El trabajo aquí descrito fue financiado por los EE.UU. Departamento de Asuntos de Veteranos Biomédica Laboratorio de Investigación y Desarrollo BX001686-1A1 y VA Rehabilitación de Investigación y Desarrollo.

1. Plating y Mantenimiento de Cultivos Celulares <ol><li> Medio Eagle Modificado de calentamiento por Dulbecco (DMEM) medio de cultivo (DMEM + 10% de FBS + 1% de penicilina / estreptomicina / neomicina) a 37 ° C. </li><li> Obtener un balance de las células congeladas A12 de -80 ° C. </li><li> Rápidamente descongelar las células en 37 ° C baño de agua; una vez descongelado, suavemente las células de pipeta para transferir a un frasco de cultivo de ventilación 75 cm2 y añadir 10 ml de medio de cultivo. </li><li> Incubar frasco durante la noche en 5% de CO 2 incubadora a 37 ° C. Aspirar los medios después de 24 horas y reemplazar con medio fresco. Continúe creciendo las células hasta que se obtenga un 70-80% de confluencia (crecimiento de 2-3 días). </li></ol> 2. Contar y células en placas <ol><li> DMEM Cálido y 1 x solución de tripsina-EDTA a 37 º C. </li><li> Aspirar los medios de las células y se lavan dos veces con solución salina tamponada con fosfato estéril (PBS). </li><li> Añadir 500 l de solución de tripsina y se incuba a 37 ° C durante 2-5 min. </li><li> Despegarcélulas del frasco utilizando una espátula y suspender las células en 5 ml de medio. Pasar las células a través del filtro y pasar a través de filtro de células en un tubo limpio de 50 ml. Puede que tenga que pasar las células a través del filtro más de una vez, pero no repita más de tres veces. </li><li> Contar las células utilizando un hemocitómetro para determinar el importe de las células a la cultura de sembrar 6.000 células / pocillo en una placa de color negro o blanco 96 pocillos de fondo claro amurallado en un volumen final de 100 l de los medios de comunicación. Incubar la placa en 5% de CO 2 a 37 ° C durante 24 horas. </li></ol> 3. La determinación de la viabilidad celular y la caspasa-3/7 Actividad <ol><li> Transferencia PA a un vial de vidrio de 5 ml estéril y solubilizar en 100% de dimetil sulfóxido (DMSO). Diluir la solución madre PA 1:10 en DMSO y añadirlo a precalentado DMEM para obtener una concentración de trabajo final de 0,1 mM PA. Para los medios de control, agregue la misma concentración de DMSO que se añade a los medios de comunicación que contienen PA. </li><li> Retire los medios de comunicación en cada pozo y reemplazar con 50 medios mu l + / -PA. Incubar la placa durante 2 horas en 5% de CO 2 a 37 ° C. Añadir a continuación 5 reactivo resazurin l y se incuba durante 10 minutos a temperatura ambiente. </li><li> El uso de un lector de microplacas multimodo, ficha fluorescencia (560 EX / 590 EM) para medir la viabilidad celular. Los resultados se presentan como unidades de fluorescencia relativa (RFU). </li><li> Para la misma placa, añadir 55 l de reactivo de caspasa y se incuba a temperatura ambiente durante 2 h. </li><li> Una vez más mediante el lector de microplacas, ficha luminiscencia para medir la actividad de la caspasa-3/7. Los resultados se presentan como unidades relativas de luminiscencia (RLU), con luminiscencia directamente proporcional a la caspasa-3/7 actividad. </li><li> Para normalizar la actividad de caspasa-3/7 para el recuento de células, dividir la viabilidad celular (RFU) por la caspasa-3/7 actividad (RLU). </li></ol>

<ol>
	<li>Akil, L., Ahmad, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationships+between+obesity+and+cardiovascular+diseases+in+four+southern+states+and+Colorado.">Relationships between obesity and cardiovascular diseases in four southern states and Colorado.</a> J. Health Care Poor Underserved. 22, 61-72 (2011).</li><li>Posey, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypothalamic+proinflammatory+lipid+accumulation,+inflammation,+and+insulin+resistance+in+rats+fed+a+high-fat+diet.">Hypothalamic proinflammatory lipid accumulation, inflammation, and insulin resistance in rats fed a high-fat diet.</a> Am. J. Physiol. Endocrinol. Metab. 296, 1003-1012 (2009).</li><li>Moraes, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-fat+diet+induces+apoptosis+of+hypothalamic+neurons.">High-fat diet induces apoptosis of hypothalamic neurons.</a> PloS one. 4, (2009).</li><li>Mayer, C. M., Belsham, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Palmitate+attenuates+insulin+signaling+and+induces+endoplasmic+reticulum+stress+and+apoptosis+in+hypothalamic+neurons:+rescue+of+resistance+and+apoptosis+through+adenosine+5'+monophosphate-activated+protein+kinase+activation.">Palmitate attenuates insulin signaling and induces endoplasmic reticulum stress and apoptosis in hypothalamic neurons: rescue of resistance and apoptosis through adenosine 5' monophosphate-activated protein kinase activation.</a> Endocrinology. 151, 576-585 (2010).</li><li>Benoit, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Palmitic+acid+mediates+hypothalamic+insulin+resistance+by+altering+PKC-theta+subcellular+localization+in+rodents.">Palmitic acid mediates hypothalamic insulin resistance by altering PKC-theta subcellular localization in rodents.</a> J. Clin. Invest. 119, 2577-2589 (2009).</li><li>Thaler, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Obesity+is+associated+with+hypothalamic+injury+in+rodents+and+humans.">Obesity is associated with hypothalamic injury in rodents and humans.</a> J. Clin. Invest. 122, 153-162 (2012).</li><li>Williams, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypothalamic+dysfunction+in+obesity.">Hypothalamic dysfunction in obesity.</a> Proc. Nutr. Soc. 71, 521-533 (2012).</li><li>Belsham, D. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+a+phenotypic+array+of+hypothalamic+neuronal+cell+models+to+study+complex+neuroendocrine+disorders.">Generation of a phenotypic array of hypothalamic neuronal cell models to study complex neuroendocrine disorders.</a> Endocrinology. 145, 393-400 (2004).</li><li>Xiao, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+of+cell+viability+and+proliferation+in+hydrogel-encapsulated+system+by+resazurin+assay.">Monitoring of cell viability and proliferation in hydrogel-encapsulated system by resazurin assay.</a> Appl. Biochem. Biotechnol. 162, 1996-2007 (2010).</li><li>Butterick, T. A., Nixon, J. P., Billington, C. J., Kotz, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orexin+A+decreases+lipid+peroxidation+and+apoptosis+in+a+novel+hypothalamic+cell+model.">Orexin A decreases lipid peroxidation and apoptosis in a novel hypothalamic cell model.</a> Neurosci. Lett. 524, 30-34 (2012).</li><li>Riss, T. L., Moravec, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+multiple+assay+endpoints+to+investigate+the+effects+of+incubation+time,+dose+of+toxin,+and+plating+density+in+cell-based+cytotoxicity+assays.+Assay+Drug+Dev.">Use of multiple assay endpoints to investigate the effects of incubation time, dose of toxin, and plating density in cell-based cytotoxicity assays. Assay Drug Dev.</a> Technol. 2, 51-62 (2004).</li><li>Niles, A. L., Moravec, R. A., Riss, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+viability+and+cytotoxicity+testing+and+same-well+multi-parametric+combinations+for+high+throughput+screening.">In vitro viability and cytotoxicity testing and same-well multi-parametric combinations for high throughput screening.</a> Curr. Chem. Genomics. 3, 33-41 (2009).</li><li>Steffen, W., Linck, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+immunoblot:+a+sensitive+technique+to+stain+proteins+and+detect+multiple+antigens+on+a+single+two-dimensional+replica.">Multiple immunoblot: a sensitive technique to stain proteins and detect multiple antigens on a single two-dimensional replica.</a> Electrophoresis. 10, 714-718 (1989).</li><li>Gingrich, J. C., Davis, D. R., Nguyen, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+detection+and+quantitation+of+proteins+on+western+blots+using+fluorescent+probes.">Multiplex detection and quantitation of proteins on western blots using fluorescent probes.</a> Biotechniques. 29, 636-642 (2000).</li><li>Riss, T. L., Moravec, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+multiple+assay+endpoints+to+investigate+the+effects+of+incubation+time,+dose+of+toxin,+and+plating+density+in+cell-based+cytotoxicity+assays.">Use of multiple assay endpoints to investigate the effects of incubation time, dose of toxin, and plating density in cell-based cytotoxicity assays.</a> Assay Drug Dev. Technol. 2, 51-62 (2004).</li><li>Smyth, P. G., Berman, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Markers+of+apoptosis:+methods+for+elucidating+the+mechanism+of+apoptotic+cell+death+from+the+nervous+system.">Markers of apoptosis: methods for elucidating the mechanism of apoptotic cell death from the nervous system.</a> Biotechniques. 32, 648-650 (2002).</li><li>Lavrik, I. N., Golks, A., Krammer, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caspases:+pharmacological+manipulation+of+cell+death.">Caspases: pharmacological manipulation of cell death.</a> J. Clin. Invest. 115, 2665-2672 (2005).</li></ol>

Los autores no tienen conflictos a revelar.

El ensayo multiplex es una técnica bien aceptada que ha sido usado por los científicos en numerosas aplicaciones tales como microarrays de PCR, inmunodetección, y otros métodos de detección basados ​​en proteínas 13, 14. Recientemente, los ensayos de multiplexado se han convertido en cada vez más utilizado en los experimentos in vitro basado en la placa y se han validado como un método preciso para determinar la citotoxicidad y viabilidad 12. En el protocolo anterior, ...

Las dietas ricas en ácidos grasos saturados, como el ácido palmítico (PA) se han relacionado con la obesidad y otras comorbilidades, como la enfermedad cardiovascular y la diabetes 1, 2. Las dietas altas en grasa también se ha demostrado que aumentar el estrés oxidativo, la apoptosis, y la degeneración neuronal en el hipotálamo, un importante regulador del apetito y el gasto de energía 3-7. Entender el mecanismo a través del cual la exposición dieta rica en grasas induce desregulación hipotalámica tanto, es importante para el desarrollo de tratamientos farmacológicos para la obesidad. Sin embargo, los mecanismos celulares a través del cual la grasa dietética afecta a la función neuronal siguen sin estar claros. Una mejor comprensión de cómo las grasas tales como PA podrían dar lugar a la aparición de las vías de apoptosis hipotálamo es un primer paso necesario hacia este objetivo. El objetivo de este artículo es describir un ensayo múltiple para las pruebas in vitro de la respuesta neuronal a la exposición PA, desarrollado para su uso en estudios de hneurodegeneración ypothalamic. Proporcionamos una descripción detallada de un formato de 96 pocillos de ensayo multiplex in vitro para medir la caspasa 3/7 actividad por el número total de células en un ratón adulto línea diferenciada inmortalizado hipotalámico celular (designado A12) después del desafío oxidativo con PA 8. En pocas palabras, la viabilidad celular se determina después de la exposición a través de un ensayo de PA resazurin basado. La resazurina es un compuesto permeable a la célula que se somete a reducción enzimática en las células metabólicamente activas, un proceso de pensamiento que se produzca a través de la mitocondria 9. Las células viables se convierten continuamente resazurina a resorufina, la producción de una señal fluorescente proporcional al número de células viables. La actividad de caspasa-3/7 se analiza a continuación, utilizando un ensayo basado lumogenic-DEVD. DEVD es una secuencia de aminoácidos (Asp-Glu-Val-Asp) escindido por la caspasa-3. Cuando esta secuencia está acoplado a una sustancia lumogenic, tras la activación de intracelular de la caspasa-3/7 y posterior escisiónde sustrato DEVD, el producto luminiscente se libera. Esta reacción es proporcional a la actividad de caspasa y por lo tanto a la inducción de la apoptosis. Como las células muertas no pueden producir la caspasa, la caspasa-3/7 actividad es de naturaleza transitoria; Por lo tanto, el análisis debe ser completado entre 30 min a 4 horas después de la exposición, en función de la eficacia de estrés celular. La viabilidad celular es inversamente proporcional a la actividad de la caspasa-3/7 y se puede utilizar para determinar los mecanismos de muerte celular. Por ejemplo, este método se ha utilizado anteriormente para mostrar que el tratamiento previo con la hormona péptido orexina reduce la apoptosis en las células hipotalámicas desafiados con peróxido de hidrógeno, lo que sugiere que los mecanismos afectados por este tratamiento son importantes en la protección contra el daño oxidativo 10. Es importante tener en cuenta estos ensayos son la línea-y celular dependiente de tejido, ya que se basan en la actividad mitocondrial para reducir los reactivos de ensayo. Este protocolo ha sido optimizado para (A12), las células del hipotálamo de ratones adultos; Sin embargo,métodos descritos pueden alterarse para ajustarse dentro del alcance de investigaciones similares. Ensayos de multiplexación de un solo pocillo de cultivo proporciona una ventaja sobre el método tradicional de realizar ensayos individuales por varias razones. Además de tiempo, muestras de células, y los reactivos de cultivo de ahorro, los ensayos de multiplexación también pueden proporcionar el conocimiento de la supervivencia celular y la muerte, proporcionar controles internos, y eliminar la necesidad de experimentos repetitivos 11, 12. Los métodos alternativos se han basado en transferencias de Western o ELISA, que son ensayos fiables, pero son caros y consumir tiempo (1-2 días), especialmente cuando se utiliza un formato de 96 pocillos. Excluyendo el tiempo que tarda en células de cultivo para el ensayo de multiplexación, el tiempo total es de menos de 3 h. Aunque este protocolo ha sido optimizado para su uso con células A12, y puede ser modificada para su uso en otros modelos teniendo en cuenta factores que pueden influir en la integridad celular. Disuadirminería si este protocolo funcionaría para una serie de experimentos depende del número de muestras o condiciones experimentales y en experimentos posteriores previstas.

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	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:268px;">Adult Mouse Hypothalamus Cell Line mHypoA-1/2&#160;</td>
			<td style="width:137px;">Cellutions Biosystems Inc.</td>
			<td style="width:151px;">CLU172</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Dulbecco&#8217;s Modified Eagle&#8217;s Medium</td>
			<td style="width:137px;">Invitrogen</td>
			<td style="width:151px;">10313-039</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Fetal bovine serum&#160;</td>
			<td style="width:137px;">PAA Labs</td>
			<td style="width:151px;">A15-751</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Penicillin/Streptomycin</td>
			<td style="width:137px;">Invitrogen</td>
			<td style="width:151px;">15070-063</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Palmitic Acid</td>
			<td style="width:137px;">Sigma-Aldrich</td>
			<td style="width:151px;">P0500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Dimethy sulfoxide&#160;</td>
			<td style="width:137px;">Sigma-Aldrich</td>
			<td style="width:151px;">D2650</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:268px;"> 
			PrestoBlue Cell Viability Reagent</td>
			<td style="width:137px;">Invitrogen</td>
			<td>A13262</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Caspase-Glo 3/7 Assay Systems</td>
			<td style="width:137px;">Promega</td>
			<td style="width:151px;">G8091</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Equipment</td>
			<td style="width:137px;"></td>
			<td style="width:151px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:268px;">96W Optical Bottom Plate, Black Polystyrene, Cell Culture Treated, with lid, Sterile</td>
			<td style="width:137px;">Thermo Fisher Scientific</td>
			<td style="width:151px;">165305</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:268px;">SpectraMax M5 Multi-Mode Microplate</td>
			<td style="width:137px;">Molecular Devices</td>
			<td style="width:151px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

use of a caspase multiplexing assay to determine apoptosis in a hypothalamic cell model

La capacidad de multiplexar ensayos en los estudios de los mecanismos celulares complejas elimina la necesidad de experimentos repetitivos, proporciona controles internos, y disminuye los residuos en los costos y los reactivos. Aquí se describe la optimización de un ensayo múltiple para evaluar la apoptosis después de un ácido palmítico (PA) en un modelo de desafío hipotalámico in vitro, usando tanto fluorescente y luminiscente basada en ensayos para medir el recuento de células viables y la caspasa-3/7 actividad en un 96-así formato de placa de microtitulación. Después de la exposición PA, las células viables se determinaron por un ensayo fluorescente basado en resazurina. La caspasa-3/7 actividad fue luego determinado utilizando un sustrato luminógeno, DEVD, y se normalizó al número de células. Este ensayo multiplexado es una técnica útil para determinar el cambio en la actividad de la caspasa después de un estímulo apoptótico, tales como desafío ácido graso saturado. El ácido graso saturado PA puede aumentar el estrés oxidativo del hipotálamo y la apoptosis, lo que indica el potencial de importaciónNCE de ensayos tales como el que se describe aquí en el estudio de la relación entre los ácidos grasos saturados y la función neuronal.

El protocolo anterior describe los resultados en dos ensayos separados de multiplexación para determinar los mecanismos de muerte celular. Figura 1 muestra una visión general del protocolo para determinar la viabilidad celular y la caspasa-3/7 actividad. Actividad caspasa fue significativamente mayor en las células estimuladas con PA después de 2 horas de incubación (Figura 2A y el Cuadro 1). La pérdida de integridad de la membrana celular es un cambio morfológic...

Watch this Scientific Journal Video about Use of a Caspase Multiplexing Assay to Determine Apoptosis in a Hypothalamic Cell Model at JoVE.com

Use of a Caspase Multiplexing Assay to Determine Apoptosis in a Hypothalamic Cell Model

Ensayos multiplex pueden proporcionar información útil para los mecanismos celulares básicos y eliminar los residuos de los reactivos y experimentos repetitivos innecesarios. Se describe aquí un múltiplex de la caspasa-3/7 ensayo de actividad, utilizando métodos fluorescentes y luminiscentes basados ​​en, para determinar la viabilidad celular en un modelo hipotalámico in vitro después de desafío oxidativo con ácido palmítico.

El uso de una Multiplexación Ensayo de la caspasa para determinar la apoptosis en un modelo de célula hipotalámica

The ability to multiplex assays in studies of complex cellular mechanisms eliminates the need for repetitive experiments, provides internal controls, and ...

use-caspase-multiplexing-assay-to-determine-apoptosis-hypothalamic

Research

JoVE Journal

Neuroscience

14.9K vistas.  Minneapolis Veterans Affairs Health Care System.  Ensayos multiplex pueden proporcionar información útil para los mecanismos celulares básicos y eliminar los residuos de los reactivos y experimentos repetitivos innecesarios. Se describe aquí un múltiplex de la caspasa-3/7 ensayo de actividad, utilizando métodos fluorescentes y luminiscentes basados ​​en, para determinar la viabilidad celular en un modelo hipotalámico in vitro después de desafío oxidativo con ácido palmítico. 

Video: Use of a Caspase Multiplexing Assay to Determine Apoptosis in a Hypothalamic Cell Model

Optic Nerve Transection: A Model of Adult Neuron Apoptosis in the Central Nervous System

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. 
The optic nerve can be accessed within the orbit of the eye and completely transected (axotomized), cutting the axons of the entire RGC population. Optic nerve 
transection is a reproducible model of apoptotic neuronal cell death in the adult CNS 1-4. This model is particularly attractive because the vitreous
 chamber of the eye acts as a capsule for drug delivery to the retina, permitting experimental manipulations via intraocular injections. The diffusion of chemicals 
through the vitreous fluid ensures that they act upon the entire RGC population. Moreover, RGCs can be selectively transfected by applying short interfering RNAs 
(siRNAs), plasmids, or viral vectors to the cut end of the optic nerve 5-7 or injecting vectors into their target, the superior colliculus 8. 
This allows researchers to study apoptotic mechanisms in the desired neuronal population without confounding effects on other bystander neurons or surrounding glia. 
An additional benefit is the ease and accuracy with which cell survival can be quantified after injury. The retina is a flat, layered tissue and RGCs are localized in 
the innermost layer, the ganglion cell layer. The survival of RGCs can be tracked over time by applying a fluorescent tracer (3% Fluorogold) to the cut end of the 
optic nerve at the time of axotomy, or by injecting the tracer into the superior colliculus (RGC target) one week prior to axotomy. The tracer is retrogradely transported, labeling 
the entire RGC population. Because the ganglion cell layer is a monolayer (one cell thick), RGC densities can be quantified in flat-mounted tissue, without the need 
for stereology. Optic nerve transection leads to the apoptotic death of 90% of injured RGCs within 14 days postaxotomy 9-11. RGC apoptosis has a 
characteristic time-course whereby cell death is delayed 3-4 days postaxotomy, after which the cells rapidly degenerate. This provides a time window for 
experimental manipulations directed against pathways involved in apoptosis.

Optic Nerve transection is a widely used model of adult CNS injury. Ninety percent of retinal ganglion cells (RGCs) whose axons are completely transected (axotomy) die within 14 days after axotomy. This model is easily amenable to experimental manipulations and highly reproducible.

La transección del nervio óptico: un modelo de apoptosis neuronal de adultos en el sistema nervioso central

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. 
The optic nerve can be ...

Investigación

Neurociencias

Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). Furthermore, this serum-free culture system has also been employed to expand stem cells and determine their frequency from a variety of tumors and normal tissues. It has been shown recently that a one-to-one relationship does not exist between neurosphere formation and NSCs. This suggests that the NSA as currently applied, overestimates the frequency of NSCs in a mixed population of neural precursor cells isolated from both the embryonic and adult mammalian brain. This video practically demonstrates a novel collagen based semi- solid assay, the neural-colony forming cell assay (N-CFCA), which has the ability to discriminate stem from progenitor cells based on their long-term proliferative potential, and thus provides a method to enumerate NSC frequency. In the N-CFCA, colonies &ge;2 mm in diameter are derived from cells that meet all the functional criteria of a NSC, while colonies &lt; 2mm are derived from progenitors. The N-CFCA procedure can be used for cells prepared from different sources including primary and cultured adult or embryonic mouse CNS cells. Here we use cells prepared from passage one neurospheres generated from embryonic day 14 mice brain to perform N-CFCA. The cultures are replenished with proliferation medium every seven days for three weeks to allow the plated cells to exhibit their full proliferative potential and then the frequency of neural progenitor and bona fide neural stem cells is calculated respectively by counting the number of colonies that are &lt; 2mm and the ones that are &ge;2mm in reference to the number of cells that were initially plated.

This video protocol demonstrates how to discriminate and enumerate bona fide neural stem cells in a mixed population of neural precursor cells using the neural colony-forming cell assay.

Neural formadoras de colonias ensayo de células: un ensayo para discriminar Bona Fide células troncales nerviosas de células progenitoras neurales

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). ...

Monitoring Cleaved Caspase-3 Activity and Apoptosis of Immortalized Oligodendroglial Cells using Live-cell Imaging and Cleaveable Fluorogenic-dye Substrates Following Potassium-induced Membrane Depolarization

The central nervous system can experience a number of stresses and neurological insults, which can have numerous adverse effects that ultimately lead to a reduction in neuronal population and function. Damaged axons can release excitatory molecules including potassium or glutamate into the extracellular matrix, which in turn, can produce further insult and injury to the supporting glial cells including astrocytes and oligodendrocytes 8, 16. If the insult persists, cells will undergo programmed cell death (apoptosis), which is regulated and activated by a number of well-established signal transduction cascades 14. Apoptosis and tissue necrosis can occur after traumatic brain injury, cerebral ischemia, and seizures. A classical example of apoptotic regulation is the family of cysteine-dependent aspartate-directed proteases, or caspases. Activated proteases including caspases have also been implicated in cell death in response to chronic neurodegenerative diseases including Alzheimer's, Huntington's, and Multiple Sclerosis 4, 14, 3, 11, 7.
In this protocol we describe the use of the NucView 488 caspase-3 substrate to measure the rate of caspase-3 mediated apoptosis in immortalized N19-oligodendrocyte (OLG) cell cultures 15, 5, following exposure to different extracellular stresses such as high concentrations of potassium or glutamate. The conditionally-immortalized N19-OLG cell line (representing the O2A progenitor) was obtained from Dr. Anthony Campagnoni (UCLA Semel Institute for Neuroscience) 15, 5, and has been previously used to study molecular mechanisms of myelin gene expression and signal transduction leading to OLG differentiation (e.g.6, 10). We have found this cell line to be robust with respect to transfection with exogenous myelin basic protein (MBP) constructs fused to either RFP or GFP (red or green fluorescent protein) 13, 12. Here, the N19-OLG cell cultures were treated with either 80 mM potassium chloride or 100 mM sodium glutamate to mimic axonal leakage into the extracellular matrix to induce apoptosis 9. We used a bi-functional caspase-3 substrate containing a DEVD (Asp-Glu-Val-Asp) caspase-3 recognition subunit and a DNA-binding dye 2. The substrate quickly enters the cytoplasm where it is cleaved by intracellular caspase-3. The dye, NucView 488 is released and enters the cell nucleus where it binds DNA and fluoresces green at 488 nm, signaling apoptosis. Use of the NucView 488 caspase-3 substrate allows for live-cell imaging in real-time 1, 10. In this video, we also describe the culturing and transfection of immortalized N19-OLG cells, as well as live-cell imaging techniques.

Monitoring Cleaved Caspase-3 Activity and Apoptosis of Immortalized Oligodendroglial Cells using Live-cell Imaging and Cleaveable Fluorogenic-dye Substrates Following Potassium-induced Membrane Depola

Live-cell imaging of caspase-3 mediated apoptosis in immortalized N19-oligodendrocyte cell cultures using the NucView 488 caspase-3 substrate. This technique is applicable for programmed cell death assays in real-time in a variety of cell types and tissues.

Monitoreo escinde actividad caspasa-3 y la apoptosis de las células inmortalizadas oligodendrogliales utilizando células vivas, imágenes y Cleaveable Sustratos Fluorogenic-dye Después de despolarización de la membrana de potasio inducida por

The central nervous system can experience a number of stresses and neurological insults, which can have numerous adverse effects that ultimately lead to a ...

Functional Interrogation of Adult Hypothalamic Neurogenesis with Focal Radiological Inhibition

The functional characterization of adult-born neurons remains a significant challenge. Approaches to inhibit adult neurogenesis via invasive viral delivery or transgenic animals have potential confounds that make interpretation of results from these studies difficult. New radiological tools are emerging, however, that allow one to noninvasively investigate the function of select groups of adult-born neurons through accurate and precise anatomical targeting in small animals. Focal ionizing radiation inhibits the birth and differentiation of new neurons, and allows targeting of specific neural progenitor regions. In order to illuminate the potential functional role that adult hypothalamic neurogenesis plays in the regulation of physiological processes, we developed a noninvasive focal irradiation technique to selectively inhibit the birth of adult-born neurons in the hypothalamic median eminence. We describe a method for Computer tomography-guided focal irradiation (CFIR) delivery to enable precise and accurate anatomical targeting in small animals. CFIR uses three-dimensional volumetric image guidance for localization and targeting of the radiation dose, minimizes radiation exposure to nontargeted brain regions, and allows for conformal dose distribution with sharp beam boundaries. This protocol allows one to ask questions regarding the function of adult-born neurons, but also opens areas to questions in areas of radiobiology, tumor biology, and immunology. These radiological tools will facilitate the translation of discoveries at the bench to the bedside.

The function of adult-born mammalian neurons remains an active area of investigation. Ionizing radiation inhibits the birth of new neurons. Using computer tomography-guided focal irradiation (CFIR), three-dimensional anatomical targeting of specific neural progenitor populations can now be used to assess the functional role of adult neurogenesis.

Interrogatorio funcional de adultos hipotalámica neurogénesis con focal Radiológica Inhibición

The functional characterization of adult-born neurons remains a significant challenge. Approaches to inhibit adult neurogenesis via invasive viral ...

A Novel Light Damage Paradigm for Use in Retinal Regeneration Studies in Adult Zebrafish

Light-induced retinal degeneration (LIRD) is commonly used in both rodents and zebrafish to damage rod and cone photoreceptors. In adult zebrafish, photoreceptor degeneration triggers M&#252;ller glial cells to re-enter the cell cycle and produce transient-amplifying progenitors. These progenitors continue to proliferate as they migrate to the damaged area, where they ultimately give rise to new photoreceptors. Currently, there are two widely-used LIRD paradigms, each of which results in varying degrees of photoreceptor loss and corresponding differences in the regeneration response. As more genetic and pharmacological tools are available to test the role of individual genes of interest during regeneration, there is a need to develop a robust LIRD paradigm. Here we describe a LIRD protocol that results in widespread and consistent loss of both rod and cone photoreceptors in which we have combined the use of two previously established LIRD techniques. Furthermore, this protocol can be extended for use in pigmented animals, which eliminates the need to maintain transgenic lines of interest on the albino background for LIRD studies.

Multiple light damage protocols have been described to damage photoreceptors and consequently induce a retinal regeneration response in adult zebrafish. This protocol describes an improved method that can be used in pigmented animals and that damages the vast majority of rod and cone photoreceptors across the entire retina.

Una novela ligera Paradigm daños para su uso en estudios de regeneración retiniana en adultos de pez cebra

Light-induced retinal degeneration (LIRD) is commonly used in both rodents and zebrafish to damage rod and cone photoreceptors. In adult zebrafish, ...

Microglia as a Surrogate Biosensor to Determine Nanoparticle Neurotoxicity

Nanoparticles found in air pollutants can alter neurotransmitter profiles, increase neuroinflammation, and alter brain function. Therefore, the assay described here will aid in elucidating the role of microglia in neuroinflammation and neurodegenerative diseases. The use of microglia, resident immune cells of the brain, as a surrogate biosensor provides novel insight into how inflammatory responses mediate neuronal insults. Here, we utilize an immortalized murine microglial cell line, designated BV2, and describe a method for nanoparticle exposure using silver nanoparticles (AgNPs) as a standard. We describe how to expose microglia to nanoparticles, how to remove nanoparticles from supernatant, and how to use supernatant from activated microglia to determine toxicity, using hypothalamic cell survival as a measure. Following AgNP exposure, BV2 microglial activation was validated using a tumor necrosis factor alpha (TNF-&#945;) enzyme linked immunosorbent assay (ELISA). The supernatant was filtered to remove the AgNP and to allow cytokines and other secreted factors to remain in the conditioned media. Hypothalamic cells were then exposed to supernatant from AgNP activated microglia and survival of neurons was determined using a resazurin-based fluorescent assay. This technique is useful for utilizing microglia as a surrogate biomarker of neuroinflammation and determining the effect of neuroinflammation on other cell types.

Microglia (immune cells of the brain), are used as a surrogate biosensor to determine how nanoparticles influence neurotoxicity. We describe a series of experiments designed to assay microglial response to nanoparticles and exposure of hypothalamic neurons to supernatant from activated microglia to determine neurotoxicity.

Microglia como una biosensor sustituto para determinar la neurotoxicidad de nanopartículas

Nanoparticles found in air pollutants can alter neurotransmitter profiles, increase neuroinflammation, and alter brain function. Therefore, the assay ...

The Use of Trace Eyeblink Classical Conditioning to Assess Hippocampal Dysfunction in a Rat Model of Fetal Alcohol Spectrum Disorders

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain undergoes rapid organizational change and is similar to accelerated brain changes that occur during the third trimester in humans. This model of fetal alcohol spectrum disorders (FASDs) produces severe brain damage, mimicking the amount and pattern of binge-drinking that occurs in some pregnant alcoholic mothers. We describe the use of trace eyeblink classical conditioning (ECC), a higher-order variant of associative learning, to assess long-term hippocampal dysfunction that is typically seen in alcohol-exposed adult offspring. At 90 days of age, rodents were surgically prepared with recording and stimulating electrodes, which measured electromyographic (EMG) blink activity from the left eyelid muscle and delivered mild shock posterior to the left eye, respectively. After a 5 day recovery period, they underwent 6 sessions of trace ECC to determine associative learning differences between alcohol-exposed and control rats. Trace ECC is one of many possible ECC procedures that can be easily modified using the same equipment and software, so that different neural systems can be assessed. ECC procedures in general, can be used as diagnostic tools for detecting neural pathology in different brain systems and different conditions that insult the brain.

Trace eyeblink classical conditioning (ECC) was used to assess hippocampal-dependent associative learning in adult rats that were administered a high concentration (11.9% v/v) of alcohol during early neonatal brain development. In general, ECC procedures are sound diagnostic tools for detecting brain dysfunction across many psychological and biomedical settings.

El uso de condicionamiento de clásico de Eyeblink rastro para evaluar disfunción hipocampal en un modelo de rata de trastornos del espectro alcohólico Fetal

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain ...

Live-cell Measurement of Odorant Receptor Activation Using a Real-time cAMP Assay

The enormous sizes of the mammalian odorant receptor (OR) families present difficulties to find their cognate ligands among numerous volatile chemicals. To efficiently and accurately deorphanize ORs, we combine the use of a heterologous cell line to express mammalian ORs and a genetically modified biosensor plasmid to measure cAMP production downstream of OR activation in real time. This assay can be used to screen odorants against ORs and vice versa. Positive odorant-receptor interactions from the screens can be subsequently confirmed by testing against various odor concentrations, generating concentration-response curves. Here we used this method to perform a high-throughput screening of an odorous compound against a human OR library expressed in Hana3A cells and confirmed that the positively-responding receptor is the cognate receptor for the compound of interest. We found this high-throughput detection method to be efficient and reliable in assessing OR activation and our data provide an example of its potential use in OR functional studies.

Characterizing the function of odorant receptors serves an indispensable part in the deorphanization process. We describe a method to measure the activation of odorant receptors in real time using a cAMP assay.

Células vivas medición de olor del Receptor activación utilizando un ensayo de campo en tiempo real

The enormous sizes of the mammalian odorant receptor (OR) families present difficulties to find their cognate ligands among numerous volatile chemicals. ...

A Protocol for Measuring Cue Reactivity in a Rat Model of Cocaine Use Disorder

Cocaine use disorder (CUD) follows a trajectory of repetitive self-administration during which previously neutral stimuli gain incentive value. Cue reactivity, the sensitivity to cues previously linked with the drug-taking experience, plays a prominent role in human craving during abstinence. Cue reactivity can be assessed as the attentional orientation toward drug-associated cues that is measurable as appetitive approach behavior in both preclinical and human studies. Herein describes an assessment of cue reactivity in rats trained to self-administer cocaine. Cocaine self-administration is paired with the presentation of discrete cues that act as conditioned reinforcers (i.e., house light, stimulus light, infusion pump sounds). Following a period of abstinence, lever presses in the cocaine self-administration context accompanied by the discrete cues previously paired with cocaine infusion are measured as cue reactivity. This model is useful to explore neurobiological mechanisms underlying cue reactivity processes as well as to assess pharmacotherapies to suppress cue reactivity and therefore, modify relapse vulnerability. Advantages of the model include its translational relevance, and its face and predictive validities.&#160;The primary limitation of the model is that the cue reactivity task can only be performed infrequently and must only be used in short duration (e.g., 1 hour), otherwise rats will begin to extinguish the pairing of the discrete cues with the cocaine stimulus. The model is extendable to any positively reinforcing stimulus paired with discrete cues; though particularly applicable to drugs of abuse, this model may hold future applications in fields such as obesity, where palatable food rewards can act as positively reinforcing stimuli.

Cue reactivity is conceptualized as sensitivity to cues linked with drug-taking experiences that contribute to craving and relapse in abstinent humans. Cue reactivity is modeled in rats by measuring attentional orientation toward drug-associated cues that results in appetitive approach behavior in a cue reactivity test following self-administration and forced abstinence.

Un protocolo para medir la reactividad de Cue en un modelo de rata de trastorno de uso de cocaína

Cocaine use disorder (CUD) follows a trajectory of repetitive self-administration during which previously neutral stimuli gain incentive value. Cue ...

A Neurosphere Assay to Evaluate Endogenous Neural Stem Cell Activation in a Mouse Model of Minimal Spinal Cord Injury

Neural stem cells (NSCs) in the adult mammalian spinal cord are a relatively mitotically quiescent population of periventricular cells that can be studied in vitro using the neurosphere assay. This colony-forming assay is a powerful tool to study the response of NSCs to exogenous factors in a dish; however, this can also be used to study the effect of in vivo manipulations with the proper understanding of the strengths and limitations of the assay. One manipulation of the clinical interest is the effect of injury on endogenous NSC activation. Current models of spinal cord injury provide a challenge to study this as the severity of common contusion, compression, and transection models cause the destruction of the NSC niche at the site of the injury where the stem cells reside. Here, we describe a minimal injury model that causes localized damage at the superficial dorsolateral surface of the lower thoracic level (T7/8) of the adult mouse spinal cord. This injury model spares the central canal at the level of injury and permits analysis of the NSCs that reside at the level of the lesion at various time points following injury. Here, we show how the neurosphere assay can be utilized to study the activation of the two distinct, lineally-related, populations of NSCs that reside in the spinal cord periventricular region - primitive and definitive NSCs (pNSCs and dNSCs, respectively). We demonstrate how to isolate and culture these NSCs from the periventricular region at the level of injury and the white matter injury site. Our post-surgical spinal cord dissections show increased numbers of pNSC and dNSC-derived neurospheres from the periventricular region of injured cords compared to controls, speaking to their activation via injury. Furthermore, following injury, dNSC-derived neurospheres can be isolated from the injury site &#8212; demonstrating the ability of NSCs to migrate from their periventricular niche to sites of injury.

Here, we demonstrate the performance of a minimal spinal cord injury model in an adult mouse that spares the central canal niche housing endogenous neural stem cells (NSCs). We show how the neurosphere assay can be used to quantify activation and migration of definitive and primitive NSCs following injury.

Un ensayo desarrolló para evaluar la activación de células neuronales endógenos en un modelo murino de lesión medular mínima

Neural stem cells (NSCs) in the adult mammalian spinal cord are a relatively mitotically quiescent population of periventricular cells that can be studied ...