la lesión de la izquierda nigro-estriado vía de ratones anestesiados por la lenta entrega de 2,0 l de 6-OHDA a través de un stereotaxically inserta micro-aguja de la jeringa. La pérdida de la entrada dopaminérgica ocurre unos días después, y el deterioro funcional puede ser monitoreado en post-operatorio semanas y meses por las rotaciones de los animales calificación inducida por agentes dopaminérgicos 5. Aquí, se muestra de cuerpo entero la rotación contralateral ocurre 10 minutos después de una sola administración subcutánea de apomorfina, mide un mes después de la lesión. Los resultados y los inconvenientes se analizan a continuación.

Watch this Scientific Journal Video about Murine Model for Parkinson's Disease: from 6-OH Dopamine Lesion to Behavioral Test at JoVE.com

Murine Model for Parkinson's Disease: from 6-OH Dopamine Lesion to Behavioral Test

La enfermedad de Parkinson es causado por la pérdida de la inervación dopaminérgica en el cuerpo estriado, que puede ser inducida experimentalmente por 6-OH-dopamina. Se describe cómo realizar una lesión estereotáxica y controlar el comportamiento inducido por la apomorfina rotación en los ratones. Este modelo es útil y confiable para probar nuevas terapias para la enfermedad de Parkinson.

Modelo murino de enfermedad de Parkinson: de la lesión 6-OH dopamina a prueba de comportamiento

Parkinson's disease (PD) affects at least 6.5 million people worldwide, irrespective of gender, social, ethnic, economic, or geographic boundaries. Key ...

murine-model-for-parkinson-s-disease-from-6-oh-dopamine-lesion-to

Research

JoVE Journal

Biology

31.5K vistas.  Universidade Federal do Rio de Janeiro, Brasil. La enfermedad de Parkinson es causado por la pérdida de la inervación dopaminérgica en el cuerpo estriado, que puede ser inducida experimentalmente por 6-OH-dopamina. Se describe cómo realizar una lesión estereotáxica y controlar el comportamiento inducido por la apomorfina rotación en los ratones. Este modelo es útil y confiable para probar nuevas terapias para la enfermedad de Parkinson.

Video: Murine Model for Parkinson's Disease: from 6-OH Dopamine Lesion to Behavioral Test

Gibberella zeae Ascospore Production and Collection for Microarray Experiments.

Fusarium graminearum Schwabe (teleomorph Gibberella zeae) is a plant pathogen causing scab disease on wheat and barley that reduces crop yield and grain quality. F. graminearum also causes stalk and ear rots of maize and is a producer of mycotoxins such as the trichothecenes that contaminate grain and are harmful to humans and livestock (Goswami and Kistler, 2004).

The fungus produces two types of spores. Ascospores, the propagules resulting from sexual reproduction, are the main source of primary infection.  These spores are forcibly discharged from mature perithecia and dispersed by wind (Francl et al 1999). Secondary infections are mainly caused by macroconidia which are produced by asexual means on the plant surface. To study the developmental processes of ascospores in this fungus, a procedure for their collection in large quantity under sterile conditions was required. Our protocol was filmed in order to generate the highest level of information for understanding and reproducibility; crucial aspects when full genome gene expression profiles are generated and interpreted. In particular, the variability of ascospore germination and biological activity are dependent on the prior manipulation of the material. The use of video for documenting every step in ascospore production is proposed in order to increase standardization, complying with the increasingly stringent requirements for microarray analysis. The procedure requires only standard laboratory equipment. Steps are shown to prevent contamination and favor time synchronization of ascospores.

To study the developmental processes of ascospores in Gibberella zeae, a procedure for collection under sterile conditions is filmed in order to generate the highest level of information for protocol description. This should facilitate the reproducibility of the experiment, a crucial aspect when full genome expression profile tests are implemented.

Gibberella zeae ascósporas de producción y de recolección de experimentos de microarrays.

Fusarium graminearum Schwabe (teleomorph Gibberella zeae) is a plant pathogen causing scab disease on wheat and barley that reduces crop yield and grain ...

Investigación

Biología

Ole Isacson: Development of New Therapies for Parkinson's Disease

Ole Isacson gives a concise overview of Parkinsons's disease, its causes, therapeutic strategies, and advances in Parkinson's research.

Ole Isacson: desarrollo de nuevas terapias para la enfermedad de Parkinson

Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation from systemic input from elsewhere in the brain. In our lab, we use these neurons to assess dopamine release kinetics using carbon fiber amperometry, as well as expression levels of dopamine related genes and proteins using quantitative PCR and immunocytochemistry. In this video, we show you how we generate these cultures from rodent neonates.
The process involves several steps, including the plating of cortical glial astrocytes, the conditioning of neuronal cell culture media by the glial substrate, the dissection of the midbrain in neonates, the digestion, extraction and plating of dopamine neurons and the addition of neurotrophic factors to ensure cell survival.
The applications suitable for such a preparation include electrophysiology, immunocytochemistry, quantitative PCR, video microscopy (i.e., of real-time vesicular fusion with the plasma membrane), cell viability assays and other toxicological screens.

Primary dissociated midbrain dopamine cell cultures allow for the study of presynaptic characteristics of dopamine neurons. They can be used to monitor real-time dopamine release kinetics and protein/mRNA levels of regulators of dopamine exocytosis. Here, we show you how to generate these cultures from rodent neonates.

Primaria disociada Mesencéfalo culturas dopamina celulares de roedores recién nacidos

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation ...

Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson's Disease Genes

Improvements to the diagnosis and treatment of Parkinson's disease (PD) are dependent upon knowledge about susceptibility factors that render populations at risk. In the process of attempting to identify novel genetic factors associated with PD, scientists have generated many lists of candidate genes, polymorphisms, and proteins that represent important advances, but these leads remain mechanistically undefined. Our work is aimed toward significantly narrowing such lists by exploiting the advantages of a simple animal model system. While humans have billions of neurons, the microscopic roundworm Caenorhabditis elegans has precisely 302, of which only eight produce dopamine (DA) in hemaphrodites. Expression of a human gene encoding the PD-associated protein, alpha-synuclein, in C. elegans DA neurons results in dosage and age-dependent neurodegeneration.
Worms expressing human alpha-synuclein in DA neurons are isogenic and express both GFP and human alpha-synuclein under the DA transporter promoter (Pdat-1). The presence of GFP serves as a readily visualized marker for following DA neurodegeneration in these animals. We initially demonstrated that alpha-synuclein-induced DA neurodegeneration could be rescued in these animals by torsinA, a protein with molecular chaperone activity 1. Further, candidate PD-related genes identified in our lab via large-scale RNAi screening efforts using an alpha-synuclein misfolding assay were then over-expressed in C. elegans DA neurons. We determined that five of seven genes tested represented significant candidate modulators of PD as they rescued alpha-synuclein-induced DA neurodegeneration 2. Additionally, the Lindquist Lab (this issue of JoVE) has performed yeast screens whereby alpha-synuclein-dependent toxicity is used as a readout for genes that can enhance or suppress cytotoxicity. We subsequently examined the yeast candidate genes in our C. elegans alpha-synuclein-induced neurodegeneration assay and successfully validated many of these targets 3, 4.
Our methodology involves generation of a C. elegans DA neuron-specific expression vector using recombinational cloning of candidate gene cDNAs under control of the Pdat-1 promoter. These plasmids are then microinjected in wild-type (N2) worms, along with a selectable marker for successful transformation. Multiple stable transgenic lines producing the candidate protein in DA neurons are obtained and then independently crossed into the alpha-synuclein degenerative strain and assessed for neurodegeneration, at both the animal and individual neuron level, over the course of aging.

This video demonstrates how to use C. elegans to assess dopaminergic neuron neurodegeneration as a model for Parkinson's disease. Furthermore, genetic screens are used to identify factors that either enhance degeneration or are neuroprotective.

Aplicación de un ensayo de C. elegans la degeneración neuronal de dopamina para la validación del potencial de la enfermedad de Parkinson genes

Improvements to the diagnosis and treatment of Parkinson's disease (PD) are dependent upon knowledge about susceptibility factors that render populations ...

Murine Model of Hindlimb Ischemia 

In the United States, peripheral arterial disease (PAD) affects about 10 million individuals, and is also prevalent worldwide. Medical therapies for symptomatic relief are limited. Surgical or endovascular interventions are useful for some individuals, but long-term results are often disappointing. As a result, there is a need for developing new therapies to treat PAD. The murine hindlimb ischemia preparation is a model of PAD, and is useful for testing new therapies. When compared to other models of tissue ischemia such as coronary or cerebral artery ligation, femoral artery ligation provides for a simpler model of ischemic tissue. Other advantages of this model are the ease of access to the femoral artery and low mortality rate. 
In this video, we demonstrate the methodology for the murine model of unilateral hindimb ischemia. The specific materials and procedures for creating and evaluating the model will be described, including the assessment of limb perfusion by laser Doppler imaging. This protocol can also be utilized for the transplantation and non-invasive tracking of cells, which is demonstrated by Huang et al.1.

Murine Model of Hindlimb Ischemia

The surgical procedure for induction of unilateral hindlimb ischemia is demonstrated, with confirmation of ischemia by laser Doppler perfusion imaging.

Modelo murino de isquemia Miembro Posterior

In the United States, peripheral arterial disease (PAD) affects about 10 million individuals, and is also prevalent worldwide.  Medical therapies for ...

Blastomere Explants to Test for Cell Fate Commitment During Embryonic Development

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal which tissues descend from each cell of the embryo. Although fate maps are very useful for identifying the precursors of an organ and for elucidating the developmental path by which the descendant cells populate that organ in the normal embryo, they do not illustrate the full developmental potential of a precursor cell or identify the mechanisms by which its fate is determined. To test for cell fate commitment, one compares a cell's normal repertoire of descendants in the intact embryo (the fate map) with those expressed after an experimental manipulation. Is the cell's fate fixed (committed) regardless of the surrounding cellular environment, or is it influenced by external factors provided by its neighbors? Using the comprehensive fate maps of the Xenopus embryo, we describe how to identify, isolate and culture single cleavage stage precursors, called blastomeres. This approach allows one to assess whether these early cells are committed to the fate they acquire in their normal environment in the intact embryo, require interactions with their neighboring cells, or can be influenced to express alternate fates if exposed to other types of signals.

The fate of an individual embryonic cell can be influenced by inherited molecules and/or by signals from neighboring cells. Utilizing fate maps of the cleavage stage Xenopus embryo, single blastomeres can be identified for culture in isolation to assess the contributions of inherited molecules versus cell-cell interactions.

Los explantes blastómeras para la Prueba de Compromiso destino celular durante el desarrollo embrionario

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal  which tissues descend from each cell of the embryo.  Although fate maps ...

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The initiation step, which involves the assembly of the ribosomal subunits at the mRNA initiation codon, involved initiation factor including eIF4G1. Defects in this rate limiting step of translation are linked to diverse disorders. To study the potential consequences of such deregulations, Xenopus laevis oocytes constitute an attractive model with high degrees of conservation of essential cellular and molecular mechanisms with human. In addition, during meiotic maturation, oocytes are transcriptionally repressed and all necessary proteins are translated from preexisting, maternally derived mRNAs. This inexpensive model enables exogenous mRNA to become perfectly integrated with an effective translation. Here is described a protocol for assessing translation with a factor of interest (here eIF4G1) using stored maternal mRNA that are the first to be polyadenylated and translated during oocyte maturation as a physiological readout. At first, mRNA synthetized by in vitro transcription of plasmids of interest (here eIF4G1) are injected in oocytes and kinetics of oocyte maturation by Germinal Vesicle Breakdown detection is determined. The studied maternal mRNA target is the serine/threonine-protein-kinase mos. Its polyadenylation and its subsequent translation are investigated together with the expression and phosphorylation of proteins of the mos signaling cascade involved in oocyte maturation. Variations of the current protocol to put forward translational defects are also proposed to emphasize its general applicability. In light of emerging evidence that aberrant protein synthesis may be involved in the pathogenesis of neurological disorders, such a model provides the opportunity to easily assess this impairment and identify new targets.

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis control occurs mainly at the translation initiation step, deficiencies in which are linked to diverse disorders. To better understand their etiology, we described here a protocol using Xenopus laevis oocytes assessing the translation of mos transcript in the presence of a mutant of translation initiation factor eIF4G1.

Xenopus laevis como modelo para identificar Traducción Deterioro

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The ...

Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for cartilage tissue engineering. MSCs from synovial fluid (SF-MSCs) have been considered promising candidates for articular regeneration, and their potential therapeutic benefit has made them an important research topic of late. SF-MSCs from the knee cavity of the New Zealand white rabbit can be employed as an optimized translational model to assess human regenerative medicine. By means of CD90-based magnetic activated cell sorting (MACS) technologies, this protocol successfully obtains rabbit SF-MSCs (rbSF-MSCs) from this rabbit model and further fully demonstrates the MSC phenotype of these cells by inducing them to differentiate to osteoblasts, adipocytes, and chondrocytes. Therefore, this approach can be applied in cell biology research and tissue engineering using simple equipment and procedures.

This article presents a simple and economic protocol for the straightforward isolation and purification of mesenchymal stem cells from New Zealand white rabbit synovial fluid.

Célula activada por el magnético clasificación estrategias para aislar y purificar sinovial líquido mesenquimales células madre derivadas de un modelo de conejo

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for ...

Protocols for Testing the Toxicity of Novel Insecticidal Chemistries to Mosquitoes

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such as Zika, dengue and malaria. Assays for rapid, high-throughput analyses of unformulated novel chemistries against mosquito larvae and adults are presented. We describe protocols for single point-dose and dose response assays to evaluate the toxicity of small molecule chemistries to the Aedes aegypti vector of Zika, dengue and yellow fever,&#160;the malaria vector, Anopheles gambiae and the northern house mosquito, Culex quinquefasciatus, on contact and via ingestion. As an example, we evaluated the toxicity of amitriptyline, a small molecule antagonist of G protein-coupled receptors, via larval, adult topical and adult blood-feeding assay. The protocols provide a starting point to investigate insecticide potential. Results are discussed in the context of additional experiments to explore product applications and mechanisms for delivery.

Protocols are described for assessing the toxicity of chemistries to immature and adult mosquitoes for development as new classes of larvicides, adulticides and endectocides. The protocols enable high-throughput testing of multiple chemistries at single-point dose and subsequent evaluation via dose response assay to determine toxicity on contact or via ingestion.

Protocolos para las pruebas de la toxicidad de la novela química insecticida para mosquitos

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such ...

Noninvasive Monitoring of Lesion Size in a Heterologous Mouse Model of Endometriosis

Here, we describe a protocol for the implementation of a heterologous mouse model in which progression of endometriosis can be assessed in real time through noninvasive monitoring of fluorescence emitted by implanted ectopic human endometrial tissue. For this purpose, biopsies of human endometrium are obtained from donor women ongoing oocyte donation. Human endometrial fragments are cultured in the presence of adenoviruses engineered to express cDNA for the reporter fluorescent protein mCherry. Upon visualization, labeled tissues with an optimal rate of fluorescence after infection are subsequently chosen for the implantation in recipient mice. One week prior to the implantation surgery, recipient mice are oophorectomized, and estradiol pellets are placed subcutaneously to sustain the survival and growth of lesions. On the day of surgery mice are anesthetized, and peritoneal cavity accessed through a small (1.5 cm) incision by the linea-alba. Fluorescently labeled implants are tweezed, briefly soaked in glue and attached to the peritoneal layer. Incisions are sutured, and animals left to recover for a couple of days. Fluorescence emitted by endometriotic implants is usually non-invasively monitored every 3 days for 4 weeks with an in vivo imaging system. Variations in the size of endometriotic implants can be estimated in real time by quantification of the mCherry signal and normalization against the initial time-point showing maximal fluorescence intensity.
Traditional preclinical rodents of models of endometriosis do not allow non-invasive monitoring of lesion in real time but rather allow evaluation of the effects of drugs assayed at the end point. This protocol allows one to track lesions in real time and is more useful to explore the therapeutic potential of drugs in preclinical models of endometriosis. The main limitation of the model thus generated is that non-invasive monitoring is not possible over long periods of time due to the episomal expression of Ad-virus.

Here, we present a protocol for live-imaging of fluorescently labeled human endometrial fragments grafted in mice. The method allows studying the effects of drugs of choice on endometriotic lesion size through monitoring and quantification of fluorescence emitted by the fluorescent reporter on real time

Monitoreo no invasivo del tamaño de la lesión en un modelo de ratón heterólogos de la Endometriosis

Here, we describe a protocol for the implementation of a heterologous mouse model in which progression of endometriosis can be assessed in real time ...