Agradecemos al Dr. Wei Qi He (Universidad de Suzhou, Suzhou, China) y el Dr. Yan Ning Qiao (Universidad Normal de Shaanxi, Xi ' an, China) para la asistencia técnica. Este trabajo fue financiado por la Fundación Nacional de Ciencias naturales de China (Grant 31272311, 81373295 y 81473420) y el proyecto financiado por la prioridad académica programa de desarrollo de Jiangsu instituciones de educación superior (Grant no. ysxk-2016).

Animal manipulaciones fueron aprobadas por el cuidado de Animal institucional y Comité uso (IACUC) de la Universidad Modelo Animal Research centro de Nankín.
1. preparación de la solución
<ol><li>Prepare solución de Tyrode HEPES (H-T) con 137,0 mM NaCl, KCl de 2,7 mM, 1,8 mM CaCl2, 1 mM MgCl2∙6H2O, 5,6 mM D-glucosa y 10 mM HEPES, pH 7.3-7.4.</li>
	<li>Preparar la solución de Tyrode HEPES sin calcio (Ca2 +-libre H-T) utilizando 140,6 mM NaCl, 2,7 mM KCl, 1 mM MgCl2∙6H2O, 5,6 mM D-glucosa y 10 mM HEPES, pH 7.3-7.4.</li>
	<li>Prepare solución de Tyrode HEPES con 124 mM KCl (alto K+) usando 15,7 mM NaCl, KCl, de 124,0 mM 1,8 mM CaCl2, 1 mM MgCl2∙6H2O 5,6 mM D-glucosa y 10 mM HEPES, pH 7.3-7.4.</li>
</ol>2. preparación del experimento
<ol><li>PRECALIENTA el High-K+ soluciones usando un baño de agua de 37 ° C y H-T.</li>
	<li>Encienda el sistema miógrafo, hardware de adquisición de datos y la computadora.</li>
	<li>Cuidadosamente llene todas las cámaras de laringe con 5 mL de solución de H-T cada uno.</li>
	<li>Llenar dos placas de Petri con 20 mL de 4 ° C H-T y Ca2 +-gratis soluciones H-T, respectivamente y almacenar en hielo.</li>
	<li>Llenar un plato de Petri cubierto de 10 cm con 20 mL de solución de H-T y mantener a temperatura ambiente.</li>
</ol>3. ratón disección de la arteria mesentérica
<ol><li>Eutanasia a un ratón de C57BL/6J 8-12 semanas de edad hombre o mujer por dislocación cervical. Prender el ratón hacia abajo con el abdomen hacia arriba.</li>
	<li>Humedezca el abdomen con etanol al 70%. A continuación, cortar la piel con tijeras a lo largo de la línea media ventral desde la ingle y hacer incisiones desde el inicio de la primera incisión hacia abajo a las piernas a ambos lados. Tire de la piel en ambos lados; hacer incisiones similares para abrir el peritoneo.</li>
	<li>Con unas tijeras, cortar el esófago, el colon y otros tejidos conectivos para aislar completamente el tracto gastrointestinal con la alimentación de vasculatura del cuerpo.</li>
	<li>Con pinzas, mover el segmento aislado en el recipiente que contiene el frío H-T preparado en el paso 2.4 y enjuagar suavemente el tejido en solución H-T varias veces para lavar la sangre.</li>
	<li>Transferir el segmento aislado en la caja Petri cubierto preparado en el paso 2.5 y realizar la disección de la arteria mesentérica a temperatura ambiente.</li>
	<li>Alisar el estómago, yeyuno, íleon y ciego en sentido horario, y fijar el estómago y el intestino ciego en el lado izquierdo y derecho, respectivamente.</li>
	<li>Estirar la cama de la vasculatura mesentérica y fijar el intestino con pernos para exponer las arterias mesentéricas disecadas. Nota: En estas condiciones, las arterias están encima de las venas.</li>
	<li>Encienda la fuente de transmisión de luz de un microscopio estereoscópico y disecar las arterias bajo el microscopio. Asegúrese de que el tejido entero se sumerge en la solución.</li>
	<li>Sujetar los tejidos adiposo alrededor de las arterias con pinzas y aislar las arterias por todos los tejidos conectivos de corte con tijeras de disección. Evitar lesionar las arterias.</li>
</ol>4. arterial de montaje
<ol><li>Transferencia y sumerja el árbol de la arteria mesentérica en el frío Ca2 +-libre H-T solución (preparada en el paso 2.4) por las arterias exceso de sujeción con pinzas.</li>
	<li>Cortar una porción de 1,4 mm de la arteria proximal a la pared intestinal de un arcade mesentérico y utilizar dos pinzas para abrir ambos lados de este segmento de la arteria con cuidado.</li>
	<li>Prepare dos segmentos de alambre de acero inoxidable 2,5 cm de longitud y colocarlos en el mismo plato.</li>
	<li>Suavemente Sujete un extremo de la arteria con unas pinzas y cuidadosamente Inserte dos cables en el lumen de la arteria uno por uno con ayuda de pinzas para otros. Asegúrese que los cables se mantengan rectos y no tocan el endotelio.</li>
	<li>Utilizando dos pinzas, sujete los dos cables de acero fuera de la nave roscada simultáneamente y transferir cuidadosamente el vaso de la placa de Petri a una cámara de laringe previamente llenada con solución de H-T (paso 2.3).</li>
	<li>Atornille las mordazas aparte para hacer espacio para el montaje. Sujete ambos lados de uno de los dos cables insertados utilizando dos pinzas y colocar el vaso en el hueco de la mandíbula (figura 1A).</li>
	<li>Envolver ambos lados del alambre afianzado con abrazadera alrededor de los tornillos de la mordaza conectado al micrómetro (figura 1B).</li>
	<li>Fijar el tornillo izquierdo girando hacia la derecha. Enderezar el alambre con unas pinzas de la derechas, y luego fijar el tornillo de la derecho girando hacia la derecha (figura 1). Asegúrese de que el buque está siempre dentro de la brecha de la mandíbula, pero no toque la mandíbula para evitar daños.</li>
	<li>Cerca de las dos mordazas utilizando el micrómetro (figura 1). Asegúrese de que las dos mandíbulas están lo suficientemente cercanas pero que no toquen entre sí y que el cable no fijado en la parte superior el alambre fijo.</li>
	<li>Utilizando la pinza derecha, cuidadosamente doblar el alambre desatornillarse en la esquina de la mandíbula conectada a transductor de la fuerza y enróllela en sentido horario alrededor del tornillo del lado derecho (Figura 1E). A continuación, fijar el tornillo. Repita este paso en el lado izquierdo del hilo y fijar el tornillo del lado izquierdo (Figura 1F).</li>
	<li>Mueva las mordazas ligeramente separadas girando con cuidado el micrómetro (figura 1). Evitar estiramiento del buque. Utilice pinzas para mover el cable en el lado del micrómetro al plano horizontal del alambre en el lado del transductor. Gire con cuidado el micrómetro para que la brecha entre las dos mordazas sólo puede acomodar los dos cables.</li>
	<li>Repita los pasos 4.2 – 4.11 para montar las arterias en las otras cámaras. Conectar todas las cámaras a la cubierta de las cámaras, conecte el suministro de oxígeno al 100% y una sonda de temperatura y comienza a calentar a 37 ° C. Abra el software de gráficos y pulse el botón iniciar en la ventana de la Vista de tabla para empezar a grabar.</li>
	<li>Equilibre durante unos 20 minutos.</li>
</ol>5. normalización
Nota: Con el fin de estandarizar las condiciones experimentales y obtener confiable respuesta fisiológica de los vasos, un procedimiento de normalización es necesario15. Según la relación entre la fuerza activa y la circunferencia interna de la nave, el sistema de laringe de alambre tiene un programa de normalización estándar para evaluar la circunferencia interna (IC) del recipiente montado5,8, 9. Brevemente, calcular IC (μm), leer el micrómetro y el valor de entrada como el valor de X y el transductor de fuerza de salida, es decir, descansar de la tensión de la pared (mN/m), como el valor Y. El programa volver una curva integral de (X, Y) y calcular el CI correspondiente a una presión transmural de 100 mmHg (IC100). El buque se ajusta a la circunferencia interna normalizada (IC1) cuando es máxima la capacidad de respuesta activa.
<ol><li>Fuerzas a cero para todos los canales en el dispositivo y equilibre durante otro 1-2 min.</li>
	<li>Seleccione configuración de la normalización de la "carta DMTy configurar los parámetros como sigue: Calibración del ocular (mm/div): 0,36; Objetivo de presión (kPa): 13,3; IC1IC100: 0,9; En línea con un promedio de tiempo (segundos): 2; Retardo de tiempo (segundos): 60. Haga clic en el botón OK para cerrar la ventana de Configuración de la normalización de DMT .</li>
	<li>Seleccione el canal de interés en el menú DMT para abrir una ventana de normalización de DMT para el canal correspondiente. Entrar los valores constantes en la ventana siguiente: tejidos terminales a1: 0,1; Tejido puntos extremos a2: 4; Diámetro de alambre (μm): 40. La ventana muestra la longitud calculada del vaso como 1,40 m.</li>
	<li>Leer el micrómetro de la cámara de tejido apropiado. Introduzca el valor en el cuadro de lectura del micrómetro y haz clic en el botón Añadir punto . Este valor es el valor inicial de X (X0). Después de un tiempo de retraso 60 s, la ventana muestra la fuerza y la presión efectiva (ERTP) correspondiente a este valor de micrómetro. Al mismo tiempo, el cuadro de lectura del micrómetro se convierte en activo.</li>
	<li>Estirar el vaso se normalizó al girar el micrómetro en agujas. Introduzca el valor del micrómetro en el cuadro de lectura del micrómetro y haz clic en el botón Añadir punto . Esperar un retraso de 60 s otra vez.</li>
	<li>Repita el paso 5.5, continuar estirar el vaso y agregar valores de micrómetro hasta que la ventana muestra el valor de "X micrómetro1", que es el ajuste del micrómetro calculado requerido para estirar el vaso a su IC1.</li>
	<li>Ajustar el micrómetro x valor de1 . Nota: La tensión normalizada es generalmente 1-2 mN.</li>
</ol>6. arteria contracción grabación
Nota: Todas las soluciones, incluyendo H-T y High-K+ solución utilizada en esta sección, se prepararon en el paso 2.1.
<ol><li>Después de la normalización, equilibrar el buque en la cámara durante 15-20 min. Nota: Estos es necesario cambiar la solución en este paso.</li>
	<li>Desafío dos veces el recipiente con la solución High-K+ .<ol>
<li>Para desafiar a la nave, reemplazar la solución H-T con 5 mL de solución de alto-K+ para inducir la contracción durante 10 min, seguido de lavado con 5 mL de solución de H-T 3 - 4 veces. Nota: Contracción típica tiene una fuerza máxima durante 3 minutos y una fuerza sostenida constante alrededor de 2,5 mN12. Si el primer desafío genera una fuerza máxima por debajo de 2,5 mN o la fuerza sostenida disminuye con el tiempo, el segundo desafío genera una fuerza mucho más baja que la dosis de la primera vez, la nave se descarta y no se utilizará para la posterior investigación.</li>
	</ol>
</li>
	<li>Desafiar la nave con 5 mL de solución de alto-K+ para inducir la contracción. Después de 5 min, Añadir 0,5 μl de la solución madre (10 mM en DMSO) neoliensinine del14 en la sala para descansar la embarcación a una concentración final de 1 μm neoliensinine.</li>
	<li>Cuando la fuerza es estable (generalmente esto tarda varios minutos), añadir otro 0,5 μl de la solución madre neoliensinine en la cámara para aumentar la concentración a 2 μm. Añadir 1 μl de la solución cada vez para aumentar la concentración de a 4, 6, 8 y 10 μm para generar t curva de dosis-respuesta. Nota: Los valores y concentraciones de trabajo varían entre medicamentos.</li>
</ol>

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Los autores no tienen nada que revelar.

La hipertensión es un reto de salud pública generalizado debido a sus graves complicaciones, incluyendo la cardiovascular y renal enfermedades16. Comprensión de la patogenesia de la hipertensión arterial y explorar más los medicamentos antihipertensivos se ha convertido en una tarea urgente en este campo. La presión arterial es generada y mantenida por la resistencia periférica de la circulación. Conforme a la ley de Poiseuille, las relativamente pequeñas arterias generan una gran proporc...

El sistema de vasos miógrafo utilizado aquí fue para medir la contracción isométrica de los vasos de resistencia pequeña con diámetros interiores que van desde 100 hasta 400 μm. aislados los vasos pequeños (de unos 2 mm) se inserta por dos cables de 40 μm de diámetro y estaban entonces montado en las mordazas del micrómetro y transductor secuencialmente. Esta técnica de laringe fue primero sugerida en 19721 y entonces desarrollada principalmente por Mulvany y sus colegas2,3,4,5,6. Es una técnica madura con equipamiento, fácil funcionamiento y un procedimiento de normalización estándar7,8,9. Utilizamos este método con algunas modificaciones para la medición en la arteria mesentérica de ratón.
Músculo liso vascular recubre las paredes de casi todos los vasos sanguíneos. Su función fundamental es generar fuerzas a través de la contracción en respuesta a diversos estímulos. La contractilidad normal del músculo liso vascular es esencial para la regulación de la presión arterial y nutrición suplemento10. Regulación anormal de la presión arterial resulta en una variedad de enfermedades, como hipertensión, insuficiencia cardíaca y la isquemia. Varios estudios han sugerido que la presión de sangre anormal siempre es asociada con el músculo liso vascular disfuncional contractilidad7,11,12,13. El método de laringe permite investigación de contractilidad isométrica de los vasos del ratón inducida por varios estímulos, incluyendo drogas, inhibidores y vasoconstrictores. Medidas exitosas de contracción nos ayudará a comprender los mecanismos de mantenimiento de la hipertensión y la patogenesia de enfermedades asociadas de músculo liso vasculares y explorar nuevos enfoques terapéuticos.
Muchas hierbas chinas se han utilizado ampliamente para el tratamiento clínico de enfermedades vasculares; sin embargo, sus ingredientes eficaces generalmente siguen siendo desconocidos. Así, el aislamiento y la identificación de los componentes eficaces es muy importante para el desarrollo de nuevos fármacos. Tecnología multifilar miógrafo ofrece un enfoque simple para la detección de componentes activos en hierbas. Hemos divulgado varios estudios utilizando el sistema de laringe vasos para investigar la contracción de la arteria mesentérica de ratón e identificar compuestos naturales con actividad anti-hipertensión12,13,14. Aquí, describimos los detalles del protocolo para el método de la laringe y evaluar el efecto relajante de neoliensinine aislado de embriones de semilla de loto (Nelumbo nucifera Gaertn.) 14.

<table>
	<tbody>
		<tr>
			<td>Multi wire myograph system</td>
			<td>DMT</td>
			<td>610-M</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless steel wire</td>
			<td>DMT</td>
			<td>400447</td>
			<td></td>
		</tr>
		<tr>
			<td>Geuder dissection scissor</td>
			<td>DMT</td>
			<td>400431</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont forceps</td>
			<td>DMT</td>
			<td>300413</td>
			<td></td>
		</tr>
		<tr>
			<td>PowerLab/8SP</td>
			<td>ADInstruments</td>
			<td>ML785</td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td>ADInstruments</td>
			<td>LabChart 5</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>SigmaAldrich</td>
			<td>S5886</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>SigmaAldrich</td>
			<td>P5405</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>SigmaAldrich</td>
			<td>C4901</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2&#183;6H2O</td>
			<td>SigmaAldrich</td>
			<td>M2393</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Glucose</td>
			<td>SigmaAldrich</td>
			<td>G6152</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sangon Biotech</td>
			<td>A100511-0250</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>SigmaAldrich</td>
			<td>S8045</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>SigmaAldrich</td>
			<td>D2650</td>
			<td></td>
		</tr>
	</tbody>
</table>

isometric contractility measurement of the mouse mesenteric artery using wire myography

La técnica de laringe de alambre se utiliza para evaluar la contractilidad de los músculos lisos vasculares en respuesta a la despolarización, agonistas/inhibidores de GPCR y drogas. Es ampliamente utilizado en muchos estudios sobre las funciones fisiológicas del músculo liso vascular, la patogenesia de enfermedades vasculares como la hipertensión y el desarrollo de drogas relajantes del músculo liso. El ratón es un animal modelo ampliamente utilizado con un gran número de modelos de la enfermedad y las cepas genéticamente modificadas. Se introdujo este método para medir la contracción isométrica de arteria mesentérica ratón en detalle. Un segmento de 1,4 mm de ratón resistencia mesentérica arteria fue aislado y montado en una cámara de laringe por pasar dos cables de acero a través de su luz. Después de conseguir el equilibrio y normalización de pasos, el segmento de buques era potenciado por una solución high-K+ dos veces antes del ensayo de contracción. Como ejemplo de la aplicación de este método en el desarrollo de fármacos, medimos el efecto relajante de una nueva sustancia natural, neoliensinine, aislado de una hierba China, embriones de la semilla de loto (Nelumbo nucifera Gaertn.) en el ratón mesentérico arterias. Los segmentos de buque montados en la cámara de la laringe fueron estimulados con una solución de alto-K+ . Cuando la tensión de fuerza alcanza una fase sostenida estable, dosis acumulativas de neoliensinine fueron agregados a la cámara. Se encontró que neoliensinine tenía un efecto relajante del dependiente de la dosis en la contracción del músculo liso, así sugiriendo que tiene actividad potencial contra la hipertensión. Además, que el segmento vascular puede sobrevivir al menos 4 horas después del montaje y mantener la contractilidad inducida por la solución high-K+ para muchas veces, nos sugieren que el sistema del miógrafo de alambre puede utilizarse para el proceso desperdiciador de tiempo de la detección de drogas.

Medimos la contractilidad isométrica de arteria mesentérica de ratón utilizando un sistema de alambres múltiples miógrafo y evaluó el efecto relajante de neoliensinine purificada a partir de embriones de semilla de loto (Nelumbo nucifera Gaertn.) 14. la arteria mesentérica resistencia de ratón fue aislada, limpiar de tejidos conectivos y cortar en segmentos 1,4 mm. El segmento de la arteria fue insertado por dos alambres de acero en Ca2 +</sup...

Watch this Scientific Journal Video about Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography at JoVE.com

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

Medición de la contractilidad isométrica de la arteria mesentérica de ratón con cable miografía

La técnica del miógrafo de alambre se utiliza para investigar las funciones del músculo liso vascular y nuevos fármacos de la pantalla. Se presenta un protocolo detallado para medir la contractilidad isométrica de la arteria mesentérica de ratón y para la detección de nuevos relajantes del músculo liso vascular.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

isometric-contractility-measurement-mouse-mesenteric-artery-using

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

14.6K vistas.  Nanjing University. La técnica del miógrafo de alambre se utiliza para investigar las funciones del músculo liso vascular y nuevos fármacos de la pantalla. Se presenta un protocolo detallado para medir la contractilidad isométrica de la arteria mesentérica de ratón y para la detección de nuevos relajantes del músculo liso vascular.

Video: Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

Mouse Model of Middle Cerebral Artery Occlusion

Stroke is the most common fatal neurological disease in the United States 1. The majority of strokes (88%) result from blockage of blood vessels in the brain (ischemic stroke) 2. Since most ischemic strokes (~80%) occur in the territory of middle cerebral artery (MCA) 3, many animal stroke models that have been developed have focused on this artery. The intraluminal monofilament model of middle cerebral artery occlusion (MCAO) involves the insertion of a surgical filament into the external carotid artery and threading it forward into the internal carotid artery (ICA) until the tip occludes the origin of the MCA, resulting in a cessation of blood flow and subsequent brain infarction in the MCA territory 4. The technique can be used to model permanent or transient occlusion 5. If the suture is removed after a certain interval (30 min, 1 h, or 2 h), reperfusion is achieved (transient MCAO); if the filament is left in place (24 h) the procedure is suitable as a model of permanent MCAO. This technique does not require craniectomy, a neurosurgical procedure to remove a portion of skull, which may affect intracranial pressure and temperature 6. It has become the most frequently used method to mimic permanent and transient focal cerebral ischemia in rats and mice 7,8. To evaluate the extent of cerebral infarction, we stain brain slices with 2,3,5-triphenyltetrazolium chloride (TTC) to identify ischemic brain tissue 9. In this video, we demonstrate the MCAO method and the determination of infarct size by TTC staining.

We demonstrate in the video a method for producing a middle cerebral artery occlusion in adult mice using an intraluminal monofilament. We also show how to evaluate the extent of cerebral infarction by 2,3,5-triphenyltetrazolium chloride (TTC) staining.

Modelo de ratón de la oclusión arteria cerebral media

Stroke is the most common fatal neurological disease in the United States 1. The majority of strokes (88%) result from blockage of blood vessels in the ...

Investigación

Medicina

The Application Of Permanent Middle Cerebral Artery Ligation in the Mouse

Focal cerebral ischemia is among the most common type of stroke seen in patients. Due to the clinical significance there has been a prolonged effort to develop suitable animal models to study the events that unfold during ischemic insult. These techniques include transient or permanent, focal or global ischemia models using many different animal models, with the most common being rodents.
The permanent MCA ligation method which is also referred as pMCAo in the literature is used extensively as a focal ischemia model in rodents 1-6. This method was originally described for rats by Tamura et al. in 1981 7. In this protocol a craniotomy was used to access the MCA and the proximal regions were occluded by electrocoagulation. The infarcts involve mostly cortical and sometimes striatal regions depending on the location of the occlusion. This technique is now well established and used in many laboratories 8-13. Early use of this technique led to the definition and description of &#x201C;infarct core&#x201D; and &#x201C;penumbra&#x201D; 14-16, and it is often used to evaluate potential neuroprotective compounds 10, 12, 13, 17. Although the initial studies were performed in rats, permanent MCA ligation has been used successfully in mice with slight modifications 18-20 .
This model yields reproducible infarcts and increased post-survival rates. Approximately 80% of the ischemic strokes in humans happen in the MCA area 21 and thus this is a very relevant model for stroke studies. Currently, there is a paucity of effective treatments available to stroke patients, and thus there is a need for good models to test potential pharmacological compounds and evaluate physiological outcomes. This method can also be used for studying intracellular hypoxia response mechanisms in vivo. 
Here, we present the MCA ligation surgery in a C57/BL6 mouse. We describe the pre-surgical preparation, MCA ligation surgery and 2,3,5 Triphenyltetrazolium chloride (TTC) staining for quantification of infarct volumes.

Middle cerebral artery (MCA) ligation is a technique to study focal cerebral ischemia in animal models. In this method, the middle cerebral artery is exposed by craniotomy and ligated by cauterization. This method gives highly reproducible infarct volumes and increased post-operative survival rates compared to other methods available.

La aplicación de la Permanente Ligadura arteria cerebral media en el ratón

Focal cerebral ischemia is among the most common type of stroke seen in patients. Due to the clinical significance there has been a prolonged effort to ...

Mesenteric Artery Contraction and Relaxation Studies Using Automated Wire Myography

Proximal resistance vessels, such as the mesenteric arteries, contribute substantially to the peripheral resistance. These small vessels of between 100-400 &mu;m in diameter function primarily in directing blood flow to various organs according to the overall requirements of the body. The rat mesenteric artery has a diameter greater than 100 &mu;m. The myography technique, first described by Mulvay and Halpern1, was based on the method proposed by Bevan and Osher2. The technique provides information about small vessels under isometric conditions, where substantial shortening of the muscle preparation is prevented. Since force production and sensitivity of vessels to different agonists is dependent on the extent of stretch, according to active tension-length relation, it is essential to conduct contraction studies under isometric conditions to prevent compliance of the mounting wires. Stainless steel wires are preferred to tungsten wires because of oxidation of the latter, which affects recorded responses3.The technique allows for the comparison of agonist-induced contractions of mounted vessels to obtain evidence for normal function of vascular smooth muscle cell receptors.
We have shown in several studies that isolated mesenteric arteries that are contracted with phenylyephrine relax upon addition of cumulative concentrations of extracellular calcium (Ca2+e). The findings led us to conclude that perivascular sensory nerves, which express the G protein-coupled Ca2+-sensing receptor (CaR), mediate this vasorelaxation response. Using an automated wire myography method, we show here that mesenteric arteries from Wistar, Dahl salt-sensitive(DS) and Dahl salt-resistant (DR) rats respond differently to Ca2+e. Tissues from Wistar rats showed higher Ca2+-sensitivity compared to those from DR and DS. Reduced CaR expression in mesenteric arteries from DS rats correlates with reduced Ca2+e-induced relaxation of isolated, pre-contracted arteries. The data suggest that the CaR is required for relaxation of mesenteric arteries under increased adrenergic tone, as occurs in hypertension, and indicate an inherent defect in the CaR signaling pathway in Dahl animals, which is much more severe in DS.
The method is useful in determining vascular reactivity ex vivo in mesenteric resistance arteries and similar small blood vessels and comparisons between different agonists and/or antagonists can be easily and consistently assessed side-by-side6,7,8.

An automated myography method for force measurements in isolated mesenteric arteries is described. It employs a Mulvany-Halpern Auto Dual Wire Myograph 510A to determine responses to phenylephrine and extracellular calcium. The method allows consistent determination of isometric responses to agonists in small vessels of diameters of 60 - 300 &mu;m, independently.

Contracción de la arteria mesentérica y Estudios de relajación con miografía cable automático

Proximal resistance vessels, such as the mesenteric arteries, contribute substantially to the peripheral resistance. These small vessels of between ...

Epidural Intracranial Pressure Measurement in Rats Using a Fiber-optic Pressure Transducer

Elevated intracranial pressure (ICP) is a significant problem in several forms of ischemic brain injury including stroke, traumatic brain injury and cardiac arrest. This elevation may result in further neurological injury, in the form of transtentorial herniation1,2,3,4, midbrain compression, neurological deficit or increased cerebral infarct2,4. Current therapies are often inadequate to control elevated ICP in the clinical setting5,6,7 . Thus there is a need for accurate methods of ICP measurement in animal models to further our understanding of the basic mechanisms and to develop new treatments for elevated ICP.
In both the clinical and experimental setting ICP cannot be estimated without direct measurement. Several methods of ICP catheter insertion currently exist. Of these the intraventricular catheter has become the clinical 'gold standard' of ICP measurement in humans8. This method involves the partial removal of skull and the instrumentation of the catheter through brain tissue. Consequently, intraventricular catheters have an infection rate of 6-11%9. For this reason, subdural and epidural cannulations have become the preferred methods in animal models of ischemic injury. 
Various ICP measurement techniques have been adapted for animal models, and of these, fluid-filled telemetry catheters10 and solid state catheters are the most frequently used11,12,13,14,15. The fluid-filled systems are prone to developing air bubbles in the line, resulting in false ICP readings. Solid state probes avoid this problem (Figure 1). An additional problem is fitting catheters under the skull or into the ventricles without causing any brain injury that might alter the experimental outcomes. Therefore, we have developed a method that places an ICP catheter contiguous with the epidural space, but avoids the need to insert it between skull and brain. 
An optic fibre pressure catheter (420LP, SAMBA Sensors, Sweden) was used to measure ICP at the epidural location because the location of the pressure sensor (at the very tip of the catheter) was found to produce a high fidelity ICP signal in this model. There are other manufacturers of similar optic fibre technologies13 that may be used with our methodology. Alternative solid state catheters, which have the pressure sensor located at the side of the catheter tip, would not be appropriate for this model as the signal would be dampened by the presence of the monitoring screw. 
Here, we present a relatively simple and accurate method to measure ICP. This method can be used across a wide range of ICP related animal models.

A novel technique to record the pressures within the skull is described. The minimally invasive method uses a fibre-optic pressure sensing system to accurately measure intracranial pressure (ICP) in anaesthetized rats without causing significant brain trauma. The technique may be used in a wide range of experimental models.

Medición de la presión intracraneal epidural en ratas, utilizando un transductor de presión de fibra óptica

Elevated intracranial pressure (ICP) is a significant problem in several forms of ischemic brain injury including stroke, traumatic brain injury and ...

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

The endothelial glycocalyx is a layer of proteoglycans and associated glycosaminoglycans lining the vascular lumen. In vivo, the glycocalyx is highly hydrated, forming a substantial endothelial surface layer (ESL) that contributes to the maintenance of endothelial function. As the endothelial glycocalyx is often aberrant in vitro and is lost during standard tissue fixation techniques, study of the ESL requires use of intravital microscopy. To best approximate the complex physiology of the alveolar microvasculature, pulmonary intravital imaging is ideally performed on a freely-moving lung. These preparations, however, typically suffer from extensive motion artifact. We demonstrate how closed-chest intravital microscopy of a freely-moving mouse lung can be used to measure glycocalyx integrity via ESL exclusion of fluorescently-labeled high molecular weight dextrans from the endothelial surface. This non-recovery surgical technique, which requires simultaneous brightfield and fluorescent imaging of the mouse lung, allows for longitudinal observation of the subpleural microvasculature without evidence of inducing confounding lung injury.

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

The endothelial glycocalyx/endothelial surface layer is ideally studied using intravital microscopy. Intravital microscopy is technically challenging in a moving organ such as the lung. We demonstrate how simultaneous brightfield and fluorescent microscopy may be used to estimate endothelial surface layer thickness in a freely-moving in vivo mouse lung.

Medición in vivo de la capa de ratón pulmonar superficie endotelial

The endothelial glycocalyx is a layer of proteoglycans and associated glycosaminoglycans lining the vascular lumen. In vivo, the glycocalyx is highly ...

Assessing Myogenic Response and Vasoactivity In Resistance Mesenteric Arteries Using Pressure Myography

Small resistance arteries constrict and dilate respectively in response to increased or decreased intraluminal pressure; this phenomenon known as myogenic response is a key regulator of local blood flow. In isobaric conditions small resistance arteries develop sustained constriction known as myogenic tone (MT), which is a major determinant of systemic vascular resistance (SVR). Hence, ex vivo&#160;pressurized preparations of small resistance arteries are major tools to study microvascular function in near-physiological states. To achieve this, a freshly isolated intact segment of a small resistance artery (diameter ~260 &#956;m) is mounted onto two small glass cannulas and pressurized. These arterial preparations retain most in vivo characteristics and permit assessment of vascular tone in real-time. Here we provide a detailed protocol for assessing vasoactivity in pressurized small resistance mesenteric arteries from rats; these arteries develop sustained vasoconstriction - approximately 25% of maximal diameter - when pressurized at 70 mmHg. These arterial preparations may be used to study the effect of investigational compounds on relationship between intra-arterial pressure and vasoactivity and determine changes in microvascular function in animal models of various diseases.

Pressure myography is used to assess vasoactivity of small arteries that develop sustained constriction when pressurized. This manuscript provides a detailed protocol to assess in isolated segments of small mesenteric arteries from rats, vasoactivity and the effect of intraluminal pressure on vascular diameter.

Evaluar Miogénica Respuesta y Vasoactividad En arterias mesentéricas Resistencia Usando Miografía Presión

Small resistance arteries constrict and dilate respectively in response to increased or decreased intraluminal pressure; this phenomenon known as myogenic ...

Isolating and Using Sections of Bovine Mesenteric Artery and Vein as a Bioassay to Test for Vasoactivity in the Small Intestine

Mammalian gastrointestinal systems are constantly exposed to compounds (desirable and undesirable) that can have an effect on blood flow to and from that system. Changes in blood flow to the small intestine can result in effects on the absorptive functions of the organ. Particular interest in toxins liberated from feedstuffs through fermentative and digestive processes has developed in ruminants as an area where productive efficiencies could be improved. The video associated with this article describes an in vitro bioassay developed to screen compounds for vasoactivity in isolated cross-sections of bovine mesenteric artery and vein using a multimyograph. Once the blood vessels are mounted and equilibrated in the myograph, the bioassay itself can be used: as a screening tool to evaluate the contractile response or vasoactivity of compounds of interest; determine the presence of receptor types by pharmacologically targeting receptors with specific agonists; determine the role of a receptor with the presence of one or more antagonists; or determine potential interactions of compounds of interest with antagonists. Through all of this, data are collected real-time, tissue collected from a single animal can be exposed to a large number of different experimental treatments (an in vitro advantage), and represents vasculature on either side of the capillary bed to provide an accurate picture of what could be happening in the afferent and efferent blood supply supporting the small intestine.

The small intestine is frequently exposed to toxins that can influence blood flow and negatively impact nutrient absorption. Using a multimyograph and mesenteric artery and vein isolates, compounds or toxins of interest can be screened for vasoactivity.

El aislamiento y uso de las Secciones de bovino mesentérica arteria y la vena como un bioensayo para la Prueba de Vasoactividad en el intestino delgado

Mammalian gastrointestinal systems are constantly exposed to compounds (desirable and undesirable) that can have an effect on blood flow to and from that ...

Measurement of the Pressure-volume Curve in Mouse Lungs

In recent decades the mouse has become the primary animal model of a variety of lung diseases. In models of emphysema or fibrosis, the essential phenotypic changes are best assessed by measurement of the changes in lung elasticity. To best understand specific mechanisms underlying such pathologies in mice, it is essential to make functional measurements that can reflect the developing pathology. Although there are many ways to measure elasticity, the classical method is that of the total lung pressure-volume (PV) curve done over the whole range of lung volumes. This measurement has been made on adult lungs from nearly all mammalian species dating back almost 100 years, and such PV curves also played a major role in the discovery and understanding of the function of pulmonary surfactant in fetal lung development. Unfortunately, such total PV curves have not been widely reported in the mouse, despite the fact that they can provide useful information on the macroscopic effects of structural changes in the lung. Although partial PV curves measuring just the changes in lung volume are sometimes reported, without a measure of absolute volume, the nonlinear nature of the total PV curve makes these partial ones very difficult to interpret. In the present study, we describe a standardized way to measure the total PV curve. We have then tested the ability of these curves to detect changes in mouse lung structure in two common lung pathologies, emphysema and fibrosis. Results showed significant changes in several variables consistent with expected structural changes with these pathologies. This measurement of the lung PV curve in mice thus provides a straightforward means to monitor the progression of the pathophysiologic changes over time and the potential effect of therapeutic procedures.

Here we present a protocol to simply and reliably measure the lung pressure-volume curve in mice, showing that it is sufficiently sensitive to detect phenotypic parenchymal changes in two common lung pathologies, pulmonary fibrosis and emphysema. This metric provides a means to quantify the lung&#8217;s structural changes with developing pathology.

Medición de la curva de presión-volumen en mouse Pulmones

In recent decades the mouse has become the primary animal model of a variety of lung diseases. In models of emphysema or fibrosis, the essential ...

Murine Model of Femoral Artery Wire Injury with Implantation of a Perivascular Drug Delivery Patch

Percutaneous interventions including balloon angioplasty and stenting have been used to restore blood flow in vessels with occlusive vascular disease. While these therapies lead to the rapid restoration of blood flow, these technologies remain limited by restenosis in the case of bare metal stents and angioplasty, or reduced healing and possibly enhanced risk of thrombosis in the case of drug eluting stents. A key pathophysiological mechanism in the formation of restenosis is intimal hyperplasia caused by the activation of vascular smooth muscle cells and inflammation due to arterial stretch and injury. Surgeries that induce arterial injury in genetically modified mice are useful for the mechanistic study of the vascular response to injury but are often technically challenging to perform in mouse models due to the their small size and lack of appropriate sized devices. We describe two approaches for a surgical technique that induces endothelial denudation and arterial stretch in the femoral artery of mice to produce robust neointimal hyperplasia. The first approach creates an arteriotomy in the muscular branch of the femoral artery to obtain vascular access. Following wire injury this arterial branch is ligated to close the arteriotomy. A second approach creates an arteriotomy in the main femoral artery that is later closed through localized cautery. This method allows for vascular access through a larger vessel and, consequently, provides a less technically demanding procedure that can be used in smaller mice. Following either method of arterial injury, a degradable drug delivery patch can be placed over or around the injured artery to deliver therapeutic agents.

We describe a surgical technique that produces wire injury in the femoral artery of mice to induce neointimal hyperplasia to serve as a model testing system for the perivascular delivery of therapeutic compounds for the inhibition of restenosis.

Modelo murino de la arteria femoral Lesiones Alambre con implantación de un perivascular Drug Delivery Patch

Percutaneous interventions including balloon angioplasty and stenting have been used to restore blood flow in vessels with occlusive vascular disease. ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

Inducción de la Aterosclerosis Acelerada en Ratones: El Modelo de "Lesiones por Alambre"

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...