Este trabajo fue financiado por los Institutos Nacionales de Salud subvenciones K08 NS061039-01A2, el Programa de Investigación de Neurología y descubrimiento, y el Instituto A. Alfred Taubman de Investigación Médica de la Universidad de Michigan. En este trabajo se utilizó la Morfología y Análisis de Imágenes Core del Michigan Diabetes Research and Training Center, financiado por los Institutos Nacionales de Salud de subvención 5P90 DK-20572 del Instituto Nacional de Diabetes y Enfermedades Digestivas y Renales. Los autores desean agradecer a Robinson Singleton y Gordon Smith (Universidad de Utah) por su generosa donación de muestras de piel humana para apoyar el desarrollo inicial de la técnica inmunohistoquímica biomarcador nociceptivo.

Parte A: La inmunohistoquímica Preparación de la placa de 96 pocillos y la prevención de la tinción de fondo biopsias en sacabocados de piel se recogen a partir de sujetos humanos y se incubaron durante 12-24 h en solución fijadora (2% de paraformaldehído con una solución de L-Lisina 0,75 M (pH 7,4) y 0,05 mM de peryodato de sodio) a 4 ° C como se ha descrito previamente 8. Las muestras son entonces cryoprotected en tampón fosfato salino (PBS) con 20% de glicerol a 4 º C durante hasta 1 semana, incrustado en un medio de montaje de temperatura óptima de corte (OCT), a continuación, se seccionó en 50 micras de espesor secciones en un criostato. El protocolo se describe a continuación está diseñado para 8 secciones de piel, el número máximo de secciones de piel posible someterse a técnicas de inmunohistoquímica de flotación libre en una placa de 96 pocillos. DÍA 1: 1. Prevención de la inmunorreactividad no específica en el estrato córneo <ol><li> Etiqueta de placa de 96 pocillos como se muestra en la Figura 1. </li&gt; <li> Añadir 150 l de Imagen FX-TI de señal (Image-TI), eficaz para el bloqueo de la tinción de fondo 11,12, en cada pocillo en la fila 1 de la placa de 96 pocillos. </li><li> Añadir 150 l de 1X PBS en cada pocillo en la fila 2 y 3 de la placa de 96 pocillos. </li><li> Adquirir dos secciones de 50 micras por paciente: una sección de la biopsia tomada en DL, una sección de la biopsia tomada en PT. Un asa de inoculación (LeLoop) se utiliza para transferir secciones de un enjuague a la siguiente para evitar daño morfológico. Transferir suavemente cada sección de 50 m, con un asa de inoculación, en un individuo y de la fila 1, que contiene imagen-IT. Incubar las secciones de imagen-IT durante 30 minutos a temperatura ambiente en un agitador plana. </li><li> Enjuague secciones dos veces con PBS 1X (filas 2 y 3) durante 10 min a TA. Coloque la placa de bien en una mecedora plana entre enjuagues. </li></ol> 2. Preparación de 5% de BSA disolución de bloqueo y 1% de solución de enjuague <ol><li> Mientras que las secciones de biopsia están incubando en la imagen-IT, prepare 5% solución de bloqueoción (5% de BSA, TX-100, 0,3% solución 0,1 M de PBS-BSA al vórtice hasta que se disuelve completamente). </li><li> Añadir 150 l de solución de bloqueo BSA al 5% en cada pocillo de la fila 4. </li><li> El uso de un asa de inoculación, transferir secciones en pocillos individuales de solución de bloqueo BSA al 5%. Incubar las secciones de solución de bloqueo BSA 5% durante 1-2 horas a temperatura ambiente en un agitador plana. </li></ol> 3. Preparación de BSA al 1% solución de lavado y dilución de los anticuerpos primarios <ol><li> Mientras que las secciones están incubando en solución de bloqueo BSA al 5%, preparar la solución de enjuague 1% (1% de BSA, TX-100, solución de PBS-Vortex 0,1 M hasta 0,3% de BSA se disuelve completamente). </li><li> Mientras que las secciones están incubando en solución de bloqueo BSA al 5%, se diluye anticuerpos primarios en 1% de solución de lavado. <ol><li> Durante ocho secciones (8 x 150 l = 1,200 l) Se necesita un total de 1.200 l. Los anticuerpos primarios se componen de 1,500 l (aproximadamente 20% del volumen extra). </li><li> Las diluciones de los anticuerpos primarios: PGP, 1:500; Trk A,1:500 en BSA al 1% de solución de lavado. </li></ol></li></ol> 4. La incubación de las biopsias seccionados en el anticuerpo primario <ol><li> Añadir 150 l de anticuerpos primarios diluidos en los pocillos designados de la placa de 96 pocillos (fila 5). </li><li> Transferencia secciones de solución de bloqueo 5% (fila 4) en los pocillos de anticuerpos primarios designados (fila 5). </li><li> Selle la placa de 96 pocillos con parafilm y papel de aluminio para evitar que se sequen y exposición a la luz. </li><li> Incubar la placa de 96 pocillos O / N a 4 ° C en un agitador plana. </li></ol> DÍA 2: 5. Enjuague biopsias en BSA al 1% de solución de enjuague <ol><li> Añadir 150 l de solución de lavado BSA al 1% en cada pocillo en la fila 6, 7, y 8. </li><li> Enjuague secciones tres veces con solución de lavado BSA 1% (Filas 6, 7, y 8) durante 1 hora cada vez a temperatura ambiente. Cubra el plato bien con papel de aluminio y colocar en rocker plano entre enjuagues. </li></ol> 6. La dilución de anticuerpos secundarios &lt;ol&gt; <li> Mientras que las secciones se incubaron en el último enjuague de solución de lavado BSA al 1% (fila 8), diluir anticuerpos secundarios en solución de lavado BSA al 1%: <ol><li> Durante ocho secciones (8 x 150 l = 1,200 l) Se necesita un total de 1.200 l. La secundaria de anticuerpos se preparan en 1500 l (aproximadamente 20% del volumen extra). </li><li> Las diluciones de los anticuerpos secundarios: para PGP (Alexa Fluor 488 burro anti-conejo, 1:250), para Trk A (Alexa Fluor 647 burro anti-cabra, 1:250) en 1% BSA solución de lavado. </li></ol></li></ol> 7. La incubación de las biopsias seccionados en el anticuerpo secundario <ol><li> Añadir 150 l de anticuerpos secundarios diluidos en sus pocillos designados de la placa de 96 pocillos (Fila 9). </li><li> Transferencia secciones de solución de bloqueo 1% de BSA (Fila 8) en anticuerpo secundario bien (Fila 9). </li><li> Selle la placa de 96 pocillos con parafilm y papel de aluminio para evitar que se sequen y exposición a la luz. </li><li> Incubar las secciones de los anticuerpos secundarios designada O/ N a 4 ° C en un agitador plana. </li></ol> 8. Enjuague biopsias en BSA al 1% de solución de enjuague <ol><li> Añadir 150 l de solución de lavado BSA al 1% en cada pocillo en la fila 10, 11, y 12. </li><li> Enjuague secciones tres veces con solución de lavado BSA 1% (filas 10, 11, y 12) durante 1 hora cada vez a temperatura ambiente. Cubra el plato bien con papel de aluminio y colocar en una mecedora plana entre enjuagues. </li></ol> 9. Preparación de preparaciones microscópicas y montaje Biopsias seccionados <ol><li> Mientras que las secciones de biopsia están incubando en el último enjuague de solución de lavado BSA 1% (fila 12), preparar portaobjetos. </li><li> Coloque 50 l de BSA al 1% de solución de lavado en una diapositiva a la vez. </li><li> Retire las secciones del 1% BSA solución de lavado (Fila 12) y colocarlo en la 50 l gota de solución de lavado BSA al 1% en el portaobjetos de un microscopio designado. Después de la optimización de la posición de la sección, eliminar el exceso de solución de lavado BSA al 1% con una pipeta de vidrio abombada, tomando las precaucionespara evitar el contacto de la muestra. </li></ol> NOTA: Asegúrese de que la sección no está doblada, la muestra debe ser plana contra la superficie del portaobjetos. <ol start="4"><li> Colocar 1 gota de reactivo prolongar Oro de montaje con DAPI cerca de la biopsia en el portaobjetos de un microscopio Antifade. Tome un microscopio mm cubreobjetos de 22x22 y suavemente colóquelo sobre una biopsia y una gota de prolongar antifade reactivo de oro con caída DAPI. Repita para cada sección. </li><li> Vamos portaobjetos seco O / N a temperatura ambiente en la oscuridad. </li></ol> NOTA: Quite las burbujas de aire utilizando una punta de pipeta. Limpie el exceso de prolongar Gold antifade reactivo. Parte B: Confocal de imágenes 10. Confocal de imágenes <ol><li> Señales de fluorescencia de imagen utilizando una FluoView 500 microscopio confocal de barrido con una inmersión en aceite (1,3 NA) objetivo y zoom dos veces con software versión 5.0 FluoView 40X láser Olympus. </li&gt; <li> Utilice Alexa Fluor 488 y Alexa Fluor 647 para excitar el 543-nm HeNe láser verde y el 633-nm láser HeNe rojo, respectivamente. La señal de Alexa Fluor 488 está representado por una mirada verde tabla de consulta (LUT) y el Alexa Fluor 647 por una LUT rojo. </li><li> Ajuste las aberturas confocal para cada detector a 400 micras para mejorar las señales. Exploraciones secuenciales deben ser tomadas con una resolución de 1024 x 1024 para maximizar la separación de señales. </li><li> Captura tridimensional z-serie utilizando 1,2 micras intervalos de z a paso (sobre la base de la unidad de escaneado óptima calculada por el software FluoView) con Kalman promedio (dos marcos). </li></ol> Parte C: La visualización tridimensional y animación 11. Tridimensional Visualización y animación (3D) <ol><li> Archivos Fluoview abiertas en software Imaris x64 (versión 7.3, Bitplane AG) para visualizar los conjuntos de imágenes en 3D. </li><li> Ajuste mostrará cuando sea necesario para mejorar el contraste de la señal específica del nervio. </li><li> Crear un surface para visualizar el límite dermo-epidérmica. Utilice la herramienta de contorno en el modo de dibujo para dibujar el contorno. </li><li> Asignar una superficie semi-transparente a la frontera con el fin de ver las señales fluorescentes subyacentes. </li><li> Captura imágenes fijas de las señales fluorescentes en 3D para crear imágenes instantáneas de la película en 3D. </li><li> Utilice la función de animación para crear una película en 3D. Las imágenes se pueden girar 360 grados varias veces para mostrar cada señal por separado y combinado (Figuras 2, 3 y 4). Establezca la animación para crear películas que consta de 200 cuadros animados a 15 fotogramas por segundo. Guarde cada película como archivo AVI prima. </li><li> Comprimir la última película AVI con el software VirtualDub (versión 1.9.4). </li></ol>

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	<li>Lauria, G., Holland, 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=Epidermal+innervation:+changes+with+aging,+topographic+location,+and+in+sensory+neuropathy.">Epidermal innervation: changes with aging, topographic location, and in sensory neuropathy.</a> J. Neurol. Sci. 164 (2), 172-178 (1999).</li><li>Sullivan, K. A., Hayes, J. M., 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=Mouse+models+of+diabetic+neuropathy.">Mouse models of diabetic neuropathy.</a> Neurobiol. Dis. 28 (3), 276-285 (2007).</li><li>McArthur, J. C., Stocks, E. A., Hauer, P., Cornblath, D. R., Griffin, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+nerve+fiber+density:+normative+reference+range+and+diagnostic+efficiency.">Epidermal nerve fiber density: normative reference range and diagnostic efficiency.</a> Arch. Neurol. 55 (12), 1513-1520 (1998).</li><li>Griffin, J. W., McArthur, J. C., Polydefkis, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+cutaneous+innervation+by+skin+biopsies.">Assessment of cutaneous innervation by skin biopsies.</a> Curr. Opin. Neurol. 14 (5), 655-659 (2001).</li><li>Cheng, H. T., Dauch, J. R., Hayes, J. M., Yanik, B. M., Feldman, E. L. <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/p38+signaling+increases+intraepidermal+nerve+fiber+densities+in+painful+neuropathy+of+type+2+diabetes.">Nerve growth factor/p38 signaling increases intraepidermal nerve fiber densities in painful neuropathy of type 2 diabetes.</a> Neurobiol. Dis. 45 (1), 280-287 (2012).</li><li>Lauria, G., Lombardi, R., Camozzi, F., Devigili, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin+biopsy+for+the+diagnosis+of+peripheral+neuropathy.">Skin biopsy for the diagnosis of peripheral neuropathy.</a> Histopathology. 54 (3), 273-285 (2009).</li><li>Vlckova-Moravcova, E., Bednarik, J., Dusek, L., Toyka, K. V., Sommer, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+validity+of+epidermal+nerve+fiber+densities+in+painful+sensory+neuropathies.">Diagnostic validity of epidermal nerve fiber densities in painful sensory neuropathies.</a> Muscle Nerve. 37 (1), 50-60 (2008).</li><li>Casanova-Molla, J., Morales, M., 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=Axonal+fluorescence+quantitation+provides+a+new+approach+to+assess+cutaneous+innervation.">Axonal fluorescence quantitation provides a new approach to assess cutaneous innervation.</a> J. Neurosci. Methods. 200 (2), 190-198 (2011).</li><li>Wang, L., Hilliges, M., Jernberg, T., Wiegleb-Edstrom, D., Johansson, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+gene+product+9.5-immunoreactive+nerve+fibres+and+cells+in+human+skin.">Protein gene product 9.5-immunoreactive nerve fibres and cells in human skin.</a> Cell Tissue Res. 261 (1), 25-33 (1990).</li><li>Lauria, G., Morbin, M., 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=Expression+of+capsaicin+receptor+immunoreactivity+in+human+peripheral+nervous+system+and+in+painful+neuropathies.">Expression of capsaicin receptor immunoreactivity in human peripheral nervous system and in painful neuropathies.</a> J. Peripher. Nerv. Syst. 11 (3), 262-271 (2006).</li><li>Penna, G., Fibbi, 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=Human+benign+prostatic+hyperplasia+stromal+cells+as+inducers+and+targets+of+chronic+immuno-mediated+inflammation.">Human benign prostatic hyperplasia stromal cells as inducers and targets of chronic immuno-mediated inflammation.</a> J. Immunol. 182 (7), 4056-4064 (2009).</li><li>Lentz, S. I., Edwards, J. L., 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=Mitochondrial+DNA+(mtDNA)+Biogenesis:+Visualization+and+Duel+Incorporation+of+BrdU+and+EdU+Into+Newly+Synthesized+mtDNA+In+Vitro.">Mitochondrial DNA (mtDNA) Biogenesis: Visualization and Duel Incorporation of BrdU and EdU Into Newly Synthesized mtDNA In Vitro.</a> J. Histochem. Cytochem. 58 (2), 207-218 (2010).</li><li>Polydefkis, M., Hauer, P., Griffin, J. W., McArthur, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin+biopsy+as+a+tool+to+assess+distal+small+fiber+innervation+in+diabetic+neuropathy.">Skin biopsy as a tool to assess distal small fiber innervation in diabetic neuropathy.</a> Diabetes Technol. Ther. 3 (1), 23-28 (2001).</li><li>Lauria, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+fibre+neuropathies.">Small fibre neuropathies.</a> Curr. Opin. Neurol. 18 (5), 591-597 (2005).</li><li>Sorensen, L., Molyneaux, L., Yue, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relationship+among+pain,+sensory+loss,+and+small+nerve+fibers+in+diabetes.">The relationship among pain, sensory loss, and small nerve fibers in diabetes.</a> Diabetes Care. 29 (4), 883-887 (2006).</li><li>Lauria, G., Morbin, M., 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=Axonal+swellings+predict+the+degeneration+of+epidermal+nerve+fibers+in+painful+neuropathies.">Axonal swellings predict the degeneration of epidermal nerve fibers in painful neuropathies.</a> Neurology. 61 (5), 631-636 (2003).</li><li>Herrmann, D. N., McDermott, M. 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=Epidermal+nerve+fiber+density,+axonal+swellings+and+QST+as+predictors+of+HIV+distal+sensory+neuropathy.">Epidermal nerve fiber density, axonal swellings and QST as predictors of HIV distal sensory neuropathy.</a> Muscle Nerve. 29 (3), 420-427 (2004).</li><li>Navarro, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+27:+Neural+plasticity+after+nerve+injury+and+regeneration.">Chapter 27: Neural plasticity after nerve injury and regeneration.</a> Int. Rev. Neurobiol. 87, 483-505 (2009).</li></ol>

No hay conflictos de intereses que declarar.

Medición de IENFD ha sido ampliamente utilizado para determinar el grado de neuropatías periféricas 13,14. En la actualidad, el protocolo más comúnmente utilizado sólo mide las densidades de las fibras nerviosas que penetran en la membrana basal de la epidermis; no toma en consideración de ramificación axonal y / o cambios morfológicos de los nervios. Además, no se ha demostrado el análisis IENFD actual para correlacionar IENFD con la presencia de dolor en PN 15. <p class="jove_conte...

En la actualidad, es una práctica común para los médicos para cuantificar la densidad de fibras nerviosas intraepidérmicos, (IENFD) a partir de biopsias de piel en sacabocados, que pueden ser utilizados para diagnosticar neuropatías de fibras pequeñas 3, 6-8. Las biopsias se toman de la pierna distal (DL), 10 cm por encima del maléolo lateral y el muslo proximal (PT), 20 cm por debajo de la espina ilíaca anterior 9. Todos IENF están etiquetados de usar el producto gen de la proteína 9.5 (PGP), un marcador neuronal bandeja 10-12. En la actualidad, es una práctica estándar para diagnosticar neuropatías de fibras pequeñas utilizando IENFD determinado por tinción de PGP con microscopía de campo claro 6. Además, varios grupos de investigación han utilizado protocolos de inmunofluorescencia para PGP inmunohistoquímica 7-9. Neuropatía de fibras pequeñas se asocia con el dolor neuropático. Para entender mejor el papel de IENF esencial para el procesamiento del dolor, se desarrolló una técnica para co-label total de IENF con fibras que generan dolor. Nocicepcióntiva IENF, específicamente Aδ y las fibras C, se pueden estudiar a través de la co-etiquetado de IENF con PGP y el marcador nociceptivo, tropomiosina-receptor-quinasa A (Trk A) 5. Trk A es el receptor de alta afinidad para el factor de crecimiento nervioso que es esencial para el desarrollo de la nocicepción. Las fibras nerviosas nociceptivas A-positivos Trk son fibras peptidérgicas que expresan la sustancia P (SP) y el péptido relacionado con el gen calcitonina (CGRP). Anteriormente, Lauria y sus colegas aplicaron la técnica de doble etiquetado para estudiar PN, co-etiquetado IENF PGP-positiva con un marcador de 10 nociceptivo. En nuestro estudio anterior, hemos demostrado que Trk IENF A positivo, pero no Trk IENF A negativo, se upregulated en un modelo animal de la neuropatía diabética dolorosa 5. Esta técnica de co-etiquetado proporciona la capacidad para comparar la cuantificación de IENFD nociceptivo al total de IENFD y la capacidad para estudiar los cambios morfológicos asociados con PN. La capacidad de visualizar nociceptivo IENF y compañerasre cuantificación de IENFD total a la IENFD nociceptivo podría proporcionar evidencia objetiva para la presencia de dolor, y, posiblemente, una idea de la severidad del dolor asociado con PN. Esta técnica es también aplicable a la piel de modelos animales. En comparación con estudios previos, el protocolo actual describe métodos para el análisis de imagen en 3D, la creación de la oportunidad de evitar errores que podrían ocurrir en el análisis de imagen 2D.

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>10X PBS</td>
			<td>Fisher Scientific</td>
			<td>BP399-4</td>
			<td>To make up 1X PBS</td>
		</tr>
		<tr>
			<td>Image-IT FX Signal</td>
			<td>Invitrogen</td>
			<td>I36933</td>
			<td>Image-IT</td>
		</tr>
		<tr>
			<td>Protein Gene Product 9.5 (Polyclonal rabbit)</td>
			<td>AbD Serotec</td>
			<td>7863-0504</td>
			<td>PGP</td>
		</tr>
		<tr>
			<td>Tropomyosin Related-Kinase A (Polyclonal goat)</td>
			<td>R&amp;D Systems</td>
			<td>AF1056</td>
			<td>Trk A</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 donkey &alpha;-rabbit</td>
			<td>Invitrogen</td>
			<td>A21206</td>
			<td>AF488 donkey &alpha;-goat</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 donkey &alpha;-goat</td>
			<td>Invitrogen</td>
			<td>A21447</td>
			<td>AF647 donkey &alpha;-goat</td>
		</tr>
		<tr>
			<td>Albumin, from Bovine Serum</td>
			<td>Sigma-Aldrich</td>
			<td>A7906-100</td>
			<td>BSA</td>
		</tr>
		<tr>
			<td>Triton X- 100</td>
			<td>Sigma-Aldrich</td>
			<td>T9284</td>
			<td>TX-100</td>
		</tr>
		<tr>
			<td>Non-calibrated Loop</td>
			<td>LeLoop</td>
			<td>MP 199025</td>
			<td>inoculating Loop</td>
		</tr>
		<tr>
			<td>96-well assay plate</td>
			<td>Corning Incorporated</td>
			<td>3603</td>
			<td>Well plate</td>
		</tr>
		<tr>
			<td>Prolong Gold antifade reagent with DAPI</td>
			<td>Invitrogen</td>
			<td>P36931</td>
			<td>DAPI</td>
		</tr>
		<tr>
			<td>Microscope Cover Glass 22x22 mm</td>
			<td>Fisher Scientific</td>
			<td>12-541-B</td>
			<td>Coverslips</td>
		</tr>
		<tr>
			<td>Superfrost Plus Microscope Slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-15</td>
			<td>Microscope Slides</td>
		</tr>
		<tr>
			<td>Olympus Fluoview Laser Scanning Confocal Microscope</td>
			<td>Olympus</td>
			<td>FV500</td>
			<td>Confocal Microscope</td>
		</tr>
		<tr>
			<td>Optimum Cutting Temperature</td>
			<td>Sakura</td>
			<td>4583</td>
			<td>OCT</td>
		</tr>
		<tr>
			<td>Leica cryostat</td>
			<td>Leica</td>
			<td>CM1850</td>
			<td>Cryostat</td>
		</tr>
	</tbody>
</table>

three-dimensional imaging of nociceptive intraepidermal nerve fibers in human skin biopsies

Un punzón de biopsia de la piel se utiliza comúnmente para cuantificar la densidad de fibras nerviosas intraepidérmicos (IENFD) para el diagnóstico de 1,2 polineuropatía periférica. En la actualidad, es una práctica común para recoger biopsias de piel 3 mm a partir de la pata distal (DL) y el muslo proximal (PT) para la evaluación de las polineuropatías dependiente de la longitud 3. Sin embargo, debido a la naturaleza multidireccional de IENFs, es un reto para examinar superposición de estructuras nerviosas a través del análisis de formación de imágenes de dos dimensiones (2D). Alternativamente, imágenes en tres dimensiones (3D) podría proporcionar una mejor solución para este dilema. En el presente informe, se presentan métodos para la aplicación de imágenes en 3D para estudiar la neuropatía dolorosa (PN). Con el fin de identificar IENFs, muestras de piel se procesan para el análisis de inmunofluorescencia de producto de gen de la proteína 9.5 (PGP), un marcador neuronal sartén. En la actualidad, es una práctica estándar para diagnosticar neuropatías de fibras pequeñas utilizando IENFD disuadirminada por inmunohistoquímica utilizando PGP microscopía de campo claro 4. En el estudio actual, se aplicó análisis de doble inmunofluorescencia para identificar IENFD total de, utilizando PGP, y IENF nociceptivo, a través del uso de anticuerpos que reconocen la tropomiosina-receptor-quinasa A (Trk A), el receptor de alta afinidad para el factor de crecimiento de los nervios 5. Las ventajas de IENF co-tinción con PGP y Trk A anticuerpos beneficia el estudio de la PN por tinción con claridad, fibras nociceptivas PGP-positivos. Estas señales fluorescentes se pueden cuantificar para determinar los cambios morfológicos y IENFD nociceptivas de IENF asociados con PN. Las imágenes fluorescentes se adquirieron por microscopía confocal y se procesaron para análisis en 3D. 3D-imagen proporciona capacidades de rotación seguir analizando los cambios morfológicos asociados a la PN. En conjunto, co-tinción fluorescente, la imagen confocal y análisis 3D se benefician claramente el estudio de la PN.

Se aplicó el protocolo actual para estudiar la morfología de IENF en PT y DL biopsias de piel de pacientes con PN. La piel, a partir de tres sujetos, se recogió en la Universidad de Utah para demostrar la patomorfología asociado con PN. Los temas incluyen: Caso 1: varón de 51 años de edad, con antecedentes de PN de la diabetes tipo 2 (duración: 14 meses, la puntuación de dolor: 51), y Caso 2: varón de 56 años de edad, con antecedentes de PN de la diabetes tipo 2 (duración: 108 meses, la puntuación de dolor: ...

Watch this Scientific Journal Video about Three-dimensional Imaging of Nociceptive Intraepidermal Nerve Fibers in Human Skin Biopsies at JoVE.com

Three-dimensional Imaging of Nociceptive Intraepidermal Nerve Fibers in Human Skin Biopsies

Con el fin de estudiar los cambios en las fibras nerviosas nociceptivas intraepidérmicos (IENFs) en neuropatías dolorosas (PN), hemos desarrollado protocolos que podrían examinar directamente los cambios morfológicos observados en tres dimensiones en IENFs nociceptivas. El análisis tridimensional de IENFs tiene el potencial para evaluar los cambios morfológicos de IENF en PN.

Imagen tridimensional de nociceptivos Fibras Nerviosas intraepidérmicas en biopsias de piel humana

A punch biopsy of the skin is commonly used to quantify intraepidermal nerve fiber densities (IENFD) for the diagnosis of peripheral polyneuropathy 1,2. ...

three-dimensional-imaging-nociceptive-intraepidermal-nerve-fibers

Research

JoVE Journal

Medicine

13.0K vistas.  University of Michigan. Con el fin de estudiar los cambios en las fibras nerviosas nociceptivas intraepidérmicos (IENFs) en neuropatías dolorosas (PN), hemos desarrollado protocolos que podrían examinar directamente los cambios morfológicos observados en tres dimensiones en IENFs nociceptivas. El análisis tridimensional de IENFs tiene el potencial para evaluar los cambios morfológicos de IENF en PN.

Video: Three-dimensional Imaging of Nociceptive Intraepidermal Nerve Fibers in Human Skin Biopsies

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

Humanos neuroendocrinos líneas de células tumorales como un modelo tridimensional para el estudio de la terapia humana tumor neuroendocrino

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

Investigación

Medicina

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods rely on the ability of cells either to attach to a solid substratum of a tissue culture dish or to grow in suspension in liquid medium. Multiple immortal cell lines have been created and grown using such approaches, however, these methods frequently fail when primary cells need to be grown ex vivo. Such failure has been attributed to the absence of the appropriate extracellular matrix components of the tissue microenvironment from the standard systems where tissue culture plastic is used as a surface for cell growth. Extracellular matrix is an integral component of the tissue microenvironment and its presence is crucial for the maintenance of physiological functions such as cell polarization, survival, and proliferation. Here we present a 3-dimensional tissue culture method where primary bone marrow cells are grown in extracellular matrix formulated to recapitulate the microenvironment of the human bone (rBM system). Embedded in the extracellular matrix, cells are supplied with nutrients through the medium supplemented with human plasma, thus providing a comprehensive system where cell survival and proliferation can be sustained for up to 30 days while maintaining the cellular composition of the primary tissue. Using the rBM system we have successfully grown primary bone marrow cells from normal donors and patients with amyloidosis, and various hematological malignancies. The rBM system allows for direct, in-matrix real time visualization of the cell behavior and evaluation of preclinical efficacy of novel therapeutics. Moreover, cells can be isolated from the rBM and subsequently used for in vivo transplantation, cell sorting, flow cytometry, and nucleic acid and protein analysis. Taken together, the rBM method provides a reliable system for the growth of primary bone marrow cells under physiological conditions.

In standard culture methods cells are taken out of their physiological environment and grown on the plastic surface of a dish. To study the behavior of primary human bone marrow cells we created a 3-D culture system where cells are grown under conditions recapitulating the native microenvironment of the tissue.

Un tejido tridimensional Cultura modelo para estudiar primaria médula ósea humana y sus malignidades

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

Tumor Ensayo tridimensional (3D) esferoide Invasion

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

Three-dimensional Alginate-bead Culture of Human Pituitary Adenoma Cells

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from brown sea algae. Briefly, the tumor tissue is cut into small pieces and submitted to an enzymatic digestion with collagenase and trypsin. Next, a cell suspension is obtained. The tumor cell suspension is mixed with 1.2% sodium alginate and dropped into a CaCl2 solution, and the alginate/cell suspension is gelled on contact with the CaCl2 to form spherical beads. The cells embedded in the alginate beads are supplied with nutrients provided by the culture media enriched with 20% FBS. Three-dimensional culture in alginate beads maintains the viability of adenoma cells for long periods of time, up to four months. Moreover, the cells can be liberated from the alginate by washing the beads with sodium citrate and seeded on glass coverslips for further immunocytochemical analyses. The use of a cell culture model allows for the fixation and visualization of the actin cytoskeleton with minimal disorganization. In summary, alginate beads provide a reliable culture system for the maintenance of pituitary adenoma cells.

Three-dimensional culture in alginate beads, due to its simplicity and reproducibility, was chosen to maintain the pituitary adenoma cells. The procedures included an initial enzymatic digestion and mechanical dissociation of the tumor tissue, and the subsequent cell suspension was encapsulated in alginate beads.

Tridimensional de alginato-grano Cultura de células humanas del adenoma pituitario

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from ...

Quantitative Visualization of Leukocyte Infiltrate in a Murine Model of Fulminant Myocarditis by Light Sheet Microscopy

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently labelled structures in large biological specimens, and is down to cellular resolution. Meanwhile, the constant refinement of underlying protocols and the enhanced availability of specialized commercial systems enable us to investigate the microstructure of whole mouse organs and even allow for the characterization of cellular behavior in various live-cell imaging approaches. Here, we describe a protocol for the spatial whole-mount visualization and quantification of the CD45+ leukocyte population in inflamed mouse hearts. The method employs a transgenic mouse strain (CD11c.DTR)that has recently been shown to serve as a robust, inducible model for the study of the development of fulminant fatal myocarditis, characterized by lethal cardiac arrhythmias. This protocol includes myocarditis induction, intravital antibody-mediated cell staining, organ preparation, and LSFM with subsequent computer-assisted image post-processing. Although presented as a highly-adapted method for our particular scientific question, the protocol represents the blueprint of an easily adjustable system that can also target completely different fluorescent structures in other organs and even in other species.

Here, we describe a light-sheet microscopy approach to visualize the cardiac CD45+ leukocyte infiltrate in a murine model of aseptic fulminant myocarditis, which is induced by the intratracheal diphtheria toxin treatment of CD11c.DTR mice.

Visualización Cuantitativa del Infiltrado de Leucocitos en un Modelo Murino de Miocarditis Fulminante por Microscopía de Hoja Ligera

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently ...

Objective Nociceptive Assessment in Ventilated ICU Patients: A Feasibility Study Using Pupillometry and the Nociceptive Flexion Reflex

The concept of objective nociceptive assessment and optimal pain management have gained increasing attention. Despite the known negative short- and long-term consequences of unresolved pain or excessive analgosedation, adequate nociceptive monitoring remains challenging in non-communicative, critically ill adults. In the intensive care unit (ICU), routine nociceptive evaluation is carried out by the attending nurse using the Behavior Pain Scale (BPS) in mechanically ventilated patients. This assessment is limited by medication use (e.g., neuromuscular blocking agents) and the inherent subjective character of nociceptive evaluation by third parties.
Here, we describe the use of two nociceptive reflex testing devices as tools for objective pain evaluation: the pupillary dilation reflex (PDR) and nociception flexion reflex (NFR). These measurement tools are non-invasive and well tolerated, providing clinicians and researchers with objective information regarding two different nociceptive processing pathways: (1)&#160;the pain-related autonomic reactivity&#160;and (2) the ascending component of the somatosensory system. The use of PDR and NFR measurements are currently limited to specialized pain clinics and research institutions because of impressions that these are technically demanding or time-consuming procedures, or even because of a lack of knowledge regarding their existence.
By focusing on the two abovementioned nociceptive reflex assessments, this study evaluated their feasibility as a physiological pain measurement method in daily practice. Pursuing novel technologies for evaluating the analgesia level in unconscious patients may further improve individual pharmacological treatment and patient related outcome measures. Therefore, future research must include large well-designed clinical trials in a real-life environment.

Pain assessment in anesthetized patients who cannot communicate with the outside world in any way remains challenging despite the development of innovative objective pain evaluation tools. In this project, the pupillary dilation reflex and the nociception flexion reflex are assessed in critically ill, mechanically ventilated adult patients.

Evaluación objetiva nociceptiva en pacientes de la UCI ventilados: un estudio de viabilidad usando pupilometría y el reflejo de flexión nociceptiva

The concept of objective nociceptive assessment and optimal pain management have gained increasing attention. Despite the known negative short- and ...

Three-Dimensional Printing of a Complex Aortic Anomaly

Complex congenital aortic anomalies include diverse types of malformations that may be clinically asymptomatic or present with respiratory or esophageal symptoms. These anomalies may be associated with other congenital heart diseases. It is hard to identify the accurate anatomic vessel location from two-dimensional imaging data, such as computed tomography. As an additive manufacturing method, three-dimensional (3-D) printing can covert the acquired imaging data into 3-D physical models. This protocol describes the procedure for modeling the volumetric DICOM imaging into 3-D data and printing it as an anatomically realistic 3-D model. Using this model, surgeons can identify the vessel location of complex aortic anomalies, which is helpful for pre-operative planning and intra-operative guidance.

Here, we present a protocol to use three dimensional printed models for pre-operative planning and intra-operative reorganization of complicated vascular locations when handling a congenital aortic anomaly.

Impresión tridimensional de una anomalía aórtica compleja

Complex congenital aortic anomalies include diverse types of malformations that may be clinically asymptomatic or present with respiratory or esophageal ...

Refined CLARITY-Based Tissue Clearing for Three-Dimensional Fibroblast Organization in Healthy and Injured Mouse Hearts

Cardiovascular disease is the most prevalent cause of mortality worldwide and is often marked by heightened cardiac fibrosis that can lead to increased ventricular stiffness with altered cardiac function. This increase in cardiac ventricular fibrosis is due to activation of resident fibroblasts, although how these cells operate within the 3-dimensional (3-D) heart, at baseline or after activation, is not well understood. To examine how fibroblasts contribute to heart disease and their dynamics in the 3-D heart, a refined CLARITY-based tissue clearing and imaging method was developed that shows fluorescently labeled cardiac fibroblasts within the entire mouse heart. Tissue resident fibroblasts were genetically labeled using Rosa26-loxP-eGFP florescent reporter mice crossed with the cardiac fibroblast expressing Tcf21-MerCreMer knock-in line. This technique was used to observe fibroblast localization dynamics throughout the entire adult left ventricle in healthy mice and in fibrotic mouse models of heart disease. Interestingly, in one injury model, unique patterns of cardiac fibroblasts were observed in the injured mouse heart that followed bands of wrapped fibers in the contractile direction. In ischemic injury models, fibroblast death occurred, followed by repopulation from the infarct border zone. Collectively, this refined cardiac tissue clarifying technique and digitized imaging system allows for 3-D visualization of cardiac fibroblasts in the heart without the limitations of antibody penetration failure or previous issues surrounding lost fluorescence due to tissue processing.

A refined method of tissue clearing was developed and applied to the adult mouse heart. This method was designed to clear dense, autofluorescent cardiac tissue, while maintaining labeled fibroblast fluorescence attributed to a genetic reporter strategy.

Refinado CLARIDAD Basada En La Limpieza De Tejidos Para La Organización De Fibroblastos TridimensionalEs En Corazones De Ratón Sanos Y Lesionados

Cardiovascular disease is the most prevalent cause of mortality worldwide and is often marked by heightened cardiac fibrosis that can lead to increased ...

High-Resolution Three-Dimensional Imaging of the Footpad Vasculature in a Murine Hindlimb Gangrene Model

Peripheral arterial disease (PAD) is a significant cause of morbidity resulting from chronic exposure to atherosclerotic risk factors. Patients suffering from its most severe form, chronic limb-threatening ischemia (CLTI), face substantial impairments to daily living, including chronic pain, limited walking distance without pain, and nonhealing wounds. Preclinical models have been developed in various animals to study PAD, but mouse hindlimb ischemia remains the most widely used. There can be significant variation in response to ischemic insult in these models depending on the mouse strain used and the site, number, and means of arterial disruption. This protocol describes a unique method combining femoral artery and vein electrocoagulation with the administration of a nitric oxide synthase (NOS) inhibitor to reliably induce footpad gangrene in Friend Virus B (FVB) mice that resembles the tissue loss of CLTI. While traditional means of assessing reperfusion such as laser Doppler perfusion imaging (LDPI) are still recommended, intracardiac perfusion of the lipophilic dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) is used to label the vasculature. Subsequent whole-mount confocal laser scanning microscopy allows for high-resolution, three-dimensional (3D) reconstruction of footpad vascular networks that complements traditional means of assessing reperfusion in hindlimb ischemia models.

The present protocol describes a unique, clinically relevant model of peripheral arterial disease that combines femoral artery and vein electrocoagulation with the administration of a nitric oxide synthase inhibitor to induce hindlimb gangrene in FVB mice. Intracardiac DiI perfusion is then used for high-resolution, three-dimensional imaging of the footpad vasculature.

Imágenes tridimensionales de alta resolución de la vasculatura de la almohadilla del pie en un modelo de gangrena de extremidades posteriores murinas

Peripheral arterial disease (PAD) is a significant cause of morbidity resulting from chronic exposure to atherosclerotic risk factors. Patients suffering ...

Guided Endodontics: Three-Dimensional Planning and Template-Aided Preparation of Endodontic Access Cavities

Pulp canal obliterations (PCO) are often a consequence of dental trauma, such as luxation injuries. Even though dentin apposition is a sign of vital pulp, pulpitis or apical periodontitis may develop in the long term. Root canal treatment of teeth with severe PCO and pulpal or periapical pathosis is challenging for general practitioners and even for well-equipped endodontic specialists. To ensure detection of the calcified root canal and avoid excessive loss of tooth structure or root perforation, static navigation using templates ("Guided Endodontics") was introduced a few years ago. The general workflow includes three-dimensional imaging using cone-beam computed tomography (CBCT), a digital surface scan, and superimposition of both in a planning software. This is followed by virtual planning of the access cavity and the design of a template that will guide the drill to the desired target point. To do this, a true-to-scale virtual image of the drill must be placed in a way that the tip of the drill reaches the orifice of the calcified root canal. Once the template has been fabricated using computer-aided design and computer-aided manufacturing (CAD/CAM) or a 3D printer, guided preparation of the access cavity can be performed clinically. For research purposes, a postoperative CBCT image can be used to quantify the accuracy of the access cavity performed. This work aims to present the technique of static guided endodontics from imaging to clinical implementation.

Guided endodontics describes a template-aided approach for access cavity preparation. The procedure requires cone-beam computed tomography and a surface scan to produce a template. An incorporated sleeve guides the drill to the target point. This allows the preparation of minimally invasive endodontic access cavities in calcified teeth.

Endodoncia guiada: planificación tridimensional y preparación asistida por plantillas de cavidades de acceso endodóntico

Pulp canal obliterations (PCO) are often a consequence of dental trauma, such as luxation injuries. Even though dentin apposition is a sign of vital pulp, ...