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...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<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>
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<td>L - Ascorbic Acid</td>
<td>&nbsp;</td>
<td>USB Corporation</td>
<td>&nbsp;</td>
<td>0.2%</td>
</tr>
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<td>Xylocaine (Lidocaine Chloridrate)</td>
<td>&nbsp;</td>
<td>ASTRA Chemistry</td>
<td>&nbsp;</td>
<td>5% 50 mg</td>
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<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>
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<td>Ibuprofen</td>
<td>&nbsp;</td>
<td>Abbott</td>
<td>&nbsp;</td>
<td>4 mg/ 100 ml drinking water</td>
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<td>Povidine</td>
<td>&nbsp;</td>
<td>JohnsonDiversey</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>R-(-)-Apomorphine hydrochloride</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>&nbsp;</td>
<td>0.5 mg/Kg</td>
</tr>		</tbody>
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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&#039;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

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

31.4K 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

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

Ole Isacson: Development of New Therapies for Parkinson&#039;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

Investigación

Biología

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.

Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson&#039;s Disease Genes

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 ...

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 ...

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 ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.

Análisis de espectrometría de masas para identificar la ubiquitylación de CENP-A con etiqueta EYFP (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer ...

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and calcium handling. For this purpose, atrial or ventricular cardiomyocytes of high quality are necessary to perform patch-clamp measurements or to explore calcium handling abnormalities. However, the limited yield of high-quality cardiomyocytes obtained by current isolation protocols does not allow both measurements in the same mouse. This article describes a method to isolate high-quality murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, for subsequent simultaneous measurements of calcium transients and L-type calcium current from one animal. Mouse hearts are obtained, and the aorta is rapidly cannulated to remove blood. Hearts are then initially perfused with a calcium-free solution (37 &#176;C) to dissociate the tissue at the level of intercalated discs and afterwards with an enzyme solution containing little calcium to disrupt extracellular matrix (37 &#176;C). The digested heart is subsequently dissected into atria and ventricles. Tissue samples are chopped into small pieces and dissolved by carefully pipetting up and down. The enzymatic digestion is stopped, and cells are stepwise reintroduced to physiologic calcium concentrations. After loading with a fluorescent Ca2+-indicator, isolated cardiomyocytes are prepared for simultaneous measurement of calcium currents and transients. Additionally, isolation pitfalls are discussed and patch-clamp protocols and representative traces of L-type calcium currents with simultaneous calcium transient measurements in atrial and ventricular murine myocytes isolated as described above are provided.

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models allow studying key mechanisms of arrhythmogenesis. For this purpose, high quality cardiomyocytes are necessary to perform patch-clamp measurements. Here, a method to isolate murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, which allows simultaneous measurements of calcium-transients and L-type calcium current, is described.

Aislamiento de miocitos aurículas y ventriculares murinas de alta calidad para mediciones simultáneas de transitorios de Ca2+ y corriente de calcio tipo L

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and ...

Rating L-DOPA-Induced Dyskinesias in the Unilaterally 6-OHDA-Lesioned Rat Model of Parkinson's Disease

L-DOPA-induced dyskinesias (LIDs) refer to motor complications that arise from prolonged L-DOPA administration to patients with Parkinson's disease (PD). The most common pattern observed in the clinic is the peak-dose dyskinesia which consists of clinical manifestations of choreiform, dystonic, and ballistic movements. The 6-hydroxydopamine (6-OHDA) rat model of PD mimics several characteristics of LIDs. After repeated L-DOPA administration, 6-OHDA-lesioned rats exhibit dyskinetic-like movements (e.g., abnormal involuntary movements, AIMs). This protocol demonstrates how to induce and analyze AIMs in 6-OHDA-lesioned rats with 90%-95% dopaminergic depletion in the nigrostriatal pathway. Repeated administration (3 weeks) of L-DOPA (5 mg/kg, combined with 12.5 mg/kg of benserazide) can induce the development of AIMs. The time course analysis reveals a significant increase in AIMs at 30-90 min (peak-dose dyskinesia). Rodent models of LIDs are an important preclinical tool to identify effective antidyskinetic interventions.

Rating L-DOPA-Induced Dyskinesias in the Unilaterally 6-OHDA-Lesioned Rat Model of Parkinson&#039;s Disease

Rodent models of L-DOPA-induced dyskinesias are invaluable tools to identify therapeutic interventions to attenuate the development or alleviate the manifestations that emerge due to the repeated administration of L-DOPA. This protocol demonstrates how to induce and analyze dyskinetic-like movements in the unilaterally 6-OHDA-lesioned rat model of Parkinson's disease.

Calificación de discinesias inducidas por L-DOPA en el modelo de rata lesionada unilateralmente con 6-OHDA de la enfermedad de Parkinson

L-DOPA-induced dyskinesias (LIDs) refer to motor complications that arise from prolonged L-DOPA administration to patients with Parkinson's disease (PD). ...

Cultivo 3D de organoides a partir de criptas intestinales murinos y células madre individuales

The 3D Culturing of Organoids from Murine Intestinal Crypts and a Single Stem Cell for Organoid Research

At present, organoid culture represents an important tool for in vitro studies of different biological aspects and diseases in different organs. Murine small intestinal crypts can form organoids that mimic the intestinal epithelium when cultured in a 3D extracellular matrix. The organoids are composed of all cell types that fulfill various intestinal homeostatic functions. These include Paneth cells, enteroendocrine cells, enterocytes, goblet cells, and tuft cells. Well-characterized molecules are added into the culture medium to enrich the intestinal stem cells (ISCs) labeled with leucine-rich repeats containing G protein-coupled receptor 5 and are used to drive differentiation down specific lineages; these molecules include epidermal growth factor, Noggin (a bone morphogenetic protein), and R-spondin 1. Additionally, a protocol to generate organoids from a single erythropoietin-producing hepatocellular receptor B2 (EphB2)-positive ISC is also detailed. In this methods article, techniques to isolate small intestinal crypts and a single ISC from tissues and ensure the efficient establishment of organoids are described.

Autor destacado: El cultivo en 3D de organoides de criptas intestinales murinas y una sola célula madre para la investigación de organoides  

We describe a protocol to isolate murine small intestinal crypts and culture intestinal 3D organoids from the crypts. Additionally, we describe a method to generate organoids from a single intestinal stem cell in the absence of a sub-epithelial cellular niche.

El cultivo 3D de organoides a partir de criptas intestinales murinas y una sola célula madre para la investigación de organoides

At present, organoid culture represents an important tool for in vitro studies of different biological aspects and diseases in different organs. Murine ...