German Research Foundation (FOR2240 "(Lymph) Angiogenesis and Cellular Immunity in Inflammatory Diseases of the Eye" to CC and LMH; HE 6743/2-1 and HE 7643/3-1 to LMH; CU47/6-1 to CC), German Cancer Aid (to LMH and CC), GEROK program University of Cologne (to SLS and LMH), and EU COST BM1302 "Joining Forces to Corneal Regeneration" (to CC).

La utilización de tejidos humanos debe ser revisado y aprobado por un comité de revisión institucional o su equivalente. El trabajo aquí descrito fue aprobado por el comité de ética local y tenía la aprobación de examen científico. Este trabajo fue realizado de acuerdo con la Declaración de Helsinki. Los especímenes esclerales humanos se obtuvieron de los ojos de los donantes de globo (máximo post-mortem de tiempo de 24 horas) en el Banco de Ojos del Departamento de Oftalmología de la Universidad de Colonia, Alemania. 1. Preparación Experimental <ol><li> Preparar 96% de etanol y solución salina tamponada con fosfato (PBS) en diferentes tubos. Preparar los anticuerpos primarios en la dilución recomendada de PBS que contiene 2% de albúmina de suero bovino (BSA) y tener todas las soluciones en hielo. Preparar 100 l por muestra. </li><li> Limpiar todos los instrumentos y usar escalpelos afilados. Después de repetir el corte con el mismo bisturí, cambiar el bisturí. </li><li> Obtener 1-2 fórceps Colibri, de rectas tijeras de disección micro, un bisturí # 10o un bisturí oftálmica micro pluma, y ​​cuatro agujas de 26 G. </li><li> Envuelva el papel de aluminio alrededor de una placa de poliestireno o usar una placa de corcho para fijar el tejido. Trabajar debajo de un microscopio estereoscópico binocular. Trabajar estéril debajo de un flujo de aire laminar si es necesario. </li></ol> 2. Preparar la esclerótica <ol><li> coloque un lado del bulbo y realizar una trepanación perforación en la parte corneoscleral, a continuación, girar el trépano. Rotar el trépano con cuidado y de manera uniforme sobre la superficie para realizar un corte por igual todo el año. Use un trépano de tamaño 15,5 mm. Cortar los archivos adjuntos restantes con tijeras curvas. Retire la trepanación corneoscleral. Esto dará lugar a una bombilla en sentido anterior abierto. <ol><li> Ponga la esclerótica en un hisopo con la parte abierta hacia arriba. Retire la retina y la úvea, el uso de fórceps colibri de lograr ambas capas (retina y Uvea) de la esclerótica. No deje la úvea pigmentada sobre la esclerótica interior. Utilice tijeras curvas para eliminar la retina y la úvea de la papila. Retire el remconjuntiva aining, músculos extraoculares, y la cápsula de Tenon de la esclerótica superficial. </li></ol></li><li> Retire la muestra escleral en el tamaño requerido por el uso de las tijeras de tipo recto y las pinzas Colibri. Tomar alrededor de 2 cm 2 muestras de tamaño esclerales de diferentes lugares. Debido a su pequeño tamaño, mantenga la esclerótica con suavidad y cortar al tamaño deseado como cuadrados usando las tijeras de tipo lineal. La trepanación corneoscleral define el margen anterior de las muestras esclerales. <ol><li> Evitar repetida agarrar con el fórceps como el área en la que el tejido fue agarrado no es adecuado para los análisis de microscopía confocal. Preparar el número requerido de muestras en función del tipo experimento (por ejemplo, en sentido anterior vs. posterior, comparar la figura 1) y la cantidad de anticuerpos usados. </li></ol></li><li> Poner las muestras en tubos de 1,5 ml para su uso posterior. Fijar las muestras en 1,5 ml 96% de etanol durante 15 min, y luego los lavar tres veces cada uno durante 5 minutosen 1,5 ml de PBS en el agitador. Si se trabaja con tejido congelado, lleve a cabo este paso antes de la congelación a presión. </li><li> Utilice tejido fresco. Sin embargo, si esto no es posible, broche congelar la esclerótica en nitrógeno líquido y mantenerlo a -20 ° C para su uso posterior. Si se trabaja con tejido congelado, descongelar antes de su uso. Evitar la descongelación repetida y ciclos de congelación. </li><li> Mantener cada muestra en PBS que contenía 1,5 ml 5% de BSA a temperatura ambiente durante 2 hr. Este paso induce una inflamación de la muestra que es útil para la laminación y también evita la unión no específica. </li><li> Después de que las muestras se han hinchado, preparar las plazas esclerales para la laminación. Trabajar por debajo de un microscopio, por ejemplo, un microscopio estéreo binocular. Colocar las muestras esclerales posteriores a la membrana de poliestireno envuelto en papel de aluminio o la placa de corcho utilizando las agujas de 26 G. </li><li> Doblar los bordes de las agujas para que no se interfieren con el campo visual debajo del microscopio. </li><li> Laminar el samp escleral de espesor totalles. <ol><li> En primer lugar, mantenga el borde anterior de la esclerótica utilizando pinzas Colibri. A continuación, cortar cuidadosamente una capa delgada de la esclerótica superficial utilizando el bisturí # 10, la pluma micro bisturí oftálmica, o una hoja de la espátula. Mantenga el bisturí horizontal y cortar la capa superficial lo más finas posible de la capa subyacente. </li><li> Diseccionar una capa delgada escleral de la plaza escleral. Este método es comparable a las técnicas quirúrgicas estándar para preparar un colgajo durante la trabeculectomía y deberían dar lugar a capas gruesas 30-80 micras (comparar Figura 2). </li></ol></li><li> Evitar el secado de la esclerótica mediante la adición de pequeñas cantidades de PBS a los tejidos, por ejemplo, 50 l de PBS utilizando una pipeta. </li><li> Poner las capas cortadas en 100 l de PBS en la placa de 96 pocillos y la etiqueta tanto en la orientación (hacia extern y pasante) y la capa (superficial y profunda), por ejemplo con un color resistente al agua. </li><li> Repita los pasos 2.7-2.9 hasta que el tejido se lamina completamente. &lt;/ Li&gt; </ol> 3. Realizar inmunohistoquímica <ol><li> Añadir 100 l de los anticuerpos primarios requeridos en la dilución recomendada. Aquí, por ejemplo, uso de CD 31 anticuerpo LYVE1 (de conejo anti humano) (ratón monoclonal anti humano) y, tanto en una dilución de 1: 100 en PBS (que contiene 2% de BSA) y se incuba a 4 ° C durante la noche. Para cambiar el medio en la placa de 96 pocillos, mantenga la pipeta a la pared del pozo y extraer el líquido. </li><li> El día siguiente, lavar las muestras tres veces cada una durante 5 minutos con 200 a 300 l de PBS en el agitador. Añadir 100 l de los anticuerpos secundarios correspondientes; aquí utilizar de cabra anti ratón isotiocianato de fluoresceína (FITC) y de cabra anti conejo colorantes de cianina 3 (Cy3), y se incuban las muestras a temperatura ambiente durante 1-2 horas. Diluir los anticuerpos secundarios 1: 300 en PBS que contenía 2% de suero de cabra. </li><li> Enjuagar las muestras de nuevo tres veces cada una durante 5 minutos en 200 a 300 l de PBS en el agitador. </li><li> Realizar tinción núcleo, por ejemplo, </em&gt;. 4 &#39;, 6-diamidino-2-fenilindol (DAPI) (dilución 1: 2000, 100 l en cada pocillo e incubar 10 min a temperatura ambiente), a continuación, lavar de nuevo 2 veces en 200 a 300 l de PBS en el agitador. </li><li> La transferencia de las muestras en portaobjetos de microscopio, incrustarlos en 1-2 gotas de medio de montaje fluorescente, añadir un cubreobjetos, sellarlo, cubriendo los bordes con barniz transparente y se almacena a 4 ° C para su uso posterior o examinar directamente las diapositivas con el confocal microscopio. </li><li> Use un microscopio confocal (o equivalente) para analizar las muestras. Utilice la ampliación deseada, por ejemplo 10-40x ampliación. </li><li> Para verificar el espesor de las muestras, medir con microscopía confocal utilizando Z-pilas. La cantidad de posibles secciones esclerales depende de la ubicación de la esclerótica, como el espesor difiere ecuatorial y posterior (comparar introducción). </li></ol>

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The authors declare no financial disclosures. The contents of this article are solely the responsibility of the authors.

Laminar la esclerótica humana es un método para realizar la microscopía confocal en este tejido. Un paso crítico en este proceso es el uso de etanol en lugar de formalina para fijar el tejido. En nuestra experiencia, los mejores resultados se obtienen cuando se utiliza etanol en lugar de formol para su fijación. escalpelos Blunt agravan el procedimiento y se deben evitar. Del mismo modo, el agotamiento de la esclerótica debe evitarse, ya que complica el procedimiento y reduce la calidad de las imágenes. En cuanto a la tinción inmunohistoquímica, otros anticuerpos que las aplicadas aquí pueden ser utilizados. En general, el colágeno muestra más autofluorescencia y trayectoria en el canal verde, por lo tanto, rojo, azul, y los canales de rojo lejano mostrar mejores resultados con menos fondo. Sin embargo, hay varias limitaciones de la técnica. Laminar la esclerótica requiere mucha práctica, ya que es un procedimiento microquirúrgico realizado bajo un Microscaire libre. Debido a que esta técnica se realiza manualmente, las rebanadas individuales laminados no son exactamente del mismo tamaño y difieren en su espesor dentro de una pieza de tejido. Para que los tejidos de exactamente el mismo tamaño, la trepanación podría ser una alternativa, aunque el espesor de las capas todavía puede variar. Para lograr un espesor exactamente definido para las secciones esclerales, un microqueratomo automatizado podría ser una alternativa para futuros experimentos. Si se realiza la laminación con capas que son demasiado gruesas, la corredera de cubierta puede dislocar, y el tejido potencialmente puede secar. Las fronteras exactas entre epiesclerótica y el estroma pueden ser difíciles de diferenciar una vez que se realizan las laminaciones. A pesar de esto, la laminación de la esclerótica tiene varias ventajas en el uso de inmunohistoquímica, particularmente cuando se compara con parafina inmunohistoquímica fijado con formalina, que es el método estándar en la situación clínica actual. Laminar la esclerótica permite Microsc confocalopy a realizar, las áreas de mayor tamaño de la esclerótica a ser analizados, diferentes capas de la esclerótica a ser examinado, así como la detección y la exploración de tejido. Por el contrario, cuando se trabaja con muestras incluidas en parafina fijados con formalina, la esclerótica cambia su consistencia de una manera poco útil durante el precalentamiento para la recuperación de antígeno. Durante todo el proceso de calentamiento, las fibras de colágeno se arrugan y el tejido pierde el contacto con la corredera. Anteriormente, el plexo buque en la epiesclerótica haber sido sólo visible mediante el uso de técnicas angiográficas o en la visión de conjunto de montaje, pero no en las secciones transversales. Además de las dificultades técnicas, la esclerótica es relativamente avascular y relativamente pobre en las células inmunes en comparación con otros tejidos en el ojo, tales como la retina. Por lo tanto, al utilizar las diapositivas incluidas en parafina fijadas con formalina, que son por lo general de 4 micras de espesor, múltiples diapositivas son necesarios para detectar células positivas en la esclerótica. Recientemente, se ha demostrado el uso de esta laminatción técnica que la esclerótica humano no es acelular, pero contiene gran cantidad de macrófagos LYVE1 + a acumularse alrededor de los vasos sanguíneos 1. En resumen, la realización de la microscopía confocal en secciones esclerales laminado es una herramienta prometedora para investigar trastornos patológicos en el futuro, tales como escleritis, uveítis, o trauma.

La esclerótica es la capa exterior rígida que cubre el ojo, que está hecha de tejido conectivo denso. Ayuda a proteger las estructuras intraoculares y para mantener la presión intraocular. Por lo tanto, la esclerótica es esencial para la visión clara. Está desprovisto de vasos linfáticos 1,2 y de ese modo forma un borde libre-linfático exterior entre él y el ojo interior libre de linfático 3-7. También proporciona sitios de unión para los músculos extraoculares, compartiendo así similitudes anatómicas con tendones. Debido a que la esclerótica se compone principalmente de densos haces de colágeno de tipo I y tiene un número menor de tipos de colágeno III, IV, V, VI, VIII 8,9 y elastina 10,11, este tejido no es fácil de usar para inmunohistoquímica. Anatómicamente, la esclerótica se puede separar en tres capas principales: (1) la epiesclerótica vascularizado superficial, que se encuentra debajo de la conjuntiva y la cápsula de Tenon y hacia los lados y THCuenta E del ojo hacia la órbita; (2) el estroma escleral, la parte principal de la esclerótica; y (3) la lámina fusca, que es una capa delgada, pigmentada situado directamente encima de la úvea. Nuestro conocimiento anatómico sobre la esclerótica se deriva principalmente de la primera mitad del siglo 20. En ese momento, los investigadores estudiaron la anatomía de la vasculatura principalmente mediante el uso de inyecciones de tinta India 12 y vasculares fundición 13-15. Más tarde, se investigó en estudios angiográficos 16-19. Desde ese momento, las técnicas más antiguas se han mejorado y otros nuevos han sido desarrollados que nos han permitido complementar los conocimientos anatómicos anterior. Por ejemplo, ha sido solamente alrededor de una década desde que hemos tenido este tipo de marcadores linfáticos fiables como el endotelio vascular linfática hialuronano específico del receptor-1 (LYVE1) 20 o 21 podoplanin. La microscopía confocal ofrece nuevas posibilidades para el estudio de las características anatómicas de los diferentes tiUNDIALES del ojo. Permite múltiples manchas a ser utilizados para diferenciar los marcadores de células o para la localización de las células en relación a los vasos sanguíneos y otras estructuras anatómicas. Proporciona una visión general cuando la muestra es de un tamaño más grande y nos permite escanear a través de una muestra cuando en busca de un tipo específico de célula. Con la tecnología Z-Stack, microscopía confocal se puede utilizar para muestras de hasta 100 a 200 micras. La esclerótica se diferencia de espesor entre 0,3 mm detrás de las inserciones musculares y 1 mm en el polo posterior 11. Debido tanto a su espesor y opacidad, la esclerótica no es adecuado para microscopía confocal utilizando métodos tradicionales. Para remediar esto, los tejidos esclerales se laminaron para permitir su análisis con el microscopio confocal. Esta tecnología es útil para obtener una mejor comprensión de las dos situaciones fisiológicas y patológicas en la esclerótica humana.

<table>
	<tbody>
		<tr>
			<td>96% ethanol</td>
			<td>Merck Chemicals, Darmstadt, Germany</td>
			<td>P075.4</td>
			<td></td>
		</tr>
		<tr>
			<td>binocular stereo microscope&#160;</td>
			<td>Motic, Hongkong, China</td>
			<td>n.a</td>
			<td></td>
		</tr>
		<tr>
			<td>26G needles&#160;</td>
			<td>Terumo, Leuven, Belgium</td>
			<td>303800</td>
			<td></td>
		</tr>
		<tr>
			<td>15.5mm trepan</td>
			<td>Geuder, Heidelberg, Germany</td>
			<td>n.a</td>
			<td></td>
		</tr>
		<tr>
			<td>no.10 scalpel&#160;</td>
			<td>Feather, pfm medical, Osaka, Japan</td>
			<td>2E+08</td>
			<td></td>
		</tr>
		<tr>
			<td>ophthalmic scalpel micro feather&#160;</td>
			<td>Feather, pfm medical, Osaka, Japan</td>
			<td colspan="2">no. 7657BR</td>
		</tr>
		<tr>
			<td>CD 31 antibody (monoclonal mouse anti human)</td>
			<td>Dako, USA</td>
			<td>IR610</td>
			<td></td>
		</tr>
		<tr>
			<td>LYVE1 antibody&#160; (polyclonal rabbit anti human)</td>
			<td>Zytomed, Germany</td>
			<td colspan="2">RBK014-05</td>
		</tr>
		<tr>
			<td>goat anti mouse FITC antibody</td>
			<td>Sigma Aldrich, Steinheim, Germany</td>
			<td>F0257</td>
			<td></td>
		</tr>
		<tr>
			<td>goat anti rabbit Cy3 antibody</td>
			<td>Dianova, Germany</td>
			<td colspan="2">111-165-003</td>
		</tr>
		<tr>
			<td>Goat Serum normal</td>
			<td>Dako, Glostrup, Denmark</td>
			<td colspan="2">X090710-8</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Carl Roth GmbH, Karlsruhe, Germany</td>
			<td>6335.1</td>
			<td></td>
		</tr>
		<tr>
			<td>microscope slides&#160;</td>
			<td>Engelbrecht, Ederm&#252;nde, Germany</td>
			<td colspan="2">WC7695002</td>
		</tr>
		<tr>
			<td>Coverslips 24x24mm</td>
			<td>Th. Gayer, Lohmar, Germany</td>
			<td>7695026</td>
			<td></td>
		</tr>
		<tr>
			<td>DAKO fluorescent mounting medium&#160;</td>
			<td>DAKO, USA</td>
			<td>S3023</td>
			<td></td>
		</tr>
		<tr>
			<td>LSM Meta 510 confocal microscopy&#160;</td>
			<td>Carl Zeiss AG, Jena, Germany</td>
			<td>n.a</td>
			<td></td>
		</tr>
	</tbody>
</table>

using a laminating technique to perform confocal microscopy of the human sclera

La esclerótica es un tejido conectivo denso que cubre y protege el ojo. Se compone principalmente de fibras de colágeno denso (tipos I, III, IV, V, VI, y VII). Debido a su autofluorescencia, opacidad, y el espesor, no se ha encontrado adecuado para microscopía confocal. Un enfoque alternativo a la presentada aquí, que utiliza esclerótica fijado con formalina se incluyeron en parafina para inmunohistoquímica, tiene desafíos técnicos, especialmente cuando el precalentamiento del tejido para la recuperación de antígeno. Desde la esclerótica es relativamente pobre en ambas células y vasos, se exploró el uso de muestras de tejido más grandes para ayudar a prevenir que las células vistas y para entender su localización en relación con los buques y otros sitios anatómicos. Para permitir el análisis de muestras de tejido más grandes en el microscopio confocal, una técnica de laminación se realizó para crear capas delgadas de la esclerótica. Tras el análisis de los resultados de los vasos sanguíneos CD31 y hyalu endotelial de vasos linfáticosRonan receptor 1 (LYVE1) células positivas, para lo cual se obtuvo la aprobación para el examen científico, se discuten las ventajas y limitaciones de este método.

En los experimentos representativos realizados aquí, hay beneficios demostrables derivados de la utilización de esta técnica de laminación particular. El primer experimento ilustra la red diversa del plexo vaso sanguíneo episcleral en tres imágenes representativas (figura 3). Los vasos son positivas para CD31. El segundo experimento muestra las células inmunes, en particular las células LYVE1 + de la epiesclerótica y su relación con los vasos sanguíneos CD31 positiva. Aquí tecnología Z-Stack se utilizó a un aumento de 10X para escanear a través del tejido y comprender las relaciones tridimensionales de los vasos sanguíneos y las células inmunes (Figura 4). La esclerótica, así como los músculos extraoculares cerrados, pueden ser analizados para la presencia de vasos sanguíneos o células inmunes. La figura 5 muestracélulas positivas CD31 LYVE1 y vasos sanguíneos positivos. <img alt="Figura 1" src="/files/ftp_upload/53920/53920fig1.jpg" /> Figura 1:.. Imagen esquemática del ojo humano Aquí es un esquema de la sección transversal del ojo humano <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53920/53920fig1large.jpg" target="_blank">favor, haga clic aquí para ver una versión más grande de esta figura.</a> <img alt="Figura 2" src="/files/ftp_upload/53920/53920fig2.jpg" /> Figura 2:. Imagen esquemática de la técnica de laminado de la esclerótica se lleva a cabo cuidadosamente con unas pinzas colibri y laminado en capas finas utilizando un bisturí. La repetición de este paso conduce a portaobjetos de tejido delgado que se pueden utilizar para la microscopía confocal. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53920/53920fig2large.jpg" target="_blank">Por favor, haga clic aquí para ver una versión más grande de esta fi</a>gura. <img alt="figura 3" src="/files/ftp_upload/53920/53920fig3.jpg" /> Figura 3:. Episcleral Plexus Vaso sanguíneo, inmunopositivas para CD31 A y B se derivan de la epiesclerótica anterior, mientras que C es desde la ubicación posterior. La barra de escala indica 100 micras. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53920/53920fig3large.jpg" target="_blank">Por favor, haga clic aquí para ver una versión más grande de esta figura.</a> <img alt="Figura 4" src="/files/ftp_upload/53920/53920fig4.jpg" /> Figura 4:. Vasos sanguíneos CD31 + y + LYVE1 células en una pila Z, Resultados las relaciones anatómicas entre ellos vasos pequeños y los más grandes pueden solaparse. La barra de escala indica 100 micras. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53920/53920fig4large.jpg" target="_blank">Por favor, haga clic aquí para ver una versión más grande de esta figura.</a> Figura 5: Encerrado en la esclerótica son los músculos extraoculares Contienen una red de vasos sanguíneos finos similar y algunas células LYVE1 +.. La barra de escala indica 200 micras. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53920/53920fig5large.jpg" target="_blank">Por favor, haga clic aquí para ver una versión más grande de esta figura.</a>

Watch this Scientific Journal Video about Using a Laminating Technique to Perform Confocal Microscopy of the Human Sclera at JoVE.com

Using a Laminating Technique to Perform Confocal Microscopy of the Human Sclera

Human sclera tissue is mainly collagen; therefore, it is not easily usable for immunohistochemistry. To achieve the goal of performing immunohistochemistry for confocal microscopy of scleral tissue, a laminating technique was used.

Usando una técnica de laminación para realizar la microscopía confocal de la esclerótica humana


The sclera is a dense connective tissue that covers and protects the eye. It mainly consists of dense collagen bundles (types I, III, IV, V, VI, and ...

using-laminating-technique-to-perform-confocal-microscopy-human

Universidad San Sebastian

Research

JoVE Journal

Medicine

7.5K vistas.  University of Cologne. Human sclera tissue is mainly collagen; therefore, it is not easily usable for immunohistochemistry. To achieve the goal of performing immunohistochemistry for confocal microscopy of scleral tissue, a laminating technique was used. 

Video: Using a Laminating Technique to Perform Confocal Microscopy of the Human Sclera

Corneal Confocal Microscopy: A Novel Non-invasive Technique to Quantify Small Fibre Pathology in Peripheral Neuropathies

The accurate quantification of peripheral neuropathy is important to define at risk patients, anticipate deterioration, and assess new therapies. Conventional methods assess neurological deficits and electrophysiology and quantitative sensory testing quantifies functional alterations to detect neuropathy. However, the earliest damage appears to be to the small fibres and yet these tests primarily assess large fibre dysfunction and have a limited ability to demonstrate regeneration and repair. The only techniques which allow a direct examination of unmyelinated nerve fibre damage and repair are sural nerve biopsy with electron microscopy and skin-punch biopsy. However, both are invasive procedures and require lengthy laboratory procedures and considerable expertise. Corneal Confocal microscopy is a non-invasive clinical technique which provides in-vivo imaging of corneal nerve fibres. We have demonstrated early nerve damage, which precedes loss of intraepidermal nerve fibres in skin biopsies together with stratification of neuropathic severity and repair following pancreas transplantation in diabetic patients. We have also demonstrated nerve damage in idiopathic small fibre neuropathy and Fabry's disease.

Corneal Confocal microscopy is a non-invasive clinical technique which may be used to quantify C fibre damage to diagnose and stratify patients with increasing neuropathic severity.

Microscopía Confocal de córnea: A Novel una técnica no invasiva para cuantificar pequeñas de fibra de Patología en neuropatías periféricas

The accurate quantification of peripheral neuropathy is important to define at risk patients, anticipate deterioration, and assess new therapies. ...

Investigación

Medicina

Utilizing Transcranial Magnetic Stimulation to Study the Human Neuromuscular System

Transcranial magnetic stimulation (TMS) has been in use for more than 20 years 1, and has grown exponentially in popularity over the past decade. While the use of TMS has expanded to the study of many systems and processes during this time, the original application and perhaps one of the most common uses of TMS involves studying the physiology, plasticity and function of the human neuromuscular system. Single pulse TMS applied to the motor cortex excites pyramidal neurons transsynaptically 2 (Figure 1) and results in a measurable electromyographic response that can be used to study and evaluate the integrity and excitability of the corticospinal tract in humans 3. Additionally, recent advances in magnetic stimulation now allows for partitioning of cortical versus spinal excitability 4,5. For example, paired-pulse TMS can be used to assess intracortical facilitatory and inhibitory properties by combining a conditioning stimulus and a test stimulus at different interstimulus intervals 3,4,6-8. In this video article we will demonstrate the methodological and technical aspects of these techniques. Specifically, we will demonstrate single-pulse and paired-pulse TMS techniques as applied to the flexor carpi radialis (FCR) muscle as well as the erector spinae (ES) musculature. Our laboratory studies the FCR muscle as it is of interest to our research on the effects of wrist-hand cast immobilization on reduced muscle performance6,9, and we study the ES muscles due to these muscles clinical relevance as it relates to low back pain8. With this stated, we should note that TMS has been used to study many muscles of the hand, arm and legs, and should iterate that our demonstrations in the FCR and ES muscle groups are only selected examples of TMS being used to study the human neuromuscular system.

Transcranial magnetic stimulation (TMS) is a non-invasive tool to gain insight on the physiology and function of the human nervous system. Here, we present our TMS techniques to study cortical excitability of the upper limb and lumbar musculature.

Utilizando estimulación magnética transcraneal para Estudiar el Sistema Neuromuscular Humanos

Transcranial magnetic stimulation (TMS) has been in use for more than 20 years 1, and has grown exponentially in popularity over the past decade.  While ...

Probe-based Confocal Laser Endomicroscopy of the Urinary Tract: The Technique

Probe-based confocal laser endomicroscopy (CLE) is an emerging optical imaging technology that enables real-time in vivo microscopy of mucosal surfaces during standard endoscopy. With applications currently in the respiratory1 and gastrointestinal tracts,2-6 CLE has also been explored in the urinary tract for bladder cancer diagnosis.7-10 Cellular morphology and tissue microarchitecture can be resolved with micron scale resolution in real time, in addition to dynamic imaging of the normal and pathological vasculature.7
The probe-based CLE system (Cellvizio, Mauna Kea Technologies, France) consists of a reusable fiberoptic imaging probe coupled to a 488 nm laser scanning unit. The imaging probe is inserted in the working channels of standard flexible and rigid endoscopes. An endoscope-based CLE system (Optiscan, Australia), in which the confocal endomicroscopy functionality is integrated onto the endoscope, is also used in the gastrointestinal tract. Given the larger scope diameter, however, application in the urinary tract is currently limited to ex vivo use.11 Confocal image acquisition is done through direct contact of the imaging probe with the target tissue and recorded as video sequences. As in the gastrointestinal tract, endomicroscopy of the urinary tract requires an exogenenous contrast agent&mdash;most commonly fluorescein, which can be administered intravenously or intravesically. Intravesical administration is a well-established method to introduce pharmacological agents locally with minimal systemic toxicity that is unique to the urinary tract. Fluorescein rapidly stains the extracellular matrix and has an established safety profile.12 Imaging probes of various diameters enable compatibility with different caliber endoscopes. To date, 1.4 and 2.6 mm probes have been evaluated with flexible and rigid cystoscopy.10 Recent availability of a &lt; 1 mm imaging probe13 opens up the possibility of CLE in the upper urinary tract during ureteroscopy. Fluorescence cystoscopy (i.e. photodynamic diagnosis) and narrow band imaging are additional endoscope-based optical imaging modalities14 that can be combined with CLE to achieve multimodal imaging of the urinary tract. In the future, CLE may be coupled with molecular contrast agents such as fluorescently labeled peptides15 and antibodies for endoscopic imaging of disease processes with molecular specificity.

Probe-based confocal laser endomicroscopy enables real-time microscopy of the human urinary tract during cystoscopy, providing dynamic, intravital imaging of pathological states such as bladder cancer with cellular resolution. Endomicroscopy may augment the diagnostic accuracy of standard white light endoscopy and provide intraoperative image guidance to improve surgical resection.

Basada en sondeos endomicroscopía Láser Confocal del tracto urinario: la técnica

Probe-based  confocal laser endomicroscopy (CLE) is an emerging optical imaging technology  that enables real-time in vivo microscopy of mucosal surfaces ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

Excitotóxica Estimulación del cerebro Microslices como<em&gt; In vitro</em&gt; Modelo de Carrera

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Human Skeletal Muscle Biopsy Procedures Using the Modified Bergstr&#246;m Technique

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly effective. This video describes the percutaneous biopsy technique using a modified Bergstr&#246;m needle to obtain skeletal muscle tissue samples from the vastus lateralis of human subjects. The Bergstr&#246;m needle consists of an outer cannula with a small opening (&#8216;window&#8217;) at the side of the tip and an inner trocar with a cutting blade at the distal end. Under local anesthesia and aseptic conditions, the needle is advanced into the skeletal muscle through an incision in the skin, subcutaneous tissue, and fascia. Next, suction is applied to the inner trocar, the outer trocar is pulled back, skeletal muscle tissue is drawn into the window of the outer cannula by the suction, and the inner trocar is rapidly closed, thus cutting or clipping the skeletal muscle tissue sample. The needle is rotated 90&#176;&#160;and another cut is made. This process may be repeated three more times. This multiple cutting technique typically produces a sample of 100-200 mg or more in healthy subjects and can be done immediately before, during, and after a bout of exercise or other intervention. Following post-biopsy dressing of the incision site, subjects typically resume their activities of daily living right away and can fully participate in vigorous physical activity within 48-72 hr. Subjects should avoid heavy resistance exercise for 48 hr to reduce the risk of herniation of the muscle through the incision in the fascia.

The purpose of this video is to present the modified Bergstr&#246;m skeletal muscle biopsy technique on human subjects.

Procedimientos de biopsia de músculo esquelético humano utilizando la técnica de Bergström Modificado

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly ...

Construction of Vapor Chambers Used to Expose Mice to Alcohol During the Equivalent of all Three Trimesters of Human Development

Exposure to alcohol during development can result in a constellation of morphological and behavioral abnormalities that are collectively known as Fetal Alcohol Spectrum Disorders (FASDs). At the most severe end of the spectrum is Fetal Alcohol Syndrome (FAS), characterized by growth retardation, craniofacial dysmorphology, and neurobehavioral deficits. Studies with animal models, including rodents, have elucidated many molecular and cellular mechanisms involved in the pathophysiology of FASDs. Ethanol administration to pregnant rodents has been used to model human exposure during the first and second trimesters of pregnancy. Third trimester ethanol consumption in humans has been modeled using neonatal rodents. However, few rodent studies have characterized the effect of ethanol exposure during the equivalent to all three trimesters of human pregnancy, a pattern of exposure that is common in pregnant women. Here, we show how to build vapor chambers from readily obtainable materials that can each accommodate up to six standard mouse cages. We describe a vapor chamber paradigm that can be used to model exposure to ethanol, with minimal handling, during all three trimesters. Our studies demonstrate that pregnant dams developed significant metabolic tolerance to ethanol. However, neonatal mice did not develop metabolic tolerance and the number of fetuses, fetus weight, placenta weight, number of pups/litter, number of dead pups/litter, and pup weight were not significantly affected by ethanol exposure. An important advantage of this paradigm is its applicability to studies with genetically-modified mice. Additionally, this paradigm minimizes handling of animals, a major confound in fetal alcohol research.

We demonstrate the construction of alcohol vapor chambers using readily available materials that simultaneously house 6 mouse cages. We further describe their use in a mouse model of fetal alcohol exposure equivalent to all 3&#160;trimesters of human pregnancy. This paradigm exposes animals during gestation and postnatal days 1-12.

Construcción de cámaras de vapor utiliza para exponer los ratones al alcohol durante el equivalente de los tres trimestres del Desarrollo Humano

Exposure to alcohol during development can result in a constellation of morphological and behavioral abnormalities that are collectively known as Fetal ...

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo: A Model of Single Vessel Stroke

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to image cellular events in an intact animal. Additionally, the ability to induce physiological disease states such as stroke in vivo increases its utility. The technique described herein allows for physiological assessment of cellular responses within the CNS following a stroke and can be adapted for other pathological conditions being studied. The technique presented uses laser excitation of the photosensitive dye Rose Bengal in vivo to induce a focal ischemic event in a single blood vessel. 
The video protocol demonstrates the preparation of a thin-skulled cranial window over the somatosensory cortex in a mouse for the induction of a Rose Bengal photothrombotic event keeping injury to the underlying dura matter and brain at a minimum. Surgical preparation is initially performed under a dissecting microscope with a custom-made surgical/imaging platform, which is then transferred to a confocal microscope equipped with an inverted objective adaptor. Representative images acquired utilizing this protocol are presented as well as time-lapse sequences of stroke induction. This technique is powerful in that the same area can be imaged repeatedly on subsequent days facilitating longitudinal in vivo studies of pathological processes following stroke.

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo:  A Model of Single Vessel Stroke

Here, we describe a semi-invasive optical microscopy approach for the induction of a Rose Bengal photothrombotic clot in the somatosensory cortex of a mouse in vivo. The technical aspects of the imaging procedure are described from induction of a photothrombotic event to application and data collection.

Rosa de Bengala Photothrombosis por imagen óptica confocal<em&gt; En Vivo</em&gt;: Un modelo de solo recipiente Stroke

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to ...

Development of an Algorithm to Perform a Comprehensive Study of Autonomic Dysreflexia in Animals with High Spinal Cord Injury Using a Telemetry Device

Spinal cord injury (SCI) is a debilitating neurological condition characterized by somatic and autonomic dysfunctions. In particular, SCI above the mid-thoracic level can lead to a potentially life-threatening hypertensive condition called autonomic dysreflexia (AD) that is often triggered by noxious or non-noxious somatic or visceral stimuli below the level of injury. One of the most common triggers of AD is the distension of pelvic viscera, such as during bladder and bowel distension or evacuation. This protocol presents a novel pattern recognition algorithm developed for a JAVA platform software to study the fluctuations of cardiovascular parameters as well as the number, severity and duration of spontaneously occurring AD events. The software is able to apply a pattern recognition algorithm on hemodynamic data such as systolic blood pressure (SBP) and heart rate (HR) extracted from telemetry recordings of conscious and unrestrained animals before and after thoracic (T3) complete transection. With this software, hemodynamic parameters and episodes of AD are able to be detected and analyzed with minimal experimenter bias.

The catheter of a telemetry device is implanted into the abdominal aorta in order to continuously collect beat-by-beat hemodynamic data from animals pre and post-high thoracic spinal cord transection. A novel JAVA software was employed to analyze hemodynamic parameters as well as frequency and intensity of spontaneous episodes of autonomic dysreflexia.

Desarrollo de un algoritmo de realizar un estudio exhaustivo de Disreflexia Autónoma en animales con lesión medular alta El uso de un dispositivo de telemetría

Spinal cord injury (SCI) is a debilitating neurological condition characterized by somatic and autonomic dysfunctions. In particular, SCI above the ...

Topical Airway Anesthesia for Awake-endoscopic Intubation Using the Spray-as-you-go Technique with High Oxygen Flow

A patient&#39;s willingness to cooperate is an absolute precondition for successful awake intubation of the trachea. Whilst drug-sedation of patients can jeopardize their spontaneous breathing, topical anesthesia of the airway is a popular technique. The spray-as-you-go technique represents one of the simplest opportunities to anesthetize the airway mucosa. The application of local anesthetic through the working channel of the flexible endoscope is a widespread practice for anesthetists as well as pulmonologists. There is neither need for additional devices nor special training as a pre-requisite to perform this technique. However, a known clinical problem is the coughing and gagging reflex that may occur when the liquid anesthetic strikes the airway mucosa and other sensitive structures like the vocal cords. This can be avoided by the use of oxygen applied through the working channel with the aim of fogging the local anesthetic into finer particles. Furthermore, the oxygen flow provides a higher oxygen supply and contributes to a better view, dispersing mucus secretions and blood away from the lens. Using an atomizer with a high oxygen flow of 10 L/min we maximized these benefits, caused less coughing and had more satisfied and therefore cooperative patients. Possible, but very rare complications of using oxygen flow including gastric insufflation, organ rupture or barotrauma did not arise. We attribute the complication-free use of high oxygen flow to the design of the set, which permits flow and pressure release.

The topical anesthetic lidocaine was atomized using a high oxygen flow through the working channel of a flexible intubating endoscope to achieve topical airway anesthesia for awake endotracheal intubation. We prefer this modified spray-as-you-go technique for endoscopic intubation to classical bolus application because of higher patient satisfaction and better compliance.

Tópica de la vía aérea La anestesia para la intubación despierto-endoscópica de usar el spray-as-you-go Técnica con alto caudal de oxígeno

A patient&#39;s willingness to cooperate is an absolute precondition for successful awake intubation of the trachea. Whilst drug-sedation of patients can ...

Second Harmonic Generation Signals in Rabbit Sclera As a Tool for Evaluation of Therapeutic Tissue Cross-linking (TXL) for Myopia

Methods to strengthen tissue by introducing chemical bonds (non-enzymatic cross-linking) into structural proteins (fibrillar collagens) for therapy include photochemical cross-linking and tissue cross-linking (TXL) methods. Such methods for inducing mechanical tissue property changes are being employed to the cornea in corneal thinning (mechanically weakened) disorders such as keratoconus as well as the sclera in progressive myopia, where thinning and weakening of the posterior sclera occurs and likely contributes to axial elongation. The primary target proteins for such tissue strengthening are fibrillar collagens which constitute the great majority of dry weight proteins in the cornea and sclera. Fortuitously, fibrillar collagens are the main source of second harmonic generation signals in the tissue extracellular space. Therefore, modifications of the collagen proteins, such as those induced through cross-linking therapies, could potentially be detected and quantitated through the use of second harmonic generation microscopy (SHGM). Monitoring SHGM signals through the use of a laser scanning microscopy system coupled with an infrared excitation light source is an exciting modern imaging method that is enjoying widespread usage in the biomedical sciences. Thus, the present study was undertaken in order to evaluate the use of SHGM microscopy as a means to measure induced cross-linking effects in ex vivo rabbit sclera, following an injection of a chemical cross-linking agent into the sub-Tenon&#39;s space (sT), an injection approach that is standard practice for causing ocular anesthesia during ophthalmologic clinical procedures. The chemical cross-linking agent, sodium hydroxymethylglycinate (SMG), is from a class of cosmetic preservatives known as formaldehyde releasing agents (FARs). Scleral changes following reaction with SMG resulted in increases in SHG signals and correlated with shifts in thermal denaturation temperature, a standard method for evaluating induced tissue cross-linking effects.

Second Harmonic Generation Signals in Rabbit Sclera As a Tool for Evaluation of Therapeutic Tissue Cross-linking TXL for Myopia

This protocol describes techniques for evaluating chemical cross-linking of the rabbit sclera using&#160;second harmonic generation imaging and differential scanning calorimetry.

Segunda generación armónica las señales en esclera de conejo como una herramienta para la evaluación del tejido terapéutico Cross-linking (TXL) para la miopía

Methods to strengthen tissue by introducing chemical bonds (non-enzymatic cross-linking) into structural proteins (fibrillar collagens) for therapy ...