Edith Suss-Toby is thanked for her assistance in the time-lapse microscopy and image analysis. Nofar Rada is thanked for the artistic work.

De cuatro a seis semanas de edad C57BL / CJ ratones (Harlan, Jerusalén, Israel) se utilizaron en el experimento de acuerdo con la Asociación para la Evaluación y Acreditación de Laboratorio Animal Care especificaciones. Todos los procedimientos experimentales se realizaron de acuerdo con el Cuidado de Animales institucional y el empleo Comisión y el Departamento de Agricultura regulaciones. 1. La recolección de la Médula Espinal <ol><li> La eutanasia del ratón usando una cámara de CO 2. Evitar dislocación cervical, ya que podría causar daños a las raíces ganglionares debido a fuerzas de cizallamiento. De aquí en adelante, realizar todos los pasos en condiciones estériles. </li><li> Remojar los animales abajo con etanol al 70%. Este paso es importante para la esterilización y para prevenir el pelo de arrastre a través de los órganos internos. </li><li> Deje el ratón para secar a partir de etanol. Evitar cualquier contacto de etanol con los órganos internos debido a la neurotoxicidad. </li><li> Después de secar el ratón, la posición que el uso de clavos en posición prona(boca abajo). Coloque los pernos en cada extremidad posterior y la extremidad anterior. </li><li> El uso de pinzas, hacer una incisión de línea media que se extiende desde el cuello craneocaudal posterior a la columna vertebral lumbar. A continuación, levante colgajos dérmicos bilateralmente con unas pinzas y exponer el tejido subcutáneo. </li><li> Se palpa el cráneo del ratón hasta llegar a la unión craneocervical. Con unas tijeras perpendiculares al animal, cortar a través de los músculos cervicales y la columna vertebral en la unión craneocervical (el 7º vértebra cervical), el uso de fórceps pesados. Diseccionar la columna vertebral caudal e Impuestos Especiales de la columna vertebral con unas pinzas pesados ​​hasta alcanzar el nivel lumbar (posterior nivel de las extremidades). </li><li> Realizar una transección caudal completa a nivel de la columna lumbar (la 5ª vértebra lumbar) con unas pinzas pesadas. </li><li> Mover el ratón sobre (boca arriba). No hay necesidad de cubrir la incisión desde el DRG se extrae de los niveles vertebrales inferiores y no el cuello uterino. </li><li> Cortar hacia abajo en la línea media del cuello para el abdomen, retraer elpiel lateralmente y luego se corta para abrir el peritoneo. </li><li> Retire los órganos internos dentro del peritoneo (hígado, bazo, páncreas, estómago, intestino) y en bloque. A continuación, retire los órganos retroperitoneales (es decir, los riñones y el páncreas). Craneal, abrir la pared torácica. </li><li> El uso de pinzas y una cuchilla quirúrgica, corte de las costillas, dejando 5 mm de costillas bilateralmente lejos de la columna vertebral. En este punto, la columna vertebral está separado del resto del cuerpo. </li><li> Lavar la columna vertebral de la sangre con (4 ° C) fresco salina tamponada con fosfato fría (PBS) dos veces. </li></ol> 2. Aislamiento de la raíz dorsal ganglios (DRG) <ol><li> Utilice un estereomicroscopio con un aumento de 4X. </li><li> Coloque la columna vertebral en una plataforma no absorbente no adherente (Telfa / nylon). Coloque la cara columna hacia arriba en la misma orientación cráneo-caudal. </li><li> Retire cualquier músculos espinales y tejido conectivo. Utilice las costillas como un punto de referencia para los nervios, ya quesalir de la columna vertebral. Costillas también protegen los nervios de los instrumentos quirúrgicos. El DRG se encuentran en la torácica, lumbar, cervical y los sitios de las vértebras. </li><li> A continuación, utilizando las tijeras cortan el cuerpo vertebral en la línea media y exponen a la médula espinal y las raíces DRG por la retracción lateral suave del cuerpo vertebral. </li><li> En el espacio intercostal, siga el nervio periférico medial a lo largo del lateral de la costilla a la DRG. </li><li> Identificar los GRD. Parece que la yema de &#39;huevo frito&#39; tumbado sobre el nervio. </li><li> Cortar el distal del nervio intercostal a la DRG dejando 2-3 mm de nervio distal a los ganglios, para ser utilizado para la retracción. Esta &quot;cola&quot; se eliminará después de la cosecha. </li><li> Agarre el nervio uso de fórceps. Pellizcar el cuerpo DRG sí causará daño celular neuronal y debe ser evitado. </li><li> El enfoque de la DRG proximal a lo largo de su apego a la médula espinal, a través de su anterior y raíces posteriores. </li><li> Aplicar la retracción suave para la DRG tirando de la EFrentes nervio (muñón nervio intercostal) lateralmente, y se corta la parte anterior y posterior raíces cerca de la DRG. </li><li> Mantenga el DRG en una placa de 35 mm de Petri llena de fresco, hielo DMEM frío suplementado con 10% de suero de ternera fetal (FCS), 1% de penicilina-estreptomicina, aminoácidos no esenciales al 1%, y 1% de piruvato de sodio. </li></ol> 3. Implantación en la placa de Petri <ol><li> Realice los siguientes pasos en el hielo utilizando puntas de pipeta pre-enfriado para evitar la solidificación del ECM. </li><li> Bajo un microscopio estereoscópico con una ampliación 2-4X, colocar una placa de Petri de vidrio inferior 35 mm en una cuadrícula de papel. IMPORTANTE: El trabajo sobre hielo con el fin de mantener el ECM en estado líquido. Crear un punto de diámetro de 1 mm (aproximadamente 20 l) de crecimiento factor de ECM-agotado en el centro de la rejilla. </li><li> Bajo visualización directa, coloque el DRG en el centro de la mancha ECM cerca de la parte inferior del plato. NOTA: Las células cancerosas se utilizan en este protocolo son murinos células del cáncer pancreático (KPC) established a partir de muestras tumorales recién aisladas de ratones KPC como se describe en otro lugar 19. </li><li> células de cáncer de cosecha 40.000 a partir de cultivos confluentes, se lavan una vez con PBS, y volver a suspender en 40 l ECM en hielo. </li><li> Bajo visualización directa de la rejilla utilizando un microscopio estereoscópico micras medida 500 en cada dirección desde el DRG. En este punto sembrar lentamente 10.000 células / l 10 ECM. Utilice una punta de preenfriado 2-10 l para inyectar las células cercanas al fondo del plato, evitando su propagación en la matriz o desprendimiento de la matriz. (Figura 1). </li><li> Deja el plato dentro de una campana de flujo laminar durante 10-15 minutos para solidificar. Evitar el secado de ECM. </li><li> Añadir lentamente DMEM, preparado como se ha mencionado previamente, contra la pared lateral de la placa. Añadir suficiente medio para cubrir la ECM (alrededor de 2 ml). </li><li> Poner el plato de nuevo a la incubadora a 37 ° C. </li><li> Sustituir el medio con medio fresco al día siguiente. </li><li> Reemplazar el medio cada 2 días. &lt;/ Li&gt; </ol> 4. Adquisición de Datos, por lapsos de tiempo videomicroscopia <ol><li> En el día 7 después de la implantación, tomar el plato para microscopía lapso de tiempo. Tenga en cuenta que ciertas células pueden requerir la adquisición antes debido a una migración más rápida. </li><li> Durante la formación de imágenes en vivo, poner la caja en un entorno cerrado a una temperatura media de 37 ± 0,1 ° C y 5% de CO2. </li><li> Registrar las células mediante el uso de una carga acoplada cámara del dispositivo colocado en el microscopio. </li><li> Tomar imágenes digitalizadas cada 10 minutos durante un máximo de 72 horas para seguir la locomoción celular de tres a seis células diferentes. </li><li> Reemplazar medio después de 24 horas. </li><li> Realizar la adquisición de datos y análisis sencilla utilizando el software microscopio. </li></ol> Análisis 5. Datos <ol><li> Para aplicaciones más avanzadas (es decir, el seguimiento de celda en 2D o 3D) utilizar un software especializado (como Imaris 4D). Nota: Este software determina las coordenadas xy de una célula acualquier momento dado, junto con la distancia desde el origen, y la velocidad de movimiento. </li><li> Calcula la velocidad media mediante la medición de la distancia de desplazamiento entre las posiciones posteriores en intervalos de 10 min. Calcular el índice de migración de Forward (FMI) que es la distancia de la parte frontal de la célula de neuritas y describe el movimiento unidireccional de las células hacia el DRG. </li></ol>

<ol>
	<li>Carter, R. L., Foster, C. S., Dinsdale, E. A., Pittam, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perineural+spread+by+squamous+carcinomas+of+the+head+and+neck,+a+morphological+study+using+antiaxonal+and+antimyelin+monoclonal+antibodies.">Perineural spread by squamous carcinomas of the head and neck, a morphological study using antiaxonal and antimyelin monoclonal antibodies.</a> J Clin Pathol. 36, 269-275 (1983).</li><li>Beard, C. 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=Perineural+invasion+is+associated+with+increased+relapse+after+external+beam+radiotherapy+for+men+with+low-risk+prostate+cancer+and+may+be+a+marker+for+occult,+high-grade+cancer.">Perineural invasion is associated with increased relapse after external beam radiotherapy for men with low-risk prostate cancer and may be a marker for occult, high-grade cancer.</a> Int J Radiat Oncol Biol Phys. 58, 19-24 (2004).</li><li>Maru, N., Ohori, M., Kattan, M. W., Scardino, P. T., Wheeler, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prognostic+significance+of+the+diameter+of+perineural+invasion+in+radical+prostatectomy+specimens.">Prognostic significance of the diameter of perineural invasion in radical prostatectomy specimens.</a> Hum Pathol. 32, 828-833 (2001).</li><li>Ceyhan, G. O., 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=Pancreatic+neuropathy+and+neuropathic+pain--a+comprehensive+pathomorphological+study+of+546+cases.">Pancreatic neuropathy and neuropathic pain--a comprehensive pathomorphological study of 546 cases.</a> Gastroenterology. 136, 177-186 (2009).</li><li>Ceyhan, G. O., 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=The+neurotrophic+factor+artemin+promotes+pancreatic+cancer+invasion.">The neurotrophic factor artemin promotes pancreatic cancer invasion.</a> Ann Surg. 244, 274-281 (2006).</li><li>Takahashi, T., 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=Perineural+invasion+by+ductal+adenocarcinoma+of+the+pancreas.">Perineural invasion by ductal adenocarcinoma of the pancreas.</a> J Surg Oncol. 65, 164-170 (1997).</li><li>Zhu, Z., 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=Nerve+growth+factor+expression+correlates+with+perineural+invasion+and+pain+in+human+pancreatic+cancer.">Nerve growth factor expression correlates with perineural invasion and pain in human pancreatic cancer.</a> J Clin Oncol. 17, 2419-2428 (1999).</li><li>Hirai, I., 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=Perineural+invasion+in+pancreatic+cancer.">Perineural invasion in pancreatic cancer.</a> Pancreas. 24, 15-25 (2005).</li><li>Mitchem, J. B., 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=Targeting+tumor-infiltrating+macrophages+decreases+tumor-initiating+cells,+relieves+immunosuppression,+and+improves+chemotherapeutic+responses.">Targeting tumor-infiltrating macrophages decreases tumor-initiating cells, relieves immunosuppression, and improves chemotherapeutic responses.</a> Cancer Res. 73, 1128-1141 (2013).</li><li>Kelly, K., 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=Attenuated+multimutated+herpes+simplex+virus-1+effectively+treats+prostate+carcinomas+with+neural+invasion+while+preserving+nerve+function.">Attenuated multimutated herpes simplex virus-1 effectively treats prostate carcinomas with neural invasion while preserving nerve function.</a> FASEB J. 22, 1839-1848 (2008).</li><li>Dai, H., 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=Enhanced+survival+in+perineural+invasion+of+pancreatic+cancer,+an+in+vitro+approach.">Enhanced survival in perineural invasion of pancreatic cancer, an in vitro approach.</a> Hum Pathol. 38, 299-307 (2007).</li><li>Ayala, G. E., 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=Cancer-related+axonogenesis+and+neurogenesis+in+prostate+cancer.">Cancer-related axonogenesis and neurogenesis in prostate cancer.</a> Clin Cancer Res. 14, 7593-7603 (2008).</li><li>Ayala, G. E., 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=Stromal+antiapoptotic+paracrine+loop+in+perineural+invasion+of+prostatic+carcinoma.">Stromal antiapoptotic paracrine loop in perineural invasion of prostatic carcinoma.</a> Cancer Res. 66, 5159-5164 (2006).</li><li>Ceyhan, G. O., 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=Neural+invasion+in+pancreatic+cancer,+a+mutual+tropism+between+neurons+and+cancer+cells.">Neural invasion in pancreatic cancer, a mutual tropism between neurons and cancer cells.</a> Biochem Biophys Res Commun. 374, 442-447 (2008).</li><li>Bapat, A. A., Hostetter, G., Von Hoff, D. D., Han, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perineural+invasion+and+associated+pain+in+pancreatic+cancer.">Perineural invasion and associated pain in pancreatic cancer.</a> Nat Rev Cancer. 11, 695-707 (2011).</li><li>Ketterer, K., 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=Reverse+transcription-PCR+analysis+of+laser-captured+cells+points+to+potential+paracrine+and+autocrine+actions+of+neurotrophins+in+pancreatic+cancer.">Reverse transcription-PCR analysis of laser-captured cells points to potential paracrine and autocrine actions of neurotrophins in pancreatic cancer.</a> Clin Cancer Res. 9, 5127-5136 (2003).</li><li>Gil, Z., 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=Nerve-sparing+therapy+with+oncolytic+herpes+virus+for+cancers+with+neural+invasion.">Nerve-sparing therapy with oncolytic herpes virus for cancers with neural invasion.</a> Clin Cancer Res. 13, 6479-6485 (2007).</li><li>Gil, Z., 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=Paracrine+regulation+of+pancreatic+cancer+cell+invasion+by+peripheral+nerves.">Paracrine regulation of pancreatic cancer cell invasion by peripheral nerves.</a> J Natl Cancer Inst. 102, 107-118 (2010).</li><li>Weizman, N., 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=Macrophages+mediate+gemcitabine+resistance+of+pancreatic+adenocarcinoma+by+upregulating+cytidinedeaminase.">Macrophages mediate gemcitabine resistance of pancreatic adenocarcinoma by upregulating cytidinedeaminase.</a> Oncogene. 33, 3812-3819 (2014).</li></ol>

This research was supported by the ICRF Barbara S. Goodman endowed research career development award (2011-601-BGPC) and a grant from the US-Israel Binational Science Foundation. No other financial disclosures from the authors are to be made.

En este artículo se presenta un modelo in vitro que recapitula el microambiente canceroso en el nicho de los nervios, el modelo DRG. El vídeo muestra todos los pasos a partir de reconocer puntos de referencia anatómicos tales como el DRG en el ratón, su extracción y, finalmente, su cultivo en ECM. También se presenta Co-cultivo de la DRG junto con células cancerosas. No existen otros modelos para la investigación invasión perineural in vitro descritos en la literatura que hacen de este modelo esencial para estudiar el nicho perineural microentorno in vitro. El protocolo presentado en el vídeo tiene dos pasos críticos. En primer lugar, se debe tener cuidado al sujetar el nervio adyacente al cuerpo DRG (paso 2.7 en el protocolo). Agarrar o pellizcar el DRG podría causar daños mecánicos o isquémica irreversible en el DRG prevenir que vuelva a crecer y brotar cuando se siembran en la placa de Petri. El segundo paso crítico es la implantaciónde las células cancerosas en la placa de Petri en el interior del ECM. Es importante sembrar las células lentamente y con precaución, para evitar la flotación de las células y su propagación a través de la placa de Petri. El objetivo de la implantación es localizar las células en un solo lugar, para facilitar el seguimiento de su movimiento (distancia y dirección). Una vez establecido el modelo microambiente puede ser modificado de acuerdo con la hipótesis de la prueba. Por ejemplo, la adición de un tercer cultivo de células de la placa de Petri (por ejemplo, células no cancerosas) permite al investigador para comparar las capacidades de migración neurotrópico de diferentes células. Además, el investigador puede aplicar diferentes condiciones (es decir, temperatura, humedad, factores solubles, etc.) y examinar su efecto en la capacidad de las células invasión. El modelo de DRG permite al investigador para estudiar la interacción entre las células cancerosas y los nervios. También se utiliza para los experimentos de lapso de tiempo en el que morfológicalos cambios en el nicho perineural se demuestran de manera temporal. Además, las células neuroinvasiva pueden ser sometidos a diversos tratamientos y su efecto en la interacción con las neuritas de los ganglios se pueden evaluar. No todas las línea celular es adecuada para el modelo de DRG. Deben ser células cancerosas con la capacidad intrínseca para invadir a través de los nervios y de degradar ECM. Además, tenga en cuenta que cada tipo de célula cancerosa tiene diferentes características de invasión por lo que el tiempo estimado de las células para hacer contacto con las neuritas DRG varía. Por ejemplo, las células KPC que utilizan necesidad cerca de 7 días para invadir a través de la ECM hacia el DRG mientras que las células MiaPaca (células de adenocarcinoma de páncreas humano) sólo necesita 72-96 horas para hacer que el contacto. Una limitación de este modelo incluye la posible incompatibilidad con las células humanas. Debido a su uso de DRG ratón, la interacción proteína-proteína de ratón-humano no siempre puede ser demostrada cuando huse utilizan células de cáncer de hombre. Siempre que piense utilizar este modelo con las dos especies diferentes, la homología de la proteína examinada debe ser probado y si hay una alta homología del ensayo se supone que es adecuado para la evaluación de las interacciones proteína-proteína. Además, debe tenerse en cuenta que el ensayo DRG como se propone en este protocolo representa el microambiente neuronal incluyendo otras células, no sólo las neuronas (es decir, células de Schwann, fibroblastos, macrófagos, etc.). Por lo tanto, las poblaciones de células específicas no pueden ser probados específicamente.

Los tumores sólidos difunden en tres formas principales: la invasión directa, diseminación linfática, y la propagación hematógena. Sin embargo, hay un cuarto medio de propagación del cáncer que con frecuencia se tiene en cuenta, la difusión a lo largo de los nervios. Invasión neural cancerosas (CNI) es una ruta bien conocida de la propagación del cáncer, especialmente en los cánceres de cabeza y cuello, próstata 1 2, 3 y el páncreas. 4-8 CNI se produce en más de 80% de individuos con adenocarcinoma de páncreas, lo que lleva a un tumor retroperitoneal propagarse a través de los nervios ganglio celiaca. Estas células cancerosas neurotrópico tienen una capacidad única para migrar unidireccionalmente a lo largo de los nervios hacia el sistema nervioso central (SNC). 9 Este hallazgo sugiere que el microambiente perineural puede ser explotado por las células cancerosas, proporcionando factores que apoyan el crecimiento maligno. Uno de los pocos modelos in vitro para la investigación CNI es el ganglio de la raíz dorsal (DRG) / modelo de célula de cáncer. este model se utiliza con frecuencia para estudiar la interacción paracrina entre las células del estroma y el cáncer neural. 10-18 En este líneas celulares de cáncer humano modelo, el ratón o se cultivan en matriz extracelular (ECM) adyacente a preparaciones de DRG cultivadas recién disociados. Este artículo de vídeo muestra la aplicación de vitro CNI en en el adenocarcinoma ductal pancreático.

<table border="0" cellpadding="0" cellspacing="0" width="710">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:417px;">Equipments:</td>
			<td style="width:121px;"></td>
			<td style="width:108px;"></td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Operating microscope</td>
			<td>Leica</td>
			<td>M205</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tiime Lapse System</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Forceps</td>
			<td>Sigma-Aldrich</td>
			<td>F4142&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Surgical blade</td>
			<td>Sigma-Aldrich</td>
			<td>Z309036</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Scissors</td>
			<td>Sigma-Aldrich</td>
			<td>S3271</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">35mm petri dishes, glass bottom</td>
			<td>de groot</td>
			<td>60-627860</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Name</td>
			<td>Company&#160;</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Materials:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">70% ethanol</td>
			<td>sigma</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cold PBS</td>
			<td>Biological industries</td>
			<td>02-023-1A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM</td>
			<td>Biological industries</td>
			<td>01-055-1A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FCS</td>
			<td>Rhenium</td>
			<td align="right">10108165</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin and streptomycin</td>
			<td>Biological industries</td>
			<td>01-031-1B</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium Pyruvate</td>
			<td>Biological industries</td>
			<td>03-042-1B</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L-Glutamine</td>
			<td>Biological industries</td>
			<td>03-020-1B</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Growth factor depleted matrigel</td>
			<td>Trevigen</td>
			<td>3433-005-01</td>
			<td></td>
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in vitro modeling of cancerous neural invasion: the dorsal root ganglion model

One way that solid tumors disseminate is through neural invasion. This route is well-known in cancers of the head and neck, prostate, and pancreas. These neurotropic cancer cells have a unique ability to migrate unidirectionally along nerves towards the central nervous system (CNS). The dorsal root ganglia (DRG)/cancer cell model is a three dimensional (3D) in vitro model frequently used for studying the interaction between neural stroma and cancer cells. In this model, mouse or human cancer cell lines are grown in ECM adjacent to preparations of freshly dissociated cultured DRG. In this article, the DRG isolation protocol from mice, and implantation in petri dishes for co-culturing with pancreatic cancer cells are demonstrated. Five days after implantation, the cancer cells made contact with the DRG neurites. Later, these cells formed bridgeheads to facilitate more extensive polarized, neurotropic migration of cancer cells.

<html> Utilizando imágenes de microscopía de vídeo, el DRG se puede ver brotar neuritas 5-7 días después de la implantación, mientras que las células de cáncer de migración lejos de sus colonias hacia el DRG. Por el 7º día después de la implantación, las células cancerosas entran en contacto con las neuritas (Figura 2). El índice de migración hacia delante de las células de cáncer pancreático utilizados en el protocolo es 3-4 veces mayor que la de otras líneas celulares (QLL2, B16F) (Figura 3a). Figura 3B presenta un representante X y la coordenada Y gráfica, que representa la trayectoria de migración de una célula de cáncer KPC en contacto con el nervio; videomicroscopy análisis de lapso de tiempo mostró diferencias en el movimiento hacia adelante, pero no la velocidad entre las células y las células KPC no invasora (Figura 3c-d). gura 1 &quot;src =&quot; / files / ftp_upload / 52990 / 52990fig1.jpg &quot;/&gt; Figura 1:. Ilustración esquemática de los pasos del protocolo <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52990/52990fig1large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a> <img alt="Figura 2" src="/files/ftp_upload/52990/52990fig2.jpg" /> Figura 2:. Cancer Cell Invasion A lo largo de las neuronas del DRG (a) DRG (arriba) y las células de cáncer (abajo) en el día 0 después de la siembra (ampliación 5X). (B) DRG y el cáncer de células en el día 7 después de la siembra (5 aumentos). Células (c) cancerosas migran a lo largo de la neurona DRG (flechas). Todas las barras representan 50 micras. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52990/52990fig2large.jpg" target="_blank">Por favor, haga clic aquí para ver una versión más grande de esta figura.</a> Figura 3:. Ganglio de raíz dorsal (GRD) neuronas inducen CNI (a) índice de nervio invasión de las células cancerosas, las células MiaPaca2 KPC, QLL2 y células NIH3T3. (B) Una coordenada gráfico que representa la ruta de migración de una célula de cáncer KPC en contacto con el nervio (rojo) y de células QLL2 (púrpura). Se muestran las coordenadas X e Y. (N = 12 a 20 en cada grupo). (C) Análisis de la distancia desde el origen de la migración de células de cáncer de QLL2 con el contacto axonal y células (d) de cáncer KPC (n = 20). La dirección de la migración era constantemente hacia el ganglio del nervio. Los valores de p en (a) se calcularon mediante la prueba de la t de Student de dos caras. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52990/52990fig3large.jpg" target="_blank">Por favor, haga clic aquí para ver una versión más grande de esta figura.</a>

Watch this Scientific Journal Video about In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model at JoVE.com

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

This video article shows the use of the dorsal root ganglia (DRG)/cancer cell model in pancreatic ductal adenocarcinoma.

En Vitro Modelado de canceroso Neural Invasion: Modelo del ganglio de la raíz dorsal

One way that solid tumors disseminate is through neural invasion. This route is well-known in cancers of the head and neck, prostate, and pancreas. These ...

in-vitro-modeling-cancerous-neural-invasion-dorsal-root-ganglion

Nanyang Technological University

Research

JoVE Journal

Neuroscience

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

9.0K vistas.  Rambam Medical Healthcare Campus. This video article shows the use of the dorsal root ganglia (DRG)/cancer cell model in pancreatic ductal adenocarcinoma.

Video: In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

Modeling Neural Immune Signaling of Episodic and Chronic Migraine Using Spreading Depression In Vitro

Migraine and its transformation to chronic migraine are healthcare burdens in need of improved treatment options. We seek to define how neural immune signaling modulates the susceptibility to migraine, modeled in vitro using spreading depression (SD), as a means to develop novel therapeutic targets for episodic and chronic migraine. SD is the likely cause of migraine aura and migraine pain. It is a paroxysmal loss of neuronal function triggered by initially increased neuronal activity, which slowly propagates within susceptible brain regions. Normal brain function is exquisitely sensitive to, and relies on, coincident low-level immune signaling. Thus, neural immune signaling likely affects electrical activity of SD, and therefore migraine. Pain perception studies of SD in whole animals are fraught with difficulties, but whole animals are well suited to examine systems biology aspects of migraine since SD activates trigeminal nociceptive pathways. However, whole animal studies alone cannot be used to decipher the cellular and neural circuit mechanisms of SD. Instead, in vitro preparations where environmental conditions can be controlled are necessary. Here, it is important to recognize limitations of acute slices and distinct advantages of hippocampal slice cultures. Acute brain slices cannot reveal subtle changes in immune signaling since preparing the slices alone triggers: pro-inflammatory changes that last days, epileptiform behavior due to high levels of oxygen tension needed to vitalize the slices, and irreversible cell injury at anoxic slice centers. 
In contrast, we examine immune signaling in mature hippocampal slice cultures since the cultures closely parallel their in vivo counterpart with mature trisynaptic function; show quiescent astrocytes, microglia, and cytokine levels; and SD is easily induced in an unanesthetized preparation. Furthermore, the slices are long-lived and SD can be induced on consecutive days without injury, making this preparation the sole means to-date capable of modeling the neuroimmune consequences of chronic SD, and thus perhaps chronic migraine. We use electrophysiological techniques and non-invasive imaging to measure neuronal cell and circuit functions coincident with SD. Neural immune gene expression variables are measured with qPCR screening, qPCR arrays, and, importantly, use of cDNA preamplification for detection of ultra-low level targets such as interferon-gamma using whole, regional, or specific cell enhanced (via laser dissection microscopy) sampling. Cytokine cascade signaling is further assessed with multiplexed phosphoprotein related targets with gene expression and phosphoprotein changes confirmed via cell-specific immunostaining. Pharmacological and siRNA strategies are used to mimic and modulate SD immune signaling.

Modeling Neural Immune Signaling of Episodic and Chronic Migraine Using Spreading Depression In Vitro

Migraine and its transformation to chronic migraine are immense healthcare burdens in need of improved treatment options. We seek to define how neural immune signaling modulates the susceptibility to migraine, modeled in vitro using spreading depression in hippocampal slice cultures, as a means to develop novel therapeutic targets.

Modelado neuronal de señalización inmune de la migraña episódica y crónica mediante la depresión propagada In Vitro</em

Migraine and its transformation to chronic migraine are healthcare burdens in need of improved treatment options.  We seek to define how neural immune ...

Investigación

Neurociencias

Live Imaging of Dorsal Root Axons after Rhizotomy

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. Regeneration of dorsal root (DR) axons into spinal cord is prevented at the dorsal root entry zone (DREZ), the interface between the CNS and PNS. Our understanding of the molecular and cellular events that prevent regeneration at DREZ is incomplete, in part because complex changes associated with nerve injury have been deduced from postmortem analyses. Dynamic cellular processes, such as axon regeneration, are best studied with techniques that capture real-time events with multiple observations of each living animal. Our ability to monitor neurons serially in vivo has increased dramatically owing to revolutionary innovations in optics and mouse transgenics. Several lines of thy1-GFP transgenic mice, in which subsets of neurons are genetically labeled in distinct fluorescent colors, permit individual neurons to be imaged in vivo1. These mice have been used extensively for in vivo imaging of muscle2-4 and brain5-7, and have provided novel insights into physiological mechanisms that static analyses could not have resolved. Imaging studies of neurons in living spinal cord have only recently begun. Lichtman and his colleagues first demonstrated their feasibility by tracking injured dorsal column (DC) axons with wide-field microscopy8,9. Multi-photon in vivo imaging of deeply positioned DC axons, microglia and blood vessels has also been accomplished10. Over the last few years, we have pioneered in applying in vivo imaging to monitor regeneration of DR axons using wide-field microscopy and H line of thy1-YFP mice. These studies have led us to a novel hypothesis about why DR axons are prevented from regenerating within the spinal cord11.
In H line of thy1-YFP mice, distinct YFP+ axons are superficially positioned, which allows several axons to be monitored simultaneously. We have learned that DR axons arriving at DREZ are better imaged in lumbar than in cervical spinal cord. In the present report we describe several strategies that we have found useful to assure successful long-term and repeated imaging of regenerating DR axons. These include methods that eliminate repeated intubation and respiratory interruption, minimize surgery-associated stress and scar formation, and acquire stable images at high resolution without phototoxicity.

An in vivo imaging protocol to monitor primary sensory axons following dorsal root crush is described. The procedures utilize wide-field fluorescence microscopy and thy1-YFP transgenic mice, and permit repeated imaging of axon regeneration over 4 cm in the PNS and axon interactions with the interface of the CNS.

Imágenes en vivo de los axones de la raíz dorsal después de rizotomía

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. ...

Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability to support axon growth and a hostile environment in the mature CNS1,2. In contrast, mature neurons in the peripheral nervous system (PNS) regenerate readily after injuries3. Adult dorsal root ganglion (DRG) neurons are well known to regenerate robustly after peripheral nerve injuries. Each DRG neuron grows one axon from the cell soma, which branches into two axonal branches: a peripheral branch innervating peripheral targets and a central branch extending into the spinal cord. Injury of the DRG peripheral axons results in substantial axon regeneration, whereas central axons in the spinal cord regenerate poorly after the injury. However, if the peripheral axonal injury occurs prior to the spinal cord injury (a process called the conditioning lesion), regeneration of central axons is greatly improved4. Moreover, the central axons of DRG neurons share the same hostile environment as descending corticospinal axons in the spinal cord. Together, it is hypothesized that the molecular mechanisms controlling axon regeneration of adult DRG neurons can be harnessed to enhance CNS axon regeneration. As a result, adult DRG neurons are now widely used as a model system to study regenerative axon growth5-7.
Here we describe a method of adult DRG neuron culture that can be used for genetic study of axon regeneration in vitro. In this model adult DRG neurons are genetically manipulated via electroporation-mediated gene transfection6,8. By transfecting neurons with DNA plasmid or si/shRNA, this approach enables both gain- and loss-of-function experiments to investigate the role of any gene-of-interest in axon growth from adult DRG neurons. When neurons are transfected with si/shRNA, the targeted endogenous protein is usually depleted after 3-4 days in culture, during which time robust axon growth has already occurred, making the loss-of-function studies less effective. To solve this problem, the method described here includes a re-suspension and re-plating step after transfection, which allows axons to re-grow from neurons in the absence of the targeted protein. Finally, we provide an example of using this in vitro model to study the role of an axon regeneration-associated gene, c-Jun, in mediating axon growth from adult DRG neurons9.

An in vitro model for genetic study of axon regeneration using cultured adult mouse dorsal root ganglion neurons is described. The method includes a re-suspension/re-plating step to allow axon re-growth from neurons undergoing genetic manipulation. This approach is especially useful for loss-of-function studies of axon regeneration using RNAi-based protein knockdown.

Estudio genético de la regeneración axonal en neuronas cultivadas de Adultos ganglio de la raíz dorsal

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability ...

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

Presynaptic inhibition is one of the most powerful inhibitory mechanisms in the spinal cord. The underlying physiological mechanism is a depolarization of primary afferent fibers mediated by GABAergic axo-axonal synapses (primary afferent depolarization). The strength of primary afferent depolarization can be measured by recording of volume-conducted potentials at the dorsal root (dorsal root potentials, DRP). Pathological changes of presynaptic inhibition are crucial in the abnormal central processing of certain pain conditions and in some disorders of motor hyperexcitability. Here, we describe a method of recording DRP in vivo in mice. The preparation of spinal cord dorsal roots in the anesthetized animal and the recording procedure using suction electrodes are explained. This method allows measuring GABAergic DRP and thereby estimating spinal presynaptic inhibition in the living mouse. In combination with transgenic mouse models, DRP recording may serve as a powerful tool to investigate disease-associated spinal pathophysiology. In vivo recording has several advantages compared to ex vivo isolated spinal cord preparations, e.g. the possibility of simultaneous recording or manipulation of supraspinal networks and induction of DRP by stimulation of peripheral nerves.

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

GABAergic presynaptic inhibition is a powerful inhibitory mechanism in the spinal cord important for motor and sensory signal integration in spinal cord networks. Underlying primary afferent depolarization can be measured by recording of dorsal root potentials (DRP). Here we demonstrate a method of in vivo recording of DRP in mice.

Medición de la columna vertebral presináptica inhibición en ratones mediante la raíz dorsal de grabación Potencial<em&gt; En Vivo</em

Presynaptic inhibition is one of the most powerful inhibitory mechanisms in the spinal cord. The underlying physiological mechanism is a depolarization of ...

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the study of nerve regeneration and myelination. The glial cells of the peripheral nervous system, Schwann cells (SC), are key facilitators of these processes; it is therefore crucial that the interactions of these cellular components are studied together. Direct contact between DRG neurons and glial cells provides additional stimuli sensed by specific membrane receptors, further improving the neuronal response. SC release growth factors and proteins in the culture medium, which enhance neuron survival and stimulate neurite sprouting and extension. However, SC require long proliferation time to be used for tissue engineering applications and the sacrifice of an healthy nerve for their sourcing. Adipose-derived stem cells (ASC) differentiated into SC phenotype are a valid alternative to SC for the set-up of a co-culture model with DRG neurons to study nerve regeneration. The present work presents a detailed and reproducible step-by-step protocol to harvest both DRG neurons and ASC from adult rats; to differentiate ASC towards a SC phenotype; and combines the two cell types in a direct co-culture system to investigate the interplay between neurons and SC in the peripheral nervous system. This tool has great potential in the optimization of tissue-engineered constructs for peripheral nerve repair.

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal root ganglia (DRG) are structures containing the sensory neurons of the peripheral nervous system. When dissociated, they can be co-cultured with SC-like adipose-derived stem cells (ASC), providing a valuable model to study in vitro nerve regeneration and myelination, mimicking the in vivo environment at the injury site.

Ganglios de raíz dorsal neuronas y las células madre derivadas de la adiposa diferenciada: Un<em&gt; In Vitro</em&gt; Co-cultivo modelo para estudiar la regeneración de los nervios periféricos

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the ...

An Approach to Enhance Alignment and Myelination of Dorsal Root Ganglion Neurons

Axon regeneration is a chaotic process due largely to unorganized axon alignment. Therefore, in order for a sufficient number of regenerated axons to bridge the lesion site, properly organized axonal alignment is required. Since demyelination after nerve injury strongly impairs the conductive capacity of surviving axons, remyelination is critical for successful functioning of regenerated nerves. Previously, we demonstrated that mesenchymal stem cells (MSCs) aligned on a pre-stretch induced anisotropic surface because the cells can sense a larger effective stiffness in the stretched direction than in the perpendicular direction. We also showed that an anisotropic surface arising from a mechanical pre-stretched surface similarly affects alignment, as well as growth and myelination of axons. Here, we provide a detailed protocol for preparing a pre-stretched anisotropic surface, the isolation and culture of dorsal root ganglion (DRG) neurons on a pre-stretched surface, and show the myelination behavior of a co-culture of DRG neurons with Schwann cells (SCs) on a pre-stretched surface.

This protocol describes the isolation of dorsal root ganglion (DRG) neurons isolated from rats and the culture of DRG neurons on a static pre-stretched cell culture system to enhance axon alignment, with subsequent co-culture of Schwann Cells (SCs) to promote myelination.

Un Enfoque para Mejorar la alineación y la mielinización de las neuronas del ganglio de la raíz dorsal

Axon regeneration is a chaotic process due largely to unorganized axon alignment. Therefore, in order for a sufficient number of regenerated axons to ...

Hydraulic Extrusion of the Spinal Cord and Isolation of Dorsal Root Ganglia in Rodents

Traditionally, the spinal cord is isolated by laminectomy, i.e. by breaking open the spinal vertebrae one at a time. This is both time consuming and may result in damage to the spinal cord caused by the dissection process. Here, we show how the spinal cord can be extruded using hydraulic pressure. Handling time is significantly reduced to only a few minutes, likely decreasing protein damage. The low risk of damage to the spinal cord tissue improves subsequent immunohistochemical analysis. By performing hydraulic spinal cord extrusion instead of traditional laminectomy, the rodents can further be used for DRG isolation, thereby lowering the number of animals and allowing analysis across tissues from the same rodent. We demonstrate a consistent method to identify and isolate the DRGs according to their localization relative to the costae. It is, however, important to adjust this method to the particular animal used, as the number of spinal cord segments, both thoracic and lumbar, may vary according to animal type and strain. In addition, we illustrate further processing examples of the isolated tissues.

Here, we present a protocol for hydraulic extrusion of the spinal cord as well as identification and isolation of specific dorsal root ganglia (DRGs) in the same rodent. Compared to standard spinal cord isolation methods, this method is significantly faster and reduces the risk of tissue damage.

Extrusión hidráulica de la médula espinal y aislamiento de los ganglios de la raíz dorsal en roedores

Traditionally, the spinal cord is isolated by laminectomy, i.e. by breaking open the spinal vertebrae one at a time. This is both time consuming and may ...

Dorsal Root Ganglion Injection and Dorsal Root Crush Injury as a Model for Sensory Axon Regeneration

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each other anatomically, it is important to identify an appropriate model to use for the study of axon regeneration. By using a suitable model, we can formulate a specific treatment based on the severity of injury, the neuronal cell type of interest, and the desired spinal tract for assessing regeneration. Within the sensory pathway, DRG neurons are responsible for relaying sensory information from the periphery to the CNS. We present here a protocol that uses a DRG injection with a viral vector and a concurrent dorsal root crush injury in the lower cervical spinal cord of an adult rat as a model to study sensory axon regeneration. As demonstrated using a control virus, AAV5-GFP, we show the effectiveness of a direct DRG injection in transducing DRG neurons and tracing sensory axons into the spinal cord. We also show the effectiveness of the dorsal root crush injury in denervating the forepaw as an injury model for evaluating axon regeneration. Despite the requirement for specialized training to perform this invasive surgical procedure, the protocol is flexible, and potential users can modify many parts to accommodate their experimental requirements. Importantly, it can serve as a foundation for those in search of a suitable animal model for their studies. We believe that this article will help new users to learn the procedure in a very efficient and effective manner.

This protocol presents the use of a dorsal root ganglion (DRG) injection with a viral vector and a concurrent dorsal root crush injury in an adult rat as a model to study sensory axon regeneration. This model is suitable for investigating the use of gene therapy to promote sensory axon regeneration.

Inyección de raíz dorsal ganglio de raíz dorsal y Lesión por aplastamiento como modelo para la regeneración del axón sensorial

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each ...

Laminotomy for Lumbar Dorsal Root Ganglion Access and Injection in Swine

Dorsal root ganglia (DRG) are anatomically well defined structures that contain all primary sensory neurons below the head. This fact makes DRG attractive targets for injection of novel therapeutics aimed at treating chronic pain. In small animal models, laminectomy has been used to facilitate DRG injection because it involves surgical removal of the vertebral bone surrounding each DRG. We demonstrate a technique for intraganglionic injection of lumbar DRG in a large animal species, namely, swine. Laminotomy is performed to allow direct access to DRG using standard neurosurgical techniques, instruments, and materials. Compared with more extensive bone removal via laminectomy, we implement laminotomy to conserve spinal anatomy while achieving sufficient DRG access. Intraoperative progress of DRG injection is monitored using a non-toxic dye. Following euthanasia on post-operative day 21, the success of injection is determined by histology for intraganglionic distribution of 4&#39;,6-diamidino-2-phenylindole (DAPI). We inject a biologically inactive solution to demonstrate the protocol. This method could be applied in future preclinical studies to target therapeutic solutions to DRG. Our methodology should facilitate testing the translatability of intraganglionic small animal paradigms in a large animal species. Additionally, this protocol may serve as a key resource for those planning preclinical studies of DRG injection in swine.

We describe a method for laminotomy in swine that provides access to lumbar dorsal root ganglia (DRG) for intraganglionic injection. Injection progress is monitored intraoperatively and histologically confirmed up to 21 days after surgery. This protocol could be used for future preclinical studies involving DRG injection.

Laminotomía para acceso de ganglio de raíz Dorsal Lumbar y de la inyección en los cerdos

Dorsal root ganglia (DRG) are anatomically well defined structures that contain all primary sensory neurons below the head. This fact makes DRG attractive ...

Analysis of Immune Cells in Single Sciatic Nerves and Dorsal Root Ganglion from a Single Mouse Using Flow Cytometry

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of the PNS are affected by injury and disease, affecting the nerve function and the capacity for regeneration. Neuronal immune cells are commonly analyzed by immunofluorescence (IF). While IF is essential for determining the location of the immune cells in the nerve, IF is only semi-quantitative and the method is limited to the number of markers that can be analyzed simultaneously and the degree of surface expression. In this study, flow cytometry was used for quantitative analysis of leukocyte infiltration into sciatic nerves or dorsal root ganglions (DRGs) of individual mice. Single cell analysis was performed using DAPI and several proteins were analyzed simultaneously for either surface or intracellular expression. Both sciatic nerves from one mouse that were treated according to this protocol generated &#8805; 30,000 single nucleated events. The proportion of leukocytes in the sciatic nerves, determined by expression of CD45, was approximately 5% of total cell content in the sciatic nerve and approximately 5-10% in the DRG. Although this protocol focuses primarily on the immune cell population within the PNS, the flexibility of flow cytometry to measure a number of markers simultaneously means that the other cells populations present within the nerve, such as Schwann cells, pericytes, fibroblasts, and endothelial cells, can also be analyzed using this method. This method therefore provides a new means for studying systemic effects on the PNS, such as neurotoxicology and genetic models of neuropathy or in chronic diseases, such as diabetes.

Quantitative analysis of cell content within the murine sciatic nerve is difficult due to the scarcity of the tissue. This protocol describes a method for tissue digestion and preparation that provides sufficient cells for flow cytometry analysis of immune cell populations from nerves of individual mice.

Análisis de las células inmunes en solo de los nervios ciático y de ganglio de raíz Dorsal de un ratón mediante citometría de flujo

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of ...