Supported by NIH award R01 DK093525 (GFG). We greatly appreciate the scientific review and grammatical editing of the manuscript by Dr. Amber Shaffer (University of Pittsburgh) and thank Michael Burcham for assistance in preparation of figures.

NOTA: Este protocolo fue revisado y aprobado por la Universidad de Pittsburgh Institucional Cuidado de Animales y el empleo Comisión. 1. Preparación de la solución de Krebs modificada y Prueba de la Droga alícuotas <ol><li> Hacer 6 L de solución de Krebs modificada que contiene (en mM): NaCl 117,9, KCl 4,7, 25 NaHCO 3, 1,3 NaH 2 PO 4, 1.2 MgSO 4, 2.5 CaCl 2, 11,1 D-glucosa, butirato de 2 de sodio, acetato de sodio 20 , 0.004 nifedipina (para bloquear las contracciones musculares espontáneas), y 0,003 de indometacina (para bloquear la síntesis de prostaglandinas endógenas). Utilice soluciones de Krebs hielo frío y caliente para la disección de tejidos y la grabación de una sola fibra, respectivamente. </li><li> Preparar las soluciones químicas (por ejemplo, IS, la capsaicina, ATP) en alícuotas a concentraciones deseadas. </li></ol> 2. La disección del nervio del tejido colorrectal- <ol><li> Anestesiar y eutanasia ratones machos (6-8 semanas de edad, 20 - 30 g) enuna cámara de CO 2 a un caudal que desplaza 10 a 30% de volumen de la cámara por minuto hasta que los ratones deje de respirar por completo como se indica por la ausencia de movimiento del pecho. </li><li> Inmediatamente después de la eutanasia, Exsanguinate cortando abierta la cámara torácica, perforando la aurícula derecha, y sumergir el cadáver del ratón en un amplio volumen (~ 500 ml) de helado de Krebs (4 ° C) solución burbujea con carbógeno (95% de O2 , 5% de CO 2). </li><li> Retire con cuidado todas las vísceras, pero los órganos de colon y de la pelvis. Transecto el ratón en un medio a través del segmento vertebral T12 ligeramente por encima del diafragma torácico y transferir la mitad caudal a una cámara de disección que contiene hielo frío, burbujear solución de Krebs. </li><li> Bajo un microscopio estereoscópico, retire la vejiga y los órganos reproductivos por transección en sus uniones a la uretra, y retire la descendente / aorta abdominal hasta que se bifurca en las arterias ilíacas comunes. Libertad para los PN o el LSN de su surrounding tejidos mediante disección roma y seguir el nervio desde fuera de la cresta ilíaca hasta su punto de entrada ventral en la L6 y S1 columna vertebral (por PN) o T13 y L1 columna vertebral (por LSN). </li><li> Cortar la sínfisis púbica y la derecha y articulaciones acetábulo izquierdo, y quitar el hueso ilíaco. Con cuidado, libre ya sea el PN o LSN de los músculos adheridos y tejido conectivo de cerca del colon y recto hasta donde el nervio entra en la columna vertebral. </li><li> Resecar con cuidado el hueso ilíaco para exponer el colon y recto distal. Diseccionar el colon y recto distal con el PN adjunto o LSN en continuo. </li><li> Transferir el colon y recto con el nervio unido al compartimento de baño de la cámara de tejido. Quitar el tejido conectivo excesivo por más de disección, y abrir el colon y recto longitudinalmente a lo largo de la frontera anti-mesentérica. </li><li> Con el lado de la mucosa hacia arriba, el pin del borde mesentérico del colon y recto junto al compartimento de registro en la base de silicona de la chamber y adjuntar la longitud del colon y recto antimesenteric a un rastrillo de ganchos conectados a un accionador de fuerza (ilustrado en la Figura 1 y se fotografió en la Figura 2A). </li><li> Extender el PN o LSN en el compartimento de la grabación, que está conectada al compartimiento de baño por un agujero del ratón y la puerta. Coloque suavemente el tronco nervioso a un pequeño espejo de cristal en el compartimiento de la grabación, lo que proporciona una superficie hidrófila para el nervio que adherirse. Superfuse el compartimiento de baño con agua caliente (30-32 ° C), solución de Krebs oxigenada y llenar el compartimento de grabación con aceite mineral. </li></ol> 3. sola fibra de grabación y localización del campo receptivo <ol><li> Retire con cuidado la espalda epineurio (vaina nerviosa) de la PN o LSN bajo el microscopio estereoscópico a gran aumento (50 - 60X). Con unas pinzas finas, se burlan del tronco nervioso en 5-8 haces nerviosos de ~ 100 micras de espesor. </li><li> Coloque la referencia de platino-iridioelectrodo en contacto con la solución de Krebs en la cámara de tejido. Colocar secuencialmente los haces de nervios individuales en el electrodo de registro hecha del mismo material. </li><li> Utilice un pincel suave para evocar APs de los aferentes colorrectales por acariciar suavemente hacia arriba y abajo de la superficie colorrectal. Busque el paquete (s) de nervios que inervan el colon a través de AP detectable (potencial de acción) grabaciones. NOTA: La PN y LSN también inervan la vejiga urinaria y otros órganos pélvicos. </li><li> Use un par de 30 puntas de las agujas G para dividir aún más el conjunto de nervios en filamentos finos fascículos de ~ 10 micras de espesor y colocar un filamento individual sobre el electrodo de registro. </li><li> Coloque el electrodo concéntrico de punta redonda perpendicular a la superficie de la mucosa para excitar eléctricamente terminaciones aferentes en la intensidad del estímulo por encima del umbral (10 mA magnitud, 0,5 mseg de duración @ 0,3 Hz), que produce un radio ~ 2 mm de propagación actual. Mueva el electrodo de forma sistemática (~ 1,5 mm pasos) A lo largo de la longitud y la anchura del colon y recto aplanado para localizar terminaciones receptivas. </li><li> Cuando se excita un final aferente, ajuste la posición del electrodo para localizar el sitio de activación (campo receptivo, RF) que requiere la intensidad del estímulo mínimo (umbral de estímulo). Deseche terminaciones con un umbral de estímulo&gt; 3 mA 19. </li><li> Calcular la velocidad de conducción (CV) a partir de 1) la distancia entre el electrodo de estimulación en el campo receptivo (RF) y el sitio de grabación y 2) el retraso de la conducción (por ejemplo, la Figura 2B) entre el artefacto del estímulo y el inicio del potencial de acción . CV (m / seg) = distancia (mm) / retraso de la conducción (ms). </li></ol> 4. Clasificación Funcional de mechanosensitive colorrectales Aferentes <ol><li> Después de localizar una RF mediante la estimulación eléctrica, aplique los tres estímulos mecánicos siguientes a la RF: <ol><li> Llevar a cabo el estímulo sondeo presionando la punta de una calibraciónmonofilamento ted von Frey-como el nylon (0,4 y 1 g fuerza) perpendicularmente hacia la RF en el colon y recto aplanada. </li><li> Llevar a cabo el estímulo acariciando por acariciar suavemente la mucosa colorrectal con un filamento de nylon fino (10 mg fuerza) para generar una pequeña tensión de corte superficial en la RF. </li><li> Realizar el tramo circunferencial utilizando una fuerza de accionamiento controlado por ordenador, que proporciona una fuerza de estiramiento en rampa (0-170 MN en 5 mN / seg) en dirección circunferencial a lo largo del borde anti-mesentérico del colon y recto a través de la comisión de ganchos que se describen en el paso 2.8 . </li></ol></li><li> Clasificar los aferentes como serosa (responder sólo a despuntar sondeo), la mucosa (responder a la mucosa acariciando y embotar sondeo), muscular (responder a tramo circunferencial y embotar sondeo) musculoso / mucosa (responder a tramo circunferencial, acariciando la mucosa y embotar sondeo), o MIA (no sensible a cualquiera de los tres estímulos mecánicos). </li><li> Para aferentes mesentéricas (sólo enla inervación LSN) que son difíciles de activar selectivamente mediante estimulación eléctrica, localizar sus terminaciones receptivas por caricias mecánica / sondeo del mesenterio. </li><li> Para aferentes sensibles al estiramiento (muscular y músculo-mucosas), determinar el umbral de respuesta, que se define como la fuerza que evoca la primera AP durante tramo en rampa. </li><li> Para los aferentes serosas, grabar sus respuestas a ascendiendo niveles de puntiforme de sondeo del campo receptivo impulsado por la fuerza del actuador controlado por ordenador. </li></ol> 5. Aplicación Química / Modulación de Endings Receptivo <ol><li> Grabar una respuesta de línea de base a un estímulo mecánico (es decir, la respuesta al tramo en rampa, punteada de sondeo, o acariciando la mucosa). </li><li> Escudo del borde inferior de un trozo de tubo (latón o acero inoxidable, 10 mm de alto y 4 x 4 mm 2 plaza o 4-5 mm de diámetro) con vaselina y colóquelo sobre el campo receptivo en el colon y recto. </li><li> Quitarla solución de Krebs dentro de la tubería, y exponer el final receptivo durante 3 - 5 min a 170 l de la solución que contiene la sustancia química (s) a ensayar. </li><li> Monitorear la respuesta de la aferente durante la aplicación química (algunas aferentes son quimiosensible). </li><li> Retire la solución química y la tubería para finalizar la operación de la sustancia química. Dentro de 4 - 6 min, probar la respuesta aferente al mismo estímulo mecánico como en la respuesta de línea base. </li><li> Volver a aplicar el estímulo mecánico de nuevo después de un período suficiente de wash-out (&gt; 15 min). </li></ol> 6. picos de grabación y Discriminar AP <ol><li> Digitalizar las señales eléctricas registradas de axones a 20 kHz y guardar los datos en una computadora. Monitorizar la señal en línea por un monitor de audio. </li><li> Analizar los picos de AP fuera de línea y discriminar unidades individuales basados ​​en el análisis de componentes principales de las formas de onda de pico individuales 25. NOTA: Un registro no debe contener más ésimoun dos unidades activas fácilmente discernibles. </li></ol>

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The authors have nothing to disclose.

El vitro preparación colon y recto-nervio en descrito aquí ha demostrado ser un poderoso enfoque para estudiar las funciones de codificación neural de aferentes colorrectales individuales, que complementa muy bien con otros métodos no funcionales (por ejemplo, estudios celulares, moleculares e histológicos) sobre las neuronas sensoriales viscerales ( ver el comentario 27 para más detalles). Mecanismos neuronales que contribuyen a la nocicepción y la hipersensibilidad colorrec...

El dolor y la hipersensibilidad son las quejas predominantes de pacientes que sufren de trastornos gastrointestinales funcionales, incluyendo el síndrome del intestino irritable (IBS), que existen en la ausencia de causa patobiológico aparente o daño tisular. Por ejemplo, los pacientes con SII presentan hipersensibilidad, incluyendo las respuestas aumentadas a la distensión rectal globo y aumento de la sensibilidad durante la función normal del intestino, así como hipersensibilidad de referencia somáticas (es decir, sensibilidad a la palpación de la zona abdominal) 1. Debido a la orientación aferentes colorrectal ha demostrado ser eficaz en el alivio de dolor e hipersensibilidad en los pacientes con SII (por ejemplo, la instilación intra-rectal de los anestésicos locales 2,3; ingestión oral de la guanilato ciclasa C agonista linaclotide 4-6), la mejora de la comprensión de la inervación aferente del colon y recto es importante. Aferentes viscerales aferentes, incluyendo colorrectal, Son capaces de responder a las modalidades químicas / en nutrientes y térmicos (por ejemplo, 7-9). Sin embargo, los aferentes viscerales que responden a estímulos mecánicos (es decir, aferentes mechanosensitive) han sido el más estudiado, porque los estímulos mecánicos (por ejemplo, distensión luminal, estrías) son los que generalmente dan lugar a sensaciones conscientes, incluyendo malestar y dolor 10-16. Además, las vísceras también están inervadas por fibras aferentes mecánicamente insensibles (MIAs), comúnmente denominadas nociceptores silenciosos o para dormir 17. En condiciones fisiológicas normales, MIAs no responden a la estimulación mecánica o tienen muy alta respuesta umbrales de 18, pero pueden llegar a ser activa y adquirir mechanosensitivity en condiciones fisiopatológicas y contribuir a la hipersensibilidad. Uso de la preparación in vitro y el protocolo descrito aquí, hemos desarrollado y empleamos una estrategia de estímulo eléctrico al mar<html> RCH para terminaciones receptivo, que permitan identificar imparcial de ambas terminaciones mechanosensitive y MIA en el colon y recto 19. La inervación colorrectal se deriva de esplácnico lumbar (LSN) y el nervio pélvico (PN) itinerarios, e incluye los aferentes colorrectal que se pueden clasificar en cinco clases mechanosensitive (serosa, mucosa, muscular, músculo-mucoso, mesentéricos) y una clase MIA 20. Usando esta preparación in vitro, se encontró que MIAs colorrectales mechanosensitivity adquirida (sensibilizar) siguiente breve exposición de sus campos receptivos a una sopa inflamatoria (IS), que sensibiliza 71% de MIAs en la vía de PN y 23% de MIAs en la vía LSN 19. También documentamos sensibilización a largo plazo (hasta 28 días) de los desaparecidos en acción en el contexto de larga duración hipersensibilidad visceral comportamiento (es decir, en los ratones que recibieron tratamientos intracolónicas con zymosan 21 o ácido 2,4,6-trinitrobencenosulfónico (TNBS) 22) . jove_content &quot;&gt; Entre los aferentes mechanosensitive, aferentes musculares y músculo-mucosas son las únicas clases que tónicamente codifican tramo circunferencial del colon y recto (es decir, son sensibles al estiramiento) y estar al servicio de la codificación de la distensión colorrectal nociva 23,24. El uso de un controlado por ordenador la fuerza del actuador, se aplicó un estándar, homogéneo, y el estiramiento en rampa reproducible en la dirección circunferencial del tejido colorrectal aplanada y clasifican además aferentes sensibles al estiramiento como de bajo umbral y de alto umbral 23. Además, el curso temporal de la sensibilización de estiramiento aferentes -sensibles después zimosan intracolónica 21 o 22 TNBS tratamiento corresponde a la aparición, persistencia y / o recuperación de la hipersensibilidad visceral de comportamiento, lo que sugiere un papel de los aferentes colorrectales sensibles al estiramiento en el dolor visceral e hipersensibilidad.

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			<td height="21" style="height:21px;width:375px;">Name of Material/ Equipment</td>
			<td style="width:291px;">Company</td>
			<td style="width:137px;">Catalog Number</td>
			<td style="width:372px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leica MZ16 stereo microscope&#160;</td>
			<td>Leica Microsystems Inc.</td>
			<td style="width:137px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leica IC D camera</td>
			<td>Leica Microsystems Inc.</td>
			<td style="width:137px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Amplifier</td>
			<td>World Precision instruments, Inc.</td>
			<td>SYS-DAM80&#160;</td>
			<td>Low-noise differential amplifier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Two-compartment tissue chamber</td>
			<td style="width:291px;">Custom made</td>
			<td style="width:137px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Power1401</td>
			<td>Cambridge Electronic Design Limited</td>
			<td style="width:137px;">Power1401</td>
			<td>Data acquisition, analog signal input/out</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Spike2 v5.02</td>
			<td>Cambridge Electronic Design Limited</td>
			<td style="width:137px;"></td>
			<td>Software package that works with the Power1401</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Audio monitor</td>
			<td>Natus</td>
			<td style="width:137px;">Am 8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Square pulse stimulator</td>
			<td>Natus</td>
			<td style="width:137px;">S48</td>
			<td>To deliver electrical stimuli</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Photoelectric isolation unit</td>
			<td>Natus</td>
			<td style="width:137px;">PSIU6</td>
			<td>Stimulus isolation to reduce noise</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Concentric bipolar microelectrode</td>
			<td style="width:291px;">FHC Inc.</td>
			<td style="width:137px;">CBFFG75</td>
			<td>To deliver electrical stimuli</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dual-mode lever system</td>
			<td>Aurora Scientific Inc.</td>
			<td style="width:137px;">Series 300C&#160;</td>
			<td>To deliver mechanical stimuli</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">forceps</td>
			<td>Fine Science Tools</td>
			<td>11252-00</td>
			<td>forceps with fine tips</td>
		</tr>
	</tbody>
</table>

in vitro functional characterization of mouse colorectal afferent endings

Este video muestra en detalle una fibra única protocolo de registro electrofisiológico in vitro usando una preparación ratón colon y recto-nervio. El enfoque imparcial permite la identificación y caracterización funcional de los aferentes colorrectales individuales. Grabaciones extracelulares de los potenciales de acción se propagan (AP) que se originan en uno o unos pocos aferente (es decir, de una sola fibra) campos receptivos (FR) en el colon y recto están hechos de fascículos de fibras nerviosas burlado. El colon y recto se retira, ya sea con la pelvis (PN) o esplácnico lumbar (LSN) nervio conectado y abierto longitudinalmente. El tejido se coloca en una cámara de registro, clavado plana y perfundidos con solución de Krebs oxigenada. Estimulación eléctrica focal se utiliza para localizar las terminaciones aferentes colorrectales, que son probados además por tres estímulos mecánicos distintos (embotar sondeo, acariciando la mucosa y estiramiento circunferencial) para clasificar funcionalmente los aferentes en cinco mechanosensiclases tivas. Endings responden a ninguno de estos estímulos mecánicos se clasifican como aferentes mecánicamente insensibles (MIAs). Tanto mechanosensitive y MIAs pueden ser evaluados para la sensibilización (es decir, la respuesta mejorada, umbral reducido, y / o adquisición de mechanosensitivity) por la exposición localizada de RFs a los productos químicos (por ejemplo, sopa inflamatoria (IS), la capsaicina, el trifosfato de adenosina (ATP)). Describimos el equipo y preparación de grabación de colon y recto-nervio, la cosecha de colon y recto con PN adjunto o LSN, la identificación de los FR en el colon y recto, la grabación de una sola fibra de los fascículos nerviosos, y la aplicación localizada de productos químicos para la RF. Además, también se discuten los retos de la preparación y la aplicación de la estimulación mecánica estandarizada.

La configuración se ilustra en la Figura 1. Incluye una cámara de tejido a medida que alberga el colon y recto en un baño de silicona compartimento forrado y el nervio adjunto en un compartimento lleno de aceite mineral contigua. La cámara de dos compartimentos fue a máquina de un bloque sólido de plástico acrílico por una máquina de CNC; la parte inferior de ambos compartimentos fue posteriormente revestido con silicona firme para permitir una fácil pin abajo del tejido colorrectal. AP extrac...

Watch this Scientific Journal Video about In vitro Functional Characterization of Mouse Colorectal Afferent Endings at JoVE.com

In vitro Functional Characterization of Mouse Colorectal Afferent Endings

This video demonstrates a protocol for conducting single-fiber electrophysiological recordings on an in vitro mouse colorectum-nerve preparation.

In vitro Caracterización funcional de ratón colorrectal aferente Endings

This video demonstrates in detail an in vitro single-fiber electrophysiological recording protocol using a mouse colorectum-nerve preparation. The ...

in-vitro-functional-characterization-mouse-colorectal-afferent

Research

JoVE Journal

Medicine

In vitro Functional Characterization of Mouse Colorectal Afferent Endings

9.5K vistas.  University of Pittsburgh. This video demonstrates a protocol for conducting single-fiber electrophysiological recordings on an in vitro mouse colorectum-nerve preparation. 

Video: In vitro Functional Characterization of Mouse Colorectal Afferent Endings

Hyperinsulinemic-euglycemic Clamps in Conscious, Unrestrained Mice

Type 2 diabetes is characterized by a defect in insulin action. The hyperinsulinemic-euglycemic clamp, or insulin clamp, is widely considered the "gold standard" method for assessing insulin action in vivo. During an insulin clamp, hyperinsulinemia is achieved by a constant insulin infusion. Euglycemia is maintained via a concomitant glucose infusion at a variable rate. This variable glucose infusion rate (GIR) is determined by measuring blood glucose at brief intervals throughout the experiment and adjusting the GIR accordingly. The GIR is indicative of whole-body insulin action, as mice with enhanced insulin action require a greater GIR. The insulin clamp can incorporate administration of isotopic 2[14C]deoxyglucose to assess tissue-specific glucose uptake and [3-3H]glucose to assess the ability of insulin to suppress the rate of endogenous glucose appearance (endoRa), a marker of hepatic glucose production, and to stimulate the rate of whole-body glucose disappearance (Rd).
The miniaturization of the insulin clamp for use in genetic mouse models of metabolic disease has led to significant advances in diabetes research. Methods for performing insulin clamps vary between laboratories. It is important to note that the manner in which an insulin clamp is performed can significantly affect the results obtained. We have published a comprehensive assessment of different approaches to performing insulin clamps in conscious mice1 as well as an evaluation of the metabolic response of four commonly used inbred mouse strains using various clamp techniques2. Here we present a protocol for performing insulin clamps on conscious, unrestrained mice developed by the Vanderbilt Mouse Metabolic Phenotyping Center (MMPC; URL: www.mc.vanderbilt.edu/mmpc). This includes a description of the method for implanting catheters used during the insulin clamp. The protocol employed by the Vanderbilt MMPC utilizes a unique two-catheter system3. One catheter is inserted into the jugular vein for infusions. A second catheter is inserted into the carotid artery, which allows for blood sampling without the need to restrain or handle the mouse. This technique provides a significant advantage to the most common method for obtaining blood samples during insulin clamps which is to sample from the severed tip of the tail. Unlike this latter method, sampling from an arterial catheter is not stressful to the mouse1. We also describe methods for using isotopic tracer infusions to assess tissue-specific insulin action. We also provide guidelines for the appropriate presentation of results obtained from insulin clamps.

The hyperinsulinemic-euglycemic clamp, or insulin clamp, is the gold standard for assessing insulin action in vivo. A method for performing insulin clamps in mice is described. This includes a method for arterial catheterization that permits experiments to be performed in conscious, unrestrained mice with minimal stress.

Hiperinsulinémico euglucémico abrazaderas en ratones conscientes, sin freno

Type 2 diabetes is characterized by a defect in insulin action. The hyperinsulinemic-euglycemic clamp, or insulin clamp, is widely considered the "gold ...

Investigación

Medicina

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson's Disease

Parkinson's disease (PD) is the second most common movement disorder and affects 1% of people over the age of 60 1. Because ageing is the most important risk factor, cases of PD will increase during the next decades 2. Next to pathological protein folding and impaired protein degradation pathways, alterations of mitochondrial function and morphology were pointed out as further hallmark of neurodegeneration in PD 3-11.
After years of research in murine and human cancer cells as in vitro models to dissect molecular pathways of Parkinsonism, the use of human fibroblasts from patients and appropriate controls as ex vivo models has become a valuable research tool, if potential caveats are considered. Other than immortalized, rather artificial cell models, primary fibroblasts from patients carrying disease-associated mutations apparently reflect important pathological features of the human disease.
Here we delineate the procedure of taking skin biopsies, culturing human fibroblasts and using detailed protocols for essential microscopic techniques to define mitochondrial phenotypes. These were used to investigate different features associated with PD that are relevant to mitochondrial function and dynamics. Ex vivo, mitochondria can be analyzed in terms of their function, morphology, colocalization with lysosomes (the organelles degrading dysfunctional mitochondria) and degradation via the lysosomal pathway. These phenotypes are highly relevant for the identification of early signs of PD and may precede clinical motor symptoms in human disease-gene carriers. Hence, the assays presented here can be utilized as valuable tools to identify pathological features of neurodegeneration and help to define new therapeutic strategies in PD.

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson&#039;s Disease

Fibroblasts from patients carrying mutations in Parkinson's disease-causing genes represent an easily accessible ex vivo model to study disease-associated phenotypes. Live cell imaging gives the opportunity to study morphological and functional parameters in living cells. Here we describe the preparation of human fibroblasts and subsequent monitoring of mitochondrial phenotypes.

El uso de fibroblastos humanos primarios para la Supervisión de fenotipos mitocondriales en el campo de la enfermedad de Parkinson

Parkinson's disease (PD) is the second most  common movement disorder and affects 1% of people over the age of 60 1. Because ageing is  the most important ...

In vitro Organoid Culture of Primary Mouse Colon Tumors

Several human and murine colon cancer cell lines have been established, physiologic integrity of colon tumors such as multiple cell layers, basal-apical polarity, ability to differentiate, and anoikis are not maintained in colon cancer derived cell lines. The present study demonstrates a method for culturing primary mouse colon tumor organoids adapted from Sato T et al. 1, which retains important physiologic features of colon tumors. This method consists of mouse colon tumor tissue collection, adjacent normal colon epithelium dissociation, colon tumor cells digestion into single cells, embedding colon tumor cells into matrigel, and selective culture based on the principle that tumor cells maintain growth on limiting nutrient conditions compared to normal epithelial cells.
The primary tumor organoids if isolated from genetically modified mice provide a very useful system to assess tumor autonomous function of specific genes. Moreover, the tumor organoids are amenable to genetic manipulation by virus meditated gene delivery; therefore signaling pathways involved in the colon tumorigenesis could also be extensively investigated by overexpression or knockdown. Primary tumor organoids culture provides a physiologic relevant and feasible means to study the mechanisms and therapeutic modalities for colon tumorigenesis.

In vitro Organoid Culture of Primary Mouse Colon Tumors

A simple method to establish primary murine colon tumor organoid is described. This method utilizes the feature that colon tumor cells survive and grow into organoids in media containing limited growth factors, whereas normal colon epithelial do not.

In vitro organoide Cultura de tumores de colon primarios Ratón

Several  human and murine colon cancer cell lines have been established, physiologic  integrity of colon tumors such as multiple cell layers, basal-apical ...

Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and membrane ion currents. Those changes are likely&#160;to be responsible for the increased arrhythmogenic risk and the contractile alterations leading to systolic and diastolic dysfunction in cardiac patients. However, most information on the alterations of myocyte function in cardiac diseases has come from animal models.
Here we describe and validate a protocol to isolate viable myocytes from small surgical samples of ventricular myocardium from patients undergoing cardiac surgery operations. The protocol is described in detail. Electrophysiological and intracellular calcium measurements are reported to demonstrate the feasibility of a number of single cell measurements in human ventricular cardiomyocytes obtained with this method.
The protocol reported here can be useful for future investigations of the cellular and molecular basis of functional alterations of the human heart in the presence of different cardiac diseases. Further, this method can be used to identify novel therapeutic targets at cellular level and to test the effectiveness of new compounds on human cardiomyocytes, with direct translational value.

Current knowledge on the cellular basis of cardiac diseases mostly relies on studies on animal models. Here we describe and validate a novel method to obtain single viable cardiomyocytes from small surgical samples of human ventricular myocardium. Human ventricular myocytes can be used for electrophysiological studies and drug testing.

Aislamiento y caracterización funcional de Human ventricular cardiomiocitos a partir de muestras quirúrgicas recientes

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and ...

Establishment and Characterization of UTI and CAUTI in a Mouse Model

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. Females are disproportionately afflicted by UTI: 50% of all women will have a UTI in their lifetime. Additionally, 20-40% of these women who have an initial UTI will suffer a recurrence with some suffering frequent recurrences with serious deterioration in the quality of life, pain and discomfort, disruption of daily activities, increased healthcare costs, and few treatment options other than long-term antibiotic prophylaxis. Uropathogenic Escherichia coli (UPEC) is the primary causative agent of community acquired UTI. Catheter-associated UTI (CAUTI) is the most common hospital acquired infection accounting for a million occurrences in the US annually and dramatic healthcare costs. While UPEC is also the primary cause of CAUTI, other causative agents are of increased significance including Enterococcus faecalis. Here we utilize two well-established mouse models that recapitulate many of the clinical characteristics of these human diseases. For UTI, a C3H/HeN model recapitulates many of the features of UPEC virulence observed in humans including host responses, IBC formation and filamentation. For CAUTI, a model using C57BL/6 mice, which retain catheter bladder implants, has been shown to be susceptible to E. faecalis bladder infection. These representative models are being used to gain striking new insights into the pathogenesis of UTI disease, which is leading to the development of novel therapeutics and management or prevention strategies.

The ability to model urinary tract infections (UTI) is crucial in order to be able to understand bacterial pathogenesis and spawn the development of novel therapeutics. This work&#8217;s goal is to demonstrate mouse models of experimental UTI and catheter associated UTI that recapitulate and predict findings seen in humans.

Establecimiento y caracterización de IU y ITUAC en un modelo de ratón

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. ...

Assessment of Cardiac Morphological and Functional Changes in Mouse Model of Transverse Aortic Constriction by Echocardiographic Imaging

Transverse aortic constriction (TAC) in mice has been used as a valuable model to study mechanisms of cardiac hypertrophy and heart failure1. A reliable noninvasive method is essential to assess real-time cardiac morphological and functional changes in animal models of heart disease. Transthoracic echocardiography represents an important tool for noninvasive assessment of cardiac structure and function2. Here we used a high-resolution ultrasound imaging system to monitor myocardial remodeling and heart failure progression over time in a mouse model of TAC. B-mode, M-mode, and Doppler imaging were used to precisely assess cardiac hypertrophy, ventricular dilatation, and functional deterioration in mice following TAC. Color and pulse wave (PW) Doppler imaging was used to noninvasively measure pressure gradient across the aortic constriction created by TAC and to assess transmitral blood flow in mice. Thus transthoracic echocardiographic imaging provides comprehensive noninvasive measurements of cardiac dimensions and function in mouse models of heart disease.

The goal of this protocol is to noninvasively assess cardiac structural and functional changes in a mouse model of heart disease created by transverse aortic constriction, using B- and M-mode echocardiography and color/pulse wave Doppler imaging.

Evaluación de la cardíaco morfológica y funcional Los cambios en el modelo de ratón de la constricción de la aorta transversa por ecocardiográfico Imaging

Transverse aortic constriction (TAC) in mice has been used as a valuable model to study mechanisms of cardiac hypertrophy and heart failure1. A reliable ...

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular plasticity or damage occurring overtime and in relation to neuronal activity or blood flow. In vivo two-photon microscopy allows the study of the structural and functional plasticity of large cellular units in the living brain. In particular, the thinned-skull window preparation allows the visualization of cortical regions of interest (ROI) without inducing significant brain inflammation. Repetitive imaging sessions of cortical ROI are feasible, providing the characterization of disease hallmarks over time during the progression of numerous CNS diseases. This technique accessing the pial structures within 250 &#956;m of the brain relies on the detection of fluorescent probes encoded by genetic cellular markers and/or vital dyes. The latter (e.g., fluorescent dextrans) are used to map the luminal compartment of cerebrovascular structures. Germane to the protocol described herein is the use of an in vivo marker of amyloid deposits, Methoxy-O4, to assess Alzheimer&#39;s disease (AD) progression. We also describe the post-acquisition image processing used to track vascular changes and amyloid depositions. While focusing presently on a model of AD, the described protocol is relevant to other CNS disorders where pathological cerebrovascular changes occur.

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

This manuscript describes a procedure to track the remodeling of the cerebrovasculature during amyloid plaque accumulation in vivo using longitudinal two-photon microscopy. A thinned-skull preparation enables the visualization of fluorescent dyes to assess the progression of cerebrovascular damage in a mouse model of Alzheimer&#39;s disease.

Longitudinal<em&gt; En Vivo</em&gt; Obtención de imágenes de la vasculatura cerebral: Importancia de las enfermedades del sistema nervioso central

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular ...

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene and cell therapies are highly promising alternative approaches for CVD treatment. However, the broad clinical application of gene therapy is greatly limited by the lack of suitable gene delivery systems. The development of appropriate gene delivery vectors can provide a solution to current challenges in cell therapy. In particular, existing drawbacks, such as limited efficiency and low cell retention in the injured organ, could be overcome by appropriate cell engineering (i.e., genetic) prior to transplantation. The presented protocol describes the efficient and safe transient modification of endothelial cells using a polyethyleneimine superparamagnetic magnetic nanoparticle (PEI/MNP)-based delivery vector. Also, the algorithm and methods for cell characterization are defined. The successful intracellular delivery of microRNA (miR) into human umbilical vein endothelial cells (HUVECs) has been achieved without affecting cell viability, functionality, or intercellular communication. Moreover, this approach was proven to cause a strong functional effect in introduced exogenous miR. Importantly, the application of this MNP-based vector ensures cell magnetization, with accompanying possibilities of magnetic targeting and non-invasive MRI tracing. This may provide a basis for magnetically guided, genetically engineered cell therapeutics that can be monitored non-invasively with MRI.

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

This manuscript describes the efficient, non-viral delivery of miR to endothelial cells by a PEI/MNP vector and their magnetization. Thus, in addition to genetic modification, this approach allows for magnetic cell guidance and MRI detectability. The technique can be used to improve the characteristics of therapeutic cell products.

Preparación y<em&gt; In Vitro</em&gt; Caracterización de Magnetized miR-modificado células endoteliales

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene ...

Development and Functional Characterization of Murine Tolerogenic Dendritic Cells

The immune system operates by maintaining a tight balance between coordinating responses against foreign antigens and maintaining an unresponsive state against self-antigens as well as antigens derived from commensal organisms. The disruption of this immune homeostasis can lead to chronic inflammation and to the development of autoimmunity. Dendritic cells (DCs) are the professional antigen-presenting cells of the innate immune system involved in activating na&#239;ve T cells to initiate immune responses against foreign antigens. However, DCs can also be differentiated into TolDCs that act to maintain and promote T cell tolerance and to suppress effector cells contributing to the development of either autoimmune or chronic inflammation conditions. The recent advancement in our understanding of TolDCs suggests that DC tolerance can be achieved by modulating their differentiation conditions. This phenomenon has led to tremendous growth in developing TolDC therapies for numerous immune disorders caused due to break in immune tolerance. Successful studies in preclinical autoimmunity murine models have further validated the immunotherapeutic utility of TolDCs in the treatment of autoimmune disorders. Today, TolDCs have become a promising immunotherapeutic tool in the clinic for reinstating immune tolerance in various immune disorders by targeting pathogenic autoimmune responses while leaving protective immunity intact. Although an array of strategies has been proposed by multiple labs to induce TolDCs, there is no consistency in characterizing the cellular and functional phenotype of these cells. This protocol provides a step-by-step guide for the development of bone marrow-derived DCs in large numbers, a unique method used to differentiate them into TolDCs with a synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid-difluoro-propyl-amide (CDDO-DFPA), and the techniques used to confirm their phenotype, including analyses of essential molecular signatures of TolDCs. Finally, we show a method to assess TolDC function by testing their immunosuppressive response in vitro and in vivo in a preclinical model of multiple sclerosis.

Here, we present a protocol to develop and characterize tolerogenic dendritic cells (TolDCs) and evaluate their immunotherapeutic utility.

Desarrollo y caracterización funcional de células dendríticas tolerogénicas murino

The immune system operates by maintaining a tight balance between coordinating responses against foreign antigens and maintaining an unresponsive state ...

Functional Characterization of Endogenously Expressed Human RYR1 Variants

More than 700 variants in the RYR1 gene have been identified in patients with different neuromuscular disorders including malignant hyperthermia susceptibility, core myopathies and centronuclear myopathy. Because of the diverse phenotypes linked to RYR1 mutations it is fundamental to characterize their functional effects to classify variants carried by patients for future therapeutic interventions and identify non-pathogenic variants. Many laboratories have been interested in developing methods to functionally characterize RYR1 mutations expressed in patients&#39; cells. This approach has numerous advantages, including: mutations are endogenously expressed, RyR1 is not over-expressed, use of heterologous RyR1 expressing cells is avoided. However, since patients may present mutations in different genes aside RYR1, it is important to compare results from biological material from individuals harboring the same mutation, with different genetic backgrounds. The present manuscript describes methods developed to study the functional effects of endogenously expressed RYR1 variants in: (a) Epstein Barr virus immortalized human B-lymphocytes and (b) satellite cells derived from muscle biopsies and differentiated into myotubes. Changes in the intracellular calcium concentration triggered by the addition of a pharmacological RyR1 activators are then monitored. The selected cell type is loaded with a ratiometric fluorescent calcium indicator and intracellular [Ca2+] changes are monitored either at the single cell level by fluorescence microscopy or in cell populations using a spectrofluorometer. The resting [Ca2+], agonist dose response curves are then compared between cells from healthy controls and patients harboring RYR1 variants leading to insight into the functional effect of a given variant.

Here methods used to study the functional effect of RYR1 mutations endogenously expressed in Epstein Barr Virus immortalized human B-lymphocytes and muscle biopsy derived satellite cells differentiated into myotubes are described.