Los estudios fueron apoyados por NIH HL75503 de KP.

Todos los experimentos realizados en animales fueron aprobadas por el Comité de la Universidad de Tennessee Health Science Center Institucional Cuidado de Animales y el uso. 1. Tubo para Preparados perfusión del pulmón de la rata <ol><li> Preparar un sistema de tubos con tubos de Tygon (# 18) para la perfusión de la sangre como se muestra en la Figura 1. Conectar transductores de presión (P23XL) y colocar el tubo en la platina del microscopio. </li><li> Ajuste la temperatura del baño de agua a 37 ° C. </li><li> Conectar la entrada y la salida de pulmón temporalmente con tubo de Tygon. </li></ol><img alt="Figura 1" fo:content-width="5in" src="/files/ftp_upload/51552/51552fig1highres.jpg" width="500" /> Figura 1. Tubos de perfusión de sangre. El esquema muestra la configuración de la tubería usada para hacer circular la sangre a través de la preparación de pulmón aislado. También se indican los componentes asociados a través del cual la tubería is enrutado. Un esquema del corazón y de pulmón se incluye para mostrar sitios de conexión entre el tubo y la arteria pulmonar (AP), y fibrilación (LA) de la cánula (líneas azules discontinuas) a la izquierda. 2. Preparación de los pulmones de ratas aislados <ol><li> Anestesie ratas (ratas Sprague-Dawley macho; 250-300 g) con ketamina (80-100 mg / kg) y con xilazina (5-10 mg / kg). Después de asegurarse de un plano quirúrgico de anestesia, colocar el animal en posición supina. </li><li> Realizar la traqueostomía, inserte una cánula traqueal (PE-90) y asegure con sutura quirúrgica. </li><li> Infundir heparina (100-200 U) en el corazón por punción cardíaca (21 G aguja de mariposa), espere 60 segundos y la sangre Exsanguinate (~ 12 a 15 ml de sangre). </li><li> Prepare dos cánulas (tubos Tygon, 3 mm de diámetro; 4 cm de largo; acampanados en un extremo) y rellenar con solución salina. </li><li> Lleve a cabo una toracotomía. Hacer una incisión (3 mm) en el ventrículo derecho y deslice el extremo acampanado de una cánula en la incisión hacia la pulmonararteria. Asegure cánula de la arteria pulmonar con suturas quirúrgicas. </li><li> Una incisión (3 mm) de la punta del ventrículo izquierdo y deslice el extremo acampanado de la segunda cánula dentro de la incisión. Guía de la cánula en la aurícula izquierda. Asegure cánula en el ventrículo izquierdo con una cinta umbilical (2 mm de ancho). </li><li> Diseccionar cualquier tejido conectivo, y quitar el pulmón y el corazón junto con las cánulas adjuntas. Coloca los pulmones y el corazón en una placa de Petri. Vuelva a colocar el pulmón para que la superficie diafragmática es en la parte superior. Las tres cánulas deben enfrentar la misma dirección. </li><li> Colocar la preparación de pulmón en un escenario que puede ser manipulado en las direcciones XY. Cualquier etapa que viene con un microscopio se puede modificar para mantener el tubo o una etapa a medida construido, si es necesario. </li></ol> 3. Perfusión sanguínea de los pulmones de ratas aisladas <ol><li> Mezclar la sangre se desangraron con una albúmina volumen igual (5%) y añadir la solución al depósito. Arranque la bomba peristáltica y establecer el flujo de rcomió a 14 ml / min. Deje que la sangre para llenar el tubo. </li><li> Con la derivación abierta, retire el conector de entrada-salida de pulmón temporal. Fije la arteria pulmonar y la cánula de la aurícula izquierda a la entrada y la salida de pulmón, respectivamente. Asegúrese de que no haya burbujas de aire en la tubería. </li><li> Conectar la cánula traqueal a un tercer transductor de presión (P23XL). Inflar los pulmones con un 30% de oxígeno a través de la cánula traqueal y mantener la presión en 5 cm H 2 O. </li><li> Cierre la derivación con una pinza para comenzar la perfusión pulmonar. Mantener la arteria pulmonar y la presión de la aurícula izquierda a ~ 10 y 3 cm H 2 O, respectivamente. </li></ol> 4. Preparación de pulmón para microvascular Infusión <ol><li> Elevar el extremo posterior del tubo de extensión para el catéter por encima del nivel del pulmón y seguro. </li><li> Preparar un catéter de infusión mediante la inserción de unos 30 cm de un tubo PE10 40 cm de largo en una tubería PE90 ~ 30 cm de largo. Conectar el extremo expuesto de la tubería PE10 a una aguja (30 g) arecapitulativos a una jeringa (1 cc). </li><li> Insertar la combinación de catéter en el extremo posterior elevada de la tubería de extensión. Guía de la combinación de catéter a través de la aurícula izquierda y en el pulmón hasta que encuentre resistencia. A continuación, empuje el catéter PE10 sola vez en el pulmón hasta que encuentre resistencia. Tenga en cuenta que la aplicación de una fuerza excesiva al insertar el catéter lesionar el pulmón. </li><li> Llene la jeringa de 1 cc con solución de Ringer y adherirse a una bomba de jeringa. Ajuste la velocidad de infusión a 10 l / min. </li><li> Lugar de la perfusión se convertirá en pálida en comparación con el resto del pulmón. </li><li> Humedezca el pulmón con solución salina y cubra el pulmón con una envoltura de plástico con un agujero lo suficientemente grande como para dejar el lugar de la infusión al descubierto. </li><li> Hisopo un poco de grasa llave de paso en la parte inferior de una junta tórica (por encargo) y colocar un cubreobjetos de vidrio (# 1,5, 22 mm de diámetro) en la parte inferior de la junta tórica. Coloque suavemente la junta tórica con el cubreobjetos sobre el lugar de la infusión. Fije la junta tórica con una hol tubo de ensayo estándarder. La combinación cubreobjetos / junta tórica no distorsiona las estructuras alveolares por debajo de ella, como se informó recientemente 15. </li><li> Coloque un objetivo (20X) de un microscopio de fluorescencia por encima de la junta tórica y se centran en la superficie del pulmón. </li></ol> 5. Imaging fluorescente dextrano tránsito por Microvasos <ol><li> Preparar el microscopio para fluorescencia FITC imágenes. </li><li> Seleccionar una región a ser fotografiado y adquirir imágenes a la velocidad de 1/minuto usando el software de adquisición de imágenes. </li><li> Comience la infusión de FITC-dextrano 20 kD (0,5 mg / ml) usando la bomba de jeringa. La fluorescencia en los vasos sanguíneos se incrementará gradualmente y alcanzar un máximo. Continuar la infusión durante 60 min. </li><li> Luego cambiar a la infusión de Ringer para lavar fluorescencia luminal. Mantener la infusión durante más de 10 min. Continuar la adquisición de imágenes en 1/min durante el lavado-off. </li></ol> 6. Análisis de Imágenes <ol><li> Abra el archivo de adquisición de imágenes. </li><li> Coloque las regionesde interés sobre los microvasos dentro del marco de la imagen. </li><li> Reproducir el archivo de imagen cuadro por cuadro y registrar la intensidad de fluorescencia en cada región de interés para todos los fotogramas. </li><li> Trazar el cambio en la intensidad de fluorescencia para cada región de interés como una función de tiempo. </li><li> Cuantificar la máxima intensidad de fluorescencia y la intensidad de la fluorescencia residual después de 10 min de washoff. </li><li> Calcular la relación de máxima a la intensidad de fluorescencia residual en cada región de interés para obtener el índice de permeabilidad para los microvasos asociados con esa región de interés. </li></ol>

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Los autores no tienen nada que revelar.

La preparación de pulmón perfundido con sangre aislado junto con imágenes en tiempo real proporciona una herramienta sencilla para la determinación de cambios de permeabilidad en los microvasos pulmonares individuales. Hemos aplicado este método para definir los cambios de permeabilidad en respuesta a la infusión de LPS. Nuestros datos sugieren claramente que la infusión de LPS provocó un aumento de la permeabilidad microvascular. Además, los datos también indican que los cambios de permeabilidad inducidos por...

Aumento de la permeabilidad microvascular en los pulmones conduce al desarrollo de edema alveolar y el intercambio de gases en peligro y es una característica importante de la lesión pulmonar aguda (ALI) 1-3. Por lo tanto, las estimaciones de la permeabilidad vascular son importantes para definir el grado de la lesión pulmonar y la eficacia de las intervenciones terapéuticas propuestas. Análisis gravimétrico, tales como la relación húmedo a seco de pulmón libre de sangre y microvascular coeficiente de filtración se utilizan ampliamente métodos para estimar 4,5 permeabilidad. Otros métodos incluyen la cuantificación de la retención de sondas radioactivas o fluorescentes en el tejido pulmonar 6-8. Sin embargo, los métodos anteriores requieren un procesamiento postexperiment de muestras de tejido pulmonar hacia la aclaración de los datos de permeabilidad. Además, dado que un animal puede ser utilizado para un solo protocolo de tratamiento, puede ser necesario el número de animales grandes para un estudio completo. Una característica común de los métodos anteriores es que determinan la permeabilidad vascular media paratodos los vasos sanguíneos dentro de la muestra de tejido. Sin embargo, está bien establecido que los micro y macro vasos pulmonares son fenotípicamente diferentes 9. Por lo tanto, las respuestas de permeabilidad pueden ser heterogéneos entre los diversos segmentos de los vasos, así 9,10. Por lo tanto, la cuantificación de la permeabilidad media de todos los vasos pulmonares en una muestra de tejido puede no reflejar adecuadamente esta heterogeneidad. En la preparación aislada de pulmón perfundido con sangre, los vasos sanguíneos en la superficie del pulmón pueden ser visualizados por un microscopio vertical 4,11,12. Esto permite que las respuestas que caracterizan en los vasos individuales y por lo tanto, abordar cualquier heterogeneidad en las respuestas 13. Además, mediante la utilización de imágenes de fluorescencia de microvasos, ensayos de fluorescencia basado se pueden incorporar. Además, un microcatéter de la aurícula izquierda se puede utilizar para suministrar agentes y sondas de fluorescencia en los vasos sanguíneos 11,14. El microcatéter limita la entrega a una región de pulmón pequeño, por lo tanto exposando sólo los vasos sanguíneos dentro de la región a los agentes infundidos y fluoróforos. Esto permite que múltiples regiones pequeñas dentro del mismo pulmón a ser utilizados para experimentos separados, que conduce a una reducción global de animales necesarios para un estudio. Imágenes en tiempo real permite la captura de cambios dinámicos en la vascular y la fluorescencia extravascular de microvasos individuales de la preparación de pulmón aislado. Por lo tanto, para cada uno de los microvasos dentro de un campo de imagen, cambios en la fluorescencia durante la infusión de fluoróforos y washoff se pueden grabar, y se cuantificaron fuera de línea 14. El uso de valores de máxima y residual fluorescencia vascular, un índice de permeabilidad para cada uno de los microvasos dentro del campo de la imagen puede ser determinada. Para determinar los cambios de permeabilidad en respuesta a los agentes inflamatorios o perjudiciales, el agente deseado puede administrarse primero y luego el índice de permeabilidad determinada. Además, el campo de imagen se puede ajustar en cualquier lugar dentro de la región de pulmón infundido por elmicrocatéter, permitiendo así un alto grado de flexibilidad en la selección de la red vascular deseada. Por lo tanto, la preparación de pulmón aislado perfundido con sangre en tándem con formación de imágenes en tiempo real proporciona un modelo experimental atractiva para cuantificar la permeabilidad en microvasos pulmonares individuales.

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	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tygon Tubing</td>
			<td style="width:108px;">Fisher Scientific</td>
			<td style="width:119px;"></td>
			<td style="width:297px;">#18</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Pressure Transducer</td>
			<td style="width:108px;">Data Sciences International</td>
			<td style="width:119px;">P23XL</td>
			<td>Need quantity 3</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Butterfly Needle</td>
			<td style="width:108px;">Greiner Bio-One</td>
			<td align="right" style="width:119px;">450081</td>
			<td>21G</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Peristaltic pump</td>
			<td style="width:108px;">Cole Parmer</td>
			<td style="width:119px;">Masterflex L/S</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PE-90 tubing</td>
			<td style="width:108px;">Becton Dickinson</td>
			<td align="right" style="width:119px;">427421</td>
			<td>30 cm needed</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PE-10 tubing</td>
			<td style="width:108px;">Becton Dickinson</td>
			<td align="right" style="width:119px;">427401</td>
			<td>40 cm needed</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Syringe Pump</td>
			<td style="width:108px;">Braintree Scientific</td>
			<td style="width:119px;">BS8000</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">O-ring</td>
			<td style="width:108px;"></td>
			<td style="width:119px;"></td>
			<td>Custom made with a 20 mm diamter hole&#160; and a handle&#160; to secure o-ring to holder</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Upright fluorescence microscope</td>
			<td style="width:108px;">Olympus America</td>
			<td style="width:119px;">BX61WI</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Image Acquisition Software</td>
			<td style="width:108px;">Molecular Devices</td>
			<td style="width:119px;">Metamorph</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FITC Dextran 20KD</td>
			<td style="width:108px;">Sigma Aldrich</td>
			<td style="width:119px;"></td>
			<td>0.5 mg/ml (A dextran of different molecular size can be selected, if trial experiments indicate its suitability based on the calculated permeability index values)&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Lipopolysaccharide</td>
			<td style="width:108px;">Sigma Aldrich</td>
			<td style="width:119px;"></td>
			<td>Serotype 0111:B4</td>
		</tr>
	</tbody>
</table>

quantifying single microvessel permeability in isolated blood-perfused rat lung preparation

La preparación aislada de pulmón perfundido con sangre es ampliamente utilizado para visualizar y definir la señalización en los microvasos individuales. Mediante el acoplamiento de esta preparación con imágenes en tiempo real, se hace factible para determinar los cambios de permeabilidad en los microvasos pulmonares individuales. Aquí se describe los pasos para aislar a los pulmones de ratas y perfundir con sangre autóloga. A continuación, se describe los pasos para infundir fluoróforos o agentes a través de un micro-catéter en una región de pulmón pequeño. El uso de estos procedimientos descritos, se determinó la permeabilidad aumenta en microvasos pulmonares de rata en respuesta a inyecciones de lipopolisacárido bacteriano. Los datos revelaron que el lipopolisacárido aumentó fuga de líquido a través de ambos segmentos venulares y microvasos capilares. Por lo tanto, este método hace posible la comparación de las respuestas de permeabilidad entre los segmentos vasculares y por lo tanto, definir cualquier heterogeneidad en la respuesta. Mientras que los métodos comúnmente utilizados para definir la permeabilidad pulmonar requieren postprocesado de muestras de tejido de los pulmones, eluso de imágenes en tiempo real evita este requisito como se desprende de la presente método. Por lo tanto, la preparación de pulmón aislado combinado con imágenes en tiempo real, ofrece varias ventajas sobre los métodos tradicionales para determinar la permeabilidad microvascular pulmonar, sin embargo, es un método sencillo de desarrollar y poner en práctica.

Una preparación de pulmón perfundido con sangre aislado conectado al tubo de perfusión y el equipo relacionado se muestra en la Figura 2. Para fines de demostración, se utilizó una rata Sprague Dawley, aunque los procedimientos descritos en el presente documento se pueden usar con cualquier especie de rata. Infusiones través de un microcatéter de la aurícula izquierda sólo llegan a una pequeña región del pulmón. La región infundido puede ser identificado por la decoloración inducida por <s...

Watch this Scientific Journal Video about Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation at JoVE.com

Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

La preparación aislada de pulmón perfundido con sangre hace que sea posible visualizar las redes de microvasos en la superficie del pulmón. A continuación se describe un método para cuantificar la permeabilidad de la microvasculatura individuales en pulmones aislados utilizando imágenes de fluorescencia en tiempo real.

La cuantificación individual Microvessel Permeabilidad en aislado de rata Blood-perfundido Preparación de pulmón

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with ...

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9.4K vistas.  The University of Tennessee Health Science Center.  La preparación aislada de pulmón perfundido con sangre hace que sea posible visualizar las redes de microvasos en la superficie del pulmón. A continuación se describe un método para cuantificar la permeabilidad de la microvasculatura individuales en pulmones aislados utilizando imágenes de fluorescencia en tiempo real. 

Video: Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

Standardized Preparation of Single-Cell Suspensions from Mouse Lung Tissue using the gentleMACS Dissociator

The preparation of single-cell suspensions from tissues is an important prerequisite for many experiments in cellular research. The process of dissociating whole organs requires specific parameters in order to obtain a high number of viable cells in a reproducible manner. The gentleMACS Dissociator optimizes this task with a simple, practical protocol. The instrument contains pre-programmed settings that are optimized for the efficient but gentle dissociation of a variety of tissue types, including mouse lungs. In this publication the use of the gentleMACS Dissociator on lung tissue derived from mice is demonstrated.

Dissociating cells from specific tissue types requires specific parameters for tissue agitation to obtain a high volume of viable, culturable cells. The Miltenyi gentleMACS dissociator optimizes this task with a simple, practical protocol. In this publication the use of this apparatus on lung tissue is explained.

La preparación estandarizada de un solo suspensiones de células de tejido pulmonar de ratón con el disociador gentleMACS

The preparation of single-cell suspensions from tissues is an important prerequisite for many experiments in cellular research. The process of ...

Investigación

Biología

Characterization of the Isolated, Ventilated, and Instrumented Mouse Lung Perfused with Pulsatile Flow

The isolated, ventilated and instrumented mouse lung preparation allows steady and pulsatile pulmonary vascular pressure-flow relationships to be measured with independent control over pulmonary arterial flow rate, flow rate waveform, airway pressure and left atrial pressure. Pulmonary vascular resistance is calculated based on multi-point, steady pressure-flow curves; pulmonary vascular impedance is calculated from pulsatile pressure-flow curves obtained at a range of frequencies. As now recognized clinically, impedance is a superior measure of right ventricular afterload than resistance because it includes the effects of vascular compliance, which are not negligible, especially in the pulmonary circulation. Three important metrics of impedance - the zero hertz impedance Z0, the characteristic impedance ZC, and the index of wave reflection RW - provide insight into distal arterial cross-sectional area available for flow, proximal arterial stiffness and the upstream-downstream impedance mismatch, respectively. All results obtained in isolated, ventilated and perfused lungs are independent of sympathetic nervous system tone, volume status and the effects of anesthesia. We have used this technique to quantify the impact of pulmonary emboli and chronic hypoxia on resistance and impedance, and to differentiate between sites of action (i.e., proximal vs. distal) of vasoactive agents and disease using the pressure dependency of ZC. Furthermore, when these techniques are used with the lungs of genetically engineered strains of mice, the effects of molecular-level defects on pulmonary vascular structure and function can be determined.

The following protocol outlines the process of isolating, ventilating and instrumenting mouse lungs to measure steady or pulsatile pulmonary vascular pressure-flow relationships in order to quantify the effects of blood flow, airflow, airway changes and vascular changes on right ventricular afterload.

Caracterización de los aislados y ventilados, y pulmón de ratón instrumentado perfundidos con flujo pulsátil

The isolated, ventilated and instrumented mouse lung preparation allows steady and pulsatile pulmonary vascular pressure-flow relationships to be measured ...

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

The isolated microvessel preparation is an ex vivo preparation that allows for examination of the different contributions of factors that control vessel diameter, and thus, perfusion resistance1-5. This is a classic experimental preparation that was, in large measure, initially described by Uchida et al.15 several decades ago. This initial description provided the basis for the techniques that was extensively modified and enhanced, primarily in the laboratory of Dr. Brian Duling at the University of Virginia6-8, and we present a current approach in the following pages. This preparation will specifically refer to the gracilis arteriole in a rat as the microvessel of choice, but the basic preparation can readily be applied to vessels isolated from nearly any other tissue or organ across species9-13. Mechanical (i.e., dimensional) changes in the isolated microvessels can easily be evaluated in response to a broad array of physiological (e.g., hypoxia, intravascular pressure, or shear) or pharmacological challenges, and can provide insight into mechanistic elements comprising integrated responses in an intact, although ex vivo, tissue. The significance of this method is that it allows for facile manipulation of the influences on the integrated regulation of microvessel diameter, while also allowing for the control of many of the contributions from other sources, including intravascular pressure (myogenic), autonomic innervation, hemodynamic (e.g., shear stress), endothelial dependent or independent stimuli, hormonal, and parenchymal influences, to provide a partial list. Under appropriate experimental conditions and with appropriate goals, this can serve as an advantage over in vivo or in situ tissue/organ preparations, which do not readily allow for the facile control of broader systemic variables. 
The major limitation of this preparation is essentially the consequence of its strengths. By definition, the behavior of these vessels is being studied under conditions where many of the most significant contributors to the regulation of vascular resistance have been removed, including neural, humoral, metabolic, etc. As such, the investigator is cautioned to avoid over-interpretation and extrapolation of the data that are collected utilizing this preparation. The other significant area of concern with regard to this preparation is that it can be very easy to damage cellular components such as the endothelial lining or the vascular smooth muscle, such that variable source of error can be introduced. It is strongly recommended that the individual investigator utilize appropriate measurements to ensure the quality of the preparation, both at the initiation of the experiment and periodically throughout the course of a protocol.

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

An ex vivo preparation is described for isolation of the largest gracilis muscle resistance arterioles for interrogation of both vascular responses to vasoactive stimuli and the assessment of basic structural properties via passive wall mechanics.

La Ex vivo Preparación aislada Microvessel esquelético para la Investigación de la reactividad vascular

The isolated  microvessel preparation is an ex vivo preparation that allows for examination of the different contributions of  factors that control vessel ...

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with a molecular mass smaller than 1.5 kDa. Although the definitive molecular identity of the pore is still under debate, proteins such as cyclophilin D, VDAC and ANT contribute to mtPTP formation. While the involvement of mtPTP opening in cell death is well established1, accumulating evidence indicates that the mtPTP serves a physiologic role during mitochondrial Ca2+ homeostasis2, bioenergetics and redox signaling 3.
mtPTP opening is triggered by matrix Ca2+ but its activity can be modulated by several other factors such as oxidative stress, adenine nucleotide depletion, high concentrations of Pi, mitochondrial membrane depolarization or uncoupling, and long chain fatty acids4. In vitro, mtPTP opening can be achieved by increasing Ca2+ concentration inside the mitochondrial matrix through exogenous additions of Ca2+ (calcium retention capacity). When Ca2+ levels inside mitochondria reach a certain threshold, the mtPTP opens and facilitates Ca2+ release, dissipation of the proton motive force, membrane potential collapse and an increase in mitochondrial matrix volume (swelling) that ultimately leads to the rupture of the outer mitochondrial membrane and irreversible loss of organelle function.
Here we describe a fluorometric assay that allows for a comprehensive characterization of mtPTP opening in isolated mouse heart mitochondria. The assay involves the simultaneous measurement of 3 mitochondrial parameters that are altered when mtPTP opening occurs: mitochondrial Ca2+ handling (uptake and release, as measured by Ca2+ concentration in the assay medium), mitochondrial membrane potential, and mitochondrial volume. The dyes employed for Ca2+ measurement in the assay medium and mitochondrial membrane potential are Fura FF, a membrane impermeant, ratiometric indicator which undergoes a shift in the excitation wavelength in the presence of Ca2+, and JC-1, a cationic, ratiometric indicator which forms green monomers or red aggregates at low and high membrane potential, respectively. Changes in mitochondrial volume are measured by recording light scattering by the mitochondrial suspension. Since high-quality, functional mitochondria are required for the mtPTP opening assay, we also describe the steps necessary to obtain intact, highly coupled and functional isolated heart mitochondria.

A spectrofluorometric protocol for the measurement of the mitochondrial permeability transition pore opening in isolated mouse heart mitochondria is presented here. The assay involves the simultaneous measurement of mitochondria Ca2+ handling, mitochondrial membrane potential and mitochondrial volume. The procedure for obtaining high-quality and functional heart mitochondria is also described.

Multi-parámetro de medición de la apertura del poro de transición de permeabilidad en el corazón del ratón mitocondrias aisladas

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with ...

Measuring the Osmotic Water Permeability Coefficient (Pf) of Spherical Cells: Isolated Plant Protoplasts as an Example

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is a simple and very efficient method for the determination of the osmotic water permeability coefficient (Pf) in plant protoplasts, applicable in principle also to other spherical cells such as frog oocytes. The first step of the assay is the isolation of protoplasts from the plant tissue of interest by enzymatic digestion into a chamber with an appropriate isotonic solution. The second step consists of an osmotic challenge assay: protoplasts immobilized on the bottom of the chamber are submitted to a constant perfusion starting with an isotonic solution and followed by a hypotonic solution. The cell swelling is video recorded. In the third step, the images are processed offline to yield volume changes, and the time course of the volume changes is correlated with the time course of the change in osmolarity of the chamber perfusion medium, using a curve fitting procedure written in Matlab (the &#8216;PfFit&#8217;), to yield Pf.

Measuring the Osmotic Water Permeability Coefficient Pf of Spherical Cells: Isolated Plant Protoplasts as an Example

Measuring the osmotic water permeability coefficient (Pf) of cells can help understand the regulatory mechanisms of aquaporins (AQPs). Pf determination in spherical plant cell protoplasts presented here involves protoplasts isolation and numerical analysis of their initial rate of volume change as a result of an osmotic challenge during constant bath perfusion.

Medición de la osmótica de agua Coeficiente de Permeabilidad (P<sub&gt; F</sub&gt;) De células esféricas: protoplastos de plantas aisladas como ejemplo

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is ...

Single-channel Analysis and Calcium Imaging in the Podocytes of the Freshly Isolated Glomeruli

Podocytes (renal glomerular epithelial cells) are known to regulate glomerular permeability and maintain glomerular structure; a key role for these cells in the pathogenesis of various renal diseases has been established since podocyte injury leads to proteinuria and foot process effacement. It was previously reported that various endogenous agents may cause a dramatic overload in intracellular Ca2+ concentration in podocytes, presumably leading to albuminuria, and this likely occurs via calcium-conducting ion channels. Therefore, it appeared important to study calcium handling in the podocytes both under normal conditions and in various pathological states. However, available experimental approaches have remained somewhat limited to cultured and transfected cells. Although they represent a good basic model for such studies, they are essentially extracted from the native environment of the glomerulus. Here we describe the methodology of studying podocytes as a part of the freshly isolated whole glomerulus. This preparation retains the functional potential of the podocytes, which are still attached to the capillaries; therefore, podocytes remain in the environment that conserves the major parts of the glomeruli filtration apparatus. The present manuscript elaborates on two experimental approaches that allow 1) real-time detection of calcium concentration changes with the help of ratiometric confocal fluorescence microscopy, and 2) the recording of the single ion channels activity in the podocytes of the freshly isolated glomeruli. These methodologies utilize the advantages of the native environment of the glomerulus that enable researchers to resolve acute changes in the intracellular calcium handling in response to applications of various agents, measure basal concentration of calcium within the cells (for instance, to evaluate disease progression), and assess and manipulate calcium conductance at the level of single ion channels.

Changes in the intracellular calcium levels in the podocytes are one of the most important means to control the filtration function of glomeruli. Here we explain a high-throughput approach that allows detection of real-time calcium handling and single ion channels activity in the podocytes of the freshly isolated glomeruli.

Análisis y calcio monocanal Imaging en los podocitos de los glomérulos recién aisladas

Podocytes (renal glomerular epithelial cells) are known to regulate glomerular permeability and maintain glomerular structure; a key role for these cells ...

Microperfusion Technique to Investigate Regulation of Microvessel Permeability in Rat Mesentery

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular mechanisms regulating vascular permeability in cultured endothelial cell monolayers and the functional exchange properties of whole microvascular beds. A method to cannulate and perfuse venular microvessels of rat mesentery and measure the hydraulic conductivity of the microvessel wall is described. The main equipment needed includes an intravital microscope with a large modified stage that supports micromanipulators to position three different microtools: (1) a beveled glass micropipette to cannulate and perfuse the microvessel; (2) a glass micro-occluder to transiently block perfusion and enable measurement of transvascular water flow movement at a measured hydrostatic pressure, and (3) a blunt glass rod to stabilize the mesenteric tissue at the site of cannulation. The modified Landis micro-occlusion technique uses red cells suspended in the artificial perfusate as markers of transvascular fluid movement, and also enables repeated measurements of these flows as experimental conditions are changed and hydrostatic and colloid osmotic pressure difference across the microvessels are carefully controlled. Measurements of hydraulic conductivity first using a control perfusate, then after re-cannulation of the same microvessel with the test perfusates enable paired comparisons of the microvessel response under these well-controlled conditions. Attempts to extend the method to microvessels in the mesentery of mice with genetic modifications expected to modify vascular permeability were severely limited because of the absence of long straight and unbranched microvessels in the mouse mesentery, but the recent availability of the rats with similar genetic modifications using the CRISPR/Cas9 technology is expected to open new areas of investigation where the methods described herein can be applied.

The modified Landis technique enables paired measurement of the hydraulic conductivity of individual microvessels in the mesentery of normal and genetically modified rats under control and test conditions using microperfusion techniques. It provides a convenient method to evaluate mechanisms that regulate microvessel permeability and transvascular exchange under physiological conditions.

Microperfusión Técnica de investigar Reglamento de Microvessel Permeabilidad en Rata Mesenterio

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular ...

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

Mesenchymal stromal cells (MSCs) are increasingly recognized for their therapeutic potential in a wide range of diseases, including lung diseases. Besides the use of bone marrow and umbilical cord MSCs for exogenous cell therapy, there is also increasing interest in the repair and regenerative potential of resident tissue MSCs. Moreover, they likely have a role in normal organ development, and have been attributed roles in disease, particularly those with a fibrotic nature. The main hurdle for the study of these resident tissue MSCs is the lack of a clear marker for the isolation and identification of these cells. The isolation technique described here applies multiple characteristics of lung resident MSCs (L-MSCs). Upon sacrifice of the rats, lungs are removed and rinsed multiple times to remove blood. Following mechanical dissociation by scalpel, the lungs are digested for 2-3 hr using a mix of collagenase type I, neutral protease and DNase type I. The obtained single cell suspension is subsequently washed and layered over density gradient medium (density 1.073 g/ml). After centrifugation, cells from the interphase are washed and plated in culture-treated flasks. Cells are cultured for 4-7 days in physiological 5% O2, 5% CO2 conditions. To deplete fibroblasts (CD146-) and to ensure a population of only L-MSCs (CD146+), positive selection for CD146+ cells is performed through magnetic bead selection. In summary, this procedure reliably produces a population of primary L-MSCs for further in vitro study and manipulation. Because of the nature of the protocol, it can easily be translated to other experimental animal models.

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

This protocol describes an isolation technique for obtaining primary lung resident mesenchymal stromal cells from rats, through the use of enzymatic digestion, density gradient separation, plastic adherence and CD146+ magnetic bead selection.

Aislamiento de CD146<sup&gt; +</sup&gt; Las células estromales mesenquimales de pulmón Residente de los pulmones de ratas

Mesenchymal stromal cells (MSCs) are increasingly recognized for their therapeutic potential in a wide range of diseases, including lung diseases. Besides ...

The Mouse Isolated Perfused Kidney Technique

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a prerequisite for studying the physiology of the isolated organ and for many innovative applications that may be possible in the future, including perfusion decellularization for kidney bioengineering or the administration of anti-rejection or genome-editing drugs in high doses to prime the kidney for transplantation. During the time of the perfusion, the kidney can be manipulated, renal function can be assessed, and various pharmaceuticals administered. After the procedure, the kidney can be transplanted or processed for molecular biology, biochemical analysis, or microscopy.
This paper describes the perfusate and the surgical technique needed for the ex vivo perfusion of mouse kidneys. Details of the perfusion apparatus are given and data are presented showing the viability of the kidney&#39;s preparation: renal blood flow, vascular resistance, and urine data as functional, transmission electron micrographs of different nephron segments as morphological readouts, and western blots of transport proteins of different nephron segments as molecular readout.

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. The buffers and surgical technique are described in detail.

La Técnica del riñón perfundido aislado del ratón

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a ...

An Improved Chemotaxis Assay for the Rapid Identification of Rhizobacterial Chemoattractants in Root Exudates

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward method to quickly identify the chemoattractants that could induce the chemotactic movement of rhizosphere growth-promoting bacteria on sterile glass slides via simple steps. Bacteria solution (OD600 = 0.5) and sterile chemoattractant aqueous solution were added dropwise on the glass slide at an interval of 1 cm. An inoculating loop was used to connect the chemoattractant aqueous solution to the bacterial solution. The slide was kept at room temperature for 20 min on the clean bench. Finally, the chemoattractant aqueous solution was collected for bacterial counting and microscopic observation. In this study, through multiple comparisons of experimental results, the method overcame multiple shortcomings of traditional bacterial chemotaxis methods. The method reduced the error of plate counting and shortened the experimental cycle. For the identification of chemoattractant substances, this new method can save 2-3 days compared to the traditional method. Additionally, this method allows any researcher to systematically complete a bacterial chemotaxis experiment within 1-2 days. The protocol can be considered a valuable resource for understanding plant-microbe interactions.

Here, we present an improved chemotaxis assay protocol. The goal of this protocol is to reduce the steps and costs of traditional bacterial chemotaxis methods and to serve as a valuable resource for understanding plant-microbe interactions.

Un ensayo de quimiotaxis mejorado para la identificación rápida de quimioatrayentes rizobacterianos en exudados radiculares

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward ...