This work was supported by National Medical Research Council (NMRC)/Cooperative Basic Research Grant (CBRG)/0078/2014.

Este estudio está de acuerdo con la Universidad Nacional de Singapur Institucional Cuidado de Animales y el empleo (protocolo aprobado ninguna. R15-0225). 1. Preparación quirúrgica del modelo animal <ol><li> los lúmenes de los vasos <ol><li> Anestesiar a ratas macho Sprague-Dawley (6 - 7 semanas de edad) con un peso (203 ± 20) g con ketamina (37,5 mg / ml) y xilazina (5 mg / ml) cóctel a través de inyección intraperitoneal (ip) (2 ml / kg) . No vuelva a tapar la aguja o quitarlo de la jeringa después de la inyección. </li><li> Una vez que el animal ha sido anestesiado (confirmado por los pies pellizcos), colocarlo sobre una almohadilla térmica para mantener su temperatura corporal a 37 ° C. afeitarse suavemente el pelo en la escápula, el cuello uterino anterior, parte baja del abdomen, pierna trasera medial y el saco escrotal. frenar suavemente las piernas utilizando cintas adhesivas de papel. </li><li> Realizar todos los procedimientos quirúrgicos con tijeras de microdisección y pinzas en ángulo mientras observa a travésun microscopio estereoscópico. Coloque todos los instrumentos quirúrgicos cortantes en una bandeja resistente a la punción para evitar lesiones durante la cirugía. </li><li> Fregar todos los sitios quirúrgicos 3 veces alternando con yodo y alcohol al 70% antes de realizar la incisión. Enjuague todos los catéteres con 30 solución de heparina-solución salina IU / ml. </li><li> Hacer un 1-1,5 cm incisión en la piel en la línea media en las escápulas usando un par de tijeras quirúrgicas sobre la vena yugular derecha. Separar la fascia mediante disección roma para exponer la vena yugular y canular con un tubo de polietileno (PE-50) lleno de solución salina de heparina usando suturas de seda 5-0. Infundir anestesia suplementaria cuando sea necesario (1/3 a 1/2 de la dosis inicial, intravenosa (iv)) durante el curso de la cirugía y el experimento. </li><li> Realizar traqueotomía para mantener la permeabilidad de la vía aérea. Hacer un 1 - 1,5 cm de la incisión en la zona cervical anterior. Canular la tráquea utilizando un tubo de polietileno (PE-205) con suturas de seda 2-0 para asegurar el catéter en su lugar. </li><li> Vigilar la presión arteriala través de la canulación de la arteria femoral. Hacer un 1 - 1,5 cm incisión en la superficie medial izquierdo de la pata trasera. Separar la arteria femoral mediante disección roma. Canular la arteria femoral con un tubo de polietileno (PE-10) lleno de solución salina de heparina usando suturas de seda 5-0. </li></ol></li><li> Cremaster músculo Preparación y flujo de visualización <ol><li> Inserte una sutura de seda 5-0 a través del vértice del saco escrotal para extenderla. Hacer una incisión a lo largo de la superficie ventral del saco escrotal. Regularmente aplicar solución caliente isotónica (37 ° C; pH 7,4) al músculo expuesto. </li><li> Retire rodea los tejidos conectivos cuidadosamente ya fondo con un aplicador con punta de algodón. </li><li> Inserte una sutura de seda 5-0 a través del ápice del músculo cremáster. Cortar la sutura en dos piezas de igual longitud y un nudo en cada lado. Cortar el músculo entre los dos nudos y se extienden sobre una plataforma de plexiglás transparente personalizado tirando suavemente de la sutura. Fijarel extremo de la sutura a la plataforma con la tachuela azul. NOTA: la eliminación general de los tejidos conectivos que rodea es fundamental para determinar un contraste de imagen óptima. </li><li> 1.2.3 repita el paso hasta que se hagan de 5 a 6 fijaciones. Retirar con cuidado el músculo cremáster del epidídimo utilizando cauterio alta temperatura. Superfuse solución isotónica caliente al músculo expuesto para prevenir la deshidratación del tejido. <ol><li> Rodea el músculo cremáster con piezas plegadas de gasa. Cubrir el músculo expuesto con una película de polivinilo. Las piezas de gasa con la película forman una cubeta poco profunda para contener solución isotónica caliente para el objetivo del microscopio de inmersión en agua (Figura 1A). </li></ol></li><li> Transferir el animal en el escenario animal de un microscopio intravital (Figura 1C). Conectar la canulación arterial a un sistema de adquisición de datos fisiológicos de control de la presión continua (Figura 1E). </li><li> Mantener la temperat muscularure a 35 ° C con un elemento de calentamiento fijado debajo de la plataforma de los animales (Figura 1B). Colocar una sonda de temperatura al lado del músculo para proporcionar retroalimentación negativa para el controlador de potencia del elemento de calentamiento (Figura 1D). </li><li> Dejar al animal en el escenario durante 15 min se equilibre con el medio ambiente. </li><li> Visualizar el flujo sanguíneo en un microscopio intravital con un objetivo de inmersión en agua de 40X y un condensador de trabajo larga. </li><li> Elija una arteriola no ramificado (&lt;60 micras), basado en un enfoque de la imagen clara y el contraste entre el núcleo de glóbulos rojos, paredes CFL y de los vasos, con el fin de enfocar el microscopio en el plano diametral del vaso sanguíneo. Girar la cámara montada en el microscopio para alinear verticalmente la pared del vaso. </li><li> Registrar el flujo de sangre usando una cámara de vídeo de alta velocidad a una velocidad de 3.000 / seg durante 1 seg. Guardar el vídeo grabado en formato AVI sin comprimir en escala de grises de 8 bits para preservar la calidad de la imagen. NOTA:<html> Se recomienda una velocidad mínima de fotogramas de grabación de 3.000 cuadros / seg para garantizar que la medición CFL se puede realizar al menos una vez por RBC en condiciones de flujo arteriolar fisiológicas. </li><li> Utilice un filtro azul con pico de transmisión a una longitud de onda de 394 nm y de paso de banda espectral a 310 - 510 nm para mejorar el contraste entre los glóbulos rojos y plasma. NOTA: Asegúrese de que el espectro de la luz que pasa a través del filtro azul de la fuente de luz microscópico (100 W lámpara halógena) es de baja intensidad de la luz para evitar cualquier daño tisular potencial. </li><li> Al final del experimento, la eutanasia a los animales con una sobredosis de pentobarbital sódico. </li></ol></li></ol> 2. Análisis de Imagen <ol><li> Procesamiento previo para la medida de ancho CFL <ol><li> Abra MATLAB y ejecute el archivo &#39;CFL_pre.m&#39;. (Este y otros archivos de MATLAB se puede encontrar en elip &quot;&gt; Suplementario Archivo MATLAB.) </li><li> Haga clic en &quot;Abrir archivo&quot; para seleccionar el archivo de vídeo para analizar. </li><li> Ajuste el deslizador hacia la &quot;rotación&quot; para alinear verticalmente las paredes de los vasos. NOTA: Los usuarios pueden visualizar las líneas de la cuadrícula de asistencia para la alineación buque seleccionando el botón de opción &#39;Guía On&#39;, y ajustar el nivel de zoom de la imagen deslizando el control deslizante &quot;Zoom&quot;. </li><li> Haga clic en el botón &quot;Confirmar Editar &#39;para confirmar la alineación del vaso. </li><li> Haga clic en el botón &#39;Set ROI de cultivo &quot;para definir la región de interés (ROI). La imagen alineada se mostrará en una ventana emergente. Ajustar el objetivo rectangular en la imagen y haga doble clic para confirmar el retorno de la inversión. Omitir este paso si no se requiere el recorte de la imagen. NOTA: solo incluye un solo barco en el retorno de la inversión para analizar el ancho de CFL del recipiente. Haga clic en el botón &quot;Reiniciar Imagen&quot; para restaurar la imagen a su forma original, si es necesario. </li><li> Haga clic en el9; &#39;botón para extraer todos los fotogramas de vídeo editado en imágenes de mapa de bits consecutivos (en escala de grises de 8 bits&#39; Extraer imágenes en formato BMP &#39;). Las imágenes extraídas se pueden encontrar en la carpeta con el mismo nombre que el archivo de vídeo seleccionado. </li></ol></li><li> La medición de la anchura de CFL <ol><li> Abra MATLAB y ejecute el archivo &#39;CFL_measure.m&#39;. </li><li> Haga clic en &quot;Seleccionar carpeta&quot; para seleccionar la carpeta que contiene las imágenes extraídas. </li><li> Haga clic en la carpeta que contiene las imágenes y haga clic en &quot;Seleccionar carpeta&quot;. El primer cuadro de imagen en la carpeta se carga y se muestra en el panel de &quot;Escala de grises imagen &#39;, junto con su gris histograma de intensidad en el panel&#39; del histograma &#39;. </li><li> Seleccione el cuadro de imagen que desee en el cuadro de lista para realizar el análisis, de lo contrario se seleccionará el primer cuadro de la imagen. </li><li> Haga clic en &quot;Encontrar paredes de los vasos &#39;para identificar la pared del vaso interno en la imagen, que se determina en el lugar donde laperfil de intensidad de luz tránsitos pico de oscuro a claro más de dos píxeles. </li><li> Check &#39;filtro de mediana&#39; para aplicar un filtro de mediana a la imagen para reducir el ruido &quot;sal y pimienta&quot;. </li><li> Consultar &quot;Contraste automático&quot; para ajustar las intensidades de imagen digital para mejorar el contraste de la imagen. </li><li> Seleccione un algoritmo de umbral en el cuadro de lista que determina automáticamente un valor de umbral (τ) que divide los niveles de gris en dos clases - los píxeles blancos con niveles de gris por encima τ (CFL), y los píxeles negros con niveles de gris por debajo de τ (RBC básicos). NOTA: Como un método alternativo, el uso de umbralización manual si ninguno de algoritmo de umbral automatizado proporciona una umbralización imagen correspondiente. Haga clic en el botón de opción &quot;Manual&quot; y ajustar el control deslizante para definir el valor de umbral manual. </li><li> Para medir la variación espacial de los anchos de CFL, introduzca la resolución de píxeles en el cuadro &#39;píxeles de resolución&#39; (la resolucióncon esta configuración experimental fue 0,42 m / píxel). </li><li> Haga clic en el botón &quot;Calcular&quot; para obtener la variación espacial de los anchos de CFL. Haga clic en &quot;.csv Exportar &#39;para exportar los datos de anchura CFL en un formato tabulado. </li><li> Para medir la variación temporal de los anchos de CFL en una línea de análisis específico a lo largo del recipiente, haga clic en el botón de opción &#39;Variación temporal&#39; e introduzca la información de velocidad de fotogramas (la frecuencia de imagen utilizada en este sistema experimental fue de 3.000 cuadros / seg). </li><li> Introduzca el primer fotograma y el último fotograma de las imágenes para el análisis de las cajas &#39;de comienzo de trama &quot;y&quot; último cuadro&#39;, respectivamente. </li><li> Seleccione la posición de la línea de análisis a lo largo del buque deslizando la barra deslizante &#39;Análisis de línea ». Confirmar la posición de la línea de análisis, que se ilustra en tanto la &#39;Escala de grises imagen&#39; y &#39;la imagen binaria&#39;. </li><li> Haga clic en &quot;Calcular&quot; para obtener la variación temporal de los anchos de CFL. Click &#39;Exportar CSV &quot;para exportar los datos de anchura CFL en un formato tabulado. </li></ol></li></ol>

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The authors declare no competing financial interests.

La medición de la anchura CFL es esencial para una mejor comprensión de la hemodinámica en la microcirculación. En particular, la medición de las anchuras CFL se ha realizado en mesentérica 6, spinotrapezius 24 y cerebrales 25 microcirculations. La medición convencional de la in vivo anchuras CFL se restringió a estimaciones por la inspección manual de los fotogramas de vídeo grabadas. Las mediciones manuales requieren el cálculo del promedio de varios fotogramas de v...

In vivo animal studies are instrumental to basic science for understanding human physiology and pathology. In particular, in vivo microhemodynamic studies can elucidate the potential impairment of microcirculatory functions altered by abnormal rheological conditions of blood. A number of previous microhemodynamic studies1 have used the rat cremaster muscle model for visualizing microvascular blood flow. The cremaster muscle is a thin layer of striated muscle surrounding the testes. Thus, the blood flow in the muscle can be visualized with a trans-illumination microscope by means of surgical exposure. This enables us to acquire the in vivo blood flow images without the use of any fluorescence or contrast agents. In addition, the entire blood perfusion of the muscle network can be controlled by reducing the upstream blood flow with abdominal aorta occlusion2. Owing to these advantages, the cremaster muscle model has been widely used to investigate the formation of cell-free layer (CFL) in microvessels1,3. 
The CFL width is a prominent hemodynamic parameter in microcirculation, which has been of great interest for its important roles in regulating microcirculatory functions. The CFL is formed by the shear-induced transverse inward migration of red blood cells (RBCs) towards the flow center4. Consequently, this migration leads to the depletion of RBCs near the vessel walls, eventually resulting in a cell-free plasma layer. Accordingly, the parietal CFL naturally becomes a diffusion barrier to oxygen (O2) delivery from the RBC core to the tissues, and to the scavenging of nitric oxide (NO) by the RBCs5,6. In addition, the production of NO can also be modulated by the dynamic variations of the CFL width7,8. Therefore, the roles of the CFL in both gas transport and the regulation of homeostasis in microcirculation need to be fully ascertained to better understand blood flow in microcirculation. Recent studies have focused on bridging the hemodynamics and gas transport functions of the CFL in the microcirculation9-12. Furthermore, a separate set of studies has also investigated how the pathological elevation in RBC aggregation modulates CFL formation and its effect on O2 and NO bioavailability in tissues13,14. 
The roles of the CFL become more significant in microcirculation where the relative size of the CFL width to the vessel diameter is prominent. This necessitates an effective approach of quantifying the CFL in in vivo blood flow. Particularly, image acquisition and image analysis are the two key components determining the accuracy of CFL width measurement. Successful visualization of tissue blood flow should be preceded by an appropriate surgical preparation of the animal model. Additionally, a proper image analysis technique is needed to overcome the limitations of conventional manual measurements that are mostly induced by human errors15,16. With advancements in optical instrumentation and computing power for digital image processing, it is now possible to achieve a more accurate and consistent measurement of the CFL width17-19. Nonetheless, the accuracy of these measurements, being image-based, still ultimately depends on the quality of the images.
Therefore, this study explores the factors influencing the measurement of the in vivo CFL width. We focused particularly on demonstrating the surgical preparation and digital image analysis for measurements of the CFL width in arterioles of the rat cremaster muscle.

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			<td height="21" style="height:21px;width:251px;">Intravital microscope</td>
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			<td height="21" style="height:21px;width:251px;">High speed camera</td>
			<td style="width:167px;">Photron</td>
			<td style="width:119px;">1024PCI</td>
			<td>Equipment</td>
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			<td height="21" style="height:21px;width:251px;">Blue filter</td>
			<td style="width:167px;">HOYA</td>
			<td style="width:119px;">B390</td>
			<td>Equipment</td>
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			<td height="21" style="height:21px;width:251px;">Pressure sensor &#38;biopac system</td>
			<td style="width:167px;">Biopac system</td>
			<td style="width:119px;">TSD104A, MP100</td>
			<td>Equipment</td>
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			<td>Equipment</td>
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			<td height="21" style="height:21px;width:251px;">Plasma Lyte A</td>
			<td style="width:167px;">Baxter</td>
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			<td>Warm in 37 &#176;C water bath before use</td>
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			<td height="21" style="height:21px;width:251px;">Heparin (5000 IU/ml)</td>
			<td style="width:167px;">LEO</td>
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			<td height="21" style="height:21px;width:251px;">PE-10 polyethylene tube</td>
			<td style="width:167px;">Becton Dickinson</td>
			<td style="width:119px;">427400</td>
			<td>.024&#34; OD X .011&#34; ID&#160;</td>
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			<td height="21" style="height:21px;width:251px;">PE-50 polyethene tube</td>
			<td style="width:167px;">Becton Dickinson</td>
			<td style="width:119px;">427411</td>
			<td>.038&#34; OD X .023&#34; ID</td>
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			<td height="21" style="height:21px;width:251px;">PE-205 polyethene tube</td>
			<td style="width:167px;">Becton Dickinson</td>
			<td style="width:119px;">427446</td>
			<td>.082&#34; OD X .062&#34; ID</td>
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			<td height="21" style="height:21px;width:251px;">2-0 non-absorbable silk suture</td>
			<td style="width:167px;">Deknatel</td>
			<td style="width:119px;">113-S</td>
			<td></td>
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			<td height="21" style="height:21px;width:251px;">5-0 non-absorbable silk suture</td>
			<td style="width:167px;">Deknatel</td>
			<td style="width:119px;">106-S</td>
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			<td height="21" style="height:21px;width:251px;">Water circulating heating pad</td>
			<td style="width:167px;">Gaymar</td>
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			<td height="21" style="height:21px;width:251px;">Water bath</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:119px;">Isotemp 205</td>
			<td>Equipment</td>
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			<td height="21" style="height:21px;width:251px;">Sterile Cotton Gauze&#160;</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:119px;">22-415-468</td>
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			<td height="21" style="height:21px;width:251px;">Cotton-tipped applicators</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:119px;">23-400-124</td>
			<td></td>
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			<td height="21" style="height:21px;width:251px;">Dumont Forceps</td>
			<td style="width:167px;">Kent Scientific</td>
			<td style="width:119px;">INS14188</td>
			<td>Surgical instrument</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Micro Dissecting forceps</td>
			<td style="width:167px;">Kent Scientific</td>
			<td style="width:119px;">INS15915</td>
			<td>Surgical instrument</td>
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			<td height="21" style="height:21px;width:251px;">Iris forceps 1x2 teeth</td>
			<td style="width:167px;">Kent Scientific</td>
			<td style="width:119px;">INS15917</td>
			<td>Surgical instrument</td>
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			<td height="21" style="height:21px;width:251px;">Vessel cannulation forceps</td>
			<td style="width:167px;">Kent Scientific</td>
			<td style="width:119px;">INS500377</td>
			<td>Surgical instrument</td>
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			<td height="21" style="height:21px;width:251px;">Micro scissor</td>
			<td style="width:167px;">Kent Scientific</td>
			<td style="width:119px;">INS14177</td>
			<td>Surgical instrument</td>
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			<td height="21" style="height:21px;width:251px;">Iris scissor</td>
			<td style="width:167px;">Kent Scientific</td>
			<td style="width:119px;">INS14225</td>
			<td>Surgical instrument</td>
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			<td height="21" style="height:21px;width:251px;">Vessel clip</td>
			<td style="width:167px;">Kent Scientific</td>
			<td style="width:119px;">INS14120</td>
			<td>Surgical instrument</td>
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			<td height="21" style="height:21px;width:251px;">Gemini cautery system</td>
			<td style="width:167px;">Braintree Scientific</td>
			<td style="width:119px;">GEM 5917</td>
			<td>Surgical instrument</td>
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visualization and quantification of the cell-free layer in arterioles of the rat cremaster muscle

The cell-free layer is defined as the parietal plasma layer in the microvessel flow, which is devoid of red blood cells. The measurement of the in vivo cell-free layer width and its spatiotemporal variations can provide a comprehensive understanding of hemodynamics in microcirculation. In this study, we used an intravital microscopic system coupled with a high-speed video camera to quantify the cell-free layer widths in arterioles in vivo. The cremaster muscle of Sprague-Dawley rats was surgically exteriorized to visualize the blood flow. A custom-built imaging script was also developed to automate the image processing and analysis of the cell-free layer width. This approach enables the quantification of spatiotemporal variations more consistently than previous manual measurements. The accuracy of the measurement, however, partly depends on the use of a blue filter and the selection of an appropriate thresholding algorithm. Specifically, we evaluated the contrast and quality of images acquired with and without the use of a blue filter. In addition, we compared five different image histogram-based thresholding algorithms (Otsu, minimum, intermode, iterative selection, and fuzzy entropic thresholding) and illustrated the differences in their determination of the cell-free layer width.

La visualización de la CFL in vivo depende en gran medida de las preparaciones quirúrgicas de la animal. pérdida excesiva de sangre o la duración de la cirugía prolongada se puede someter al animal a golpes y aberraciones del flujo sanguíneo. El mantenimiento de la temperatura del tejido usando una almohadilla de calefacción, así como una plataforma personalizada durante la cirugía y el experimento también es crucial para el mantenimiento de las condiciones fisiológicas de la rata. Mediante el uso de...

Watch this Scientific Journal Video about Visualization and Quantification of the Cell-free Layer in Arterioles of the Rat Cremaster Muscle at JoVE.com

Visualization and Quantification of the Cell-free Layer in Arterioles of the Rat Cremaster Muscle

This study demonstrates the surgical preparation of the rat cremaster muscle for the visualization of the in vivo cell-free layer. Considerable factors affecting the accuracy of the cell-free layer width measurement are discussed in this study.

La visualización y cuantificación de la Capa libre de células en las arteriolas del músculo cremáster Rata

The cell-free layer is defined as the parietal plasma layer in the microvessel flow, which is devoid of red blood cells. The measurement of the in vivo ...

visualization-quantification-cell-free-layer-arterioles-rat-cremaster

Research

JoVE Journal

Biology

6.3K vistas.  National University of Singapore. This study demonstrates the surgical preparation of the rat cremaster muscle for the visualization of the in vivo cell-free layer. Considerable factors affecting the accuracy of the cell-free layer width measurement are discussed in this study.

Video: Visualization and Quantification of the Cell-free Layer in Arterioles of the Rat Cremaster Muscle

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

La cuantificación de γH2AX focos en respuesta a la radiación ionizante

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Investigación

Biología

Purification of Progenitors from Skeletal Muscle

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing in a "satellite cell position" between the myofiber plasma membrane and the laminin-rich basement membrane that ensheaths it, are self-renewing cells that are solely committed to the myogenic lineage1,2. We have recently described a second class of vessel associated progenitors that can generate myofibroblasts and white adipocytes, which responds to damage by efficiently entering proliferation and provides trophic support to myogenic cells during tissue regeneration3,4. One of the most trusted assays to determine the developmental and regenerative potential of a given cell population relies on their isolation and transplantation5-7. To this end we have optimized protocols for their purification by flow cytometry from enzymatically dissociated muscle, which we will outline in this article. The populations obtained using this method will contain either myogenic or fibro/adipogenic colony forming cells: no other cell types are capable of expanding in vitro or surviving in vivo delivery. However, when these populations are used immediately after the sort for molecular analysis (e.g qRT-PCR) one must keep in mind that the freshly sorted subsets may contain other contaminant cells that lack the ability of forming colonies or engrafting recipients.

Method for the enzymatic dissociation, surface labeling and purification by flow cytometry of fibro/adipogenic and myogenic progenitors from murine skeletal muscle.

Purificación de los progenitores del músculo esquelético

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing ...

The Mouse Cremaster Muscle Preparation for Intravital Imaging of the Microcirculation

Throughout the body, the maintenance of homeostasis requires the constant supply of oxygen and nutrients concomitant with removal of metabolic by-products. This balance is achieved by the movement of blood through the microcirculation, which encompasses the smallest branches of the vascular supply throughout all tissues and organs. Arterioles branch from arteries to form networks that control the distribution and magnitude of oxygenated blood flowing into the multitude of capillaries intimately associated with parenchymal cells. Capillaries provide a large surface area for diffusional exchange between tissue cells and the blood supply. Venules collect capillary effluent and converge as they return deoxygenated blood towards the heart. To observe these processes in real time requires an experimental approach for visualizing and manipulating the living microcirculation.
The cremaster muscle of rats was first used as a model for studying inflammation using histology and electron microscopy post mortem1,2. 
The first in vivo report of the exposed intact rat cremaster muscle investigated microvascular responses to vasoactive drugs using reflected light3. However 
curvature of the muscle and lack of focused illumination limited the usefulness of this preparation. The major breakthrough entailed opening the muscle, 
detaching it from the testicle and spreading it radially as a flat sheet for transillumination under a compound microscope4. While shown to be a valuable 
preparation to study the physiology of the microcirculation in rats5 and hamsters6, the cremaster muscle in mice7 has proven particularly useful in dissecting 
cellular pathways involved in regulating microvascular function8-11 and real-time imaging of intercellular signaling12. 
The cremaster muscle is derived from the internal oblique and transverse abdominus muscles as the testes descend through the inguinal canal13. 
It serves to support (Greek: cremaster = suspender) and maintain temperature of the testes. As described here, the cremaster muscle is prepared as a thin flat sheet 
for outstanding optical resolution. With the mouse maintained at a stable body temperature and plane of anesthesia, surgical preparation involves freeing the muscle 
from surrounding tissue and the testes, spreading it onto transparent pedestal of silastic rubber and securing the edges with insect pins while irrigating it 
continuously with physiological salt solution. The present protocol utilizes transgenic mice expressing GCaMP2 in arteriolar endothelial cells. GCaMP2 is a 
genetically encoded fluorescent calcium indicator molecule12. Widefield imaging and an intensified charge-coupled device camera enable in vivo study of 
calcium signaling in the arteriolar endothelium.

A tissue preparation is described for visualization and experimental manipulation of the living microcirculation. In anesthetized male mice, the thin, highly vascularized cremaster muscle is prepared for intravital microscopy to study microvascular networks including arterioles, capillaries and venules. This preparation is readily adapted for rats and hamsters.

El ratón Cremaster preparación muscular for Imaging intravital de la microcirculación

Throughout the body, the maintenance of homeostasis requires the constant supply of oxygen and nutrients concomitant with removal of metabolic ...

Imaging of Estrogen Receptor-&alpha; in Rat Pial Arterioles using a Digital Immunofluorescent Microscope

Many of estrogen's effects on vascular reactivity are mediated through interaction with estrogen receptors 1, 2, 3. Although two sub-types exist (estrogen receptor -&alpha; and &beta;),estrogen receptor-&alpha; has been identified in both the smooth muscle and in endothelial cells of pial arterial segments using fluorescent staining combined with confocal laser scanning microscopy 4. Furthermore, ER-&alpha; is located in the nuclei and in the cytoplasm of rat basilar arteries 5. The receptors are abundant and fluoresce brightly, but clear visualization of discrete groups of receptors is difficult likely due to the numbers located in many cell layers of pial vessel segments. Additionally, many reports using immunohistochemical techniques paired with confocal microscopy poorly detail the requirements critical for reproduction of experiments 6. Our purpose for this article is to describe a simple technique to optimize the staining and visualization of ER-&alpha; using cross-sectional slices of pial arterioles obtain from female rat brains. We first perfuse rats with Evans blue dye to easily identify surface pial arteries which we isolate under a dissecting microscope. Use of a cryostat to slice 8 &mu;m cross sections of the arteries allows us to obtain thin vessel sections so that different vessel planes are more clearly visualized. Cutting across the vessel rather than use of a small vessel segment has the advantage of easier viewing of the endothelial and smooth muscle layers. In addition, use of a digital immunofluorescent microscope with extended depth software produces clear images of ten to twelve different vessel planes and is less costly than use of a confocal laser scanning microscope.

Imaging of Estrogen Receptor-&#945; in Rat Pial Arterioles using a Digital Immunofluorescent Microscope

The goal of this article is to demonstrate a method to optimize immunofluorescent detection of estrogen receptor-&#945; (ER&#945;) in rat pial arterial slices using a digital immunofluorescent microscope.

Imágenes de los receptores de estrógenos-α en ratas arteriolas Pial usando un microscopio de inmunofluorescencia Digital

Many of estrogen's effects on vascular reactivity are mediated through interaction with estrogen receptors 1, 2, 3. Although two sub-types exist (estrogen ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

Visualización y Análisis de las moléculas de ARNm mediante fluorescencia<em&gt; In Situ</em&gt; La hibridación en<em&gt; Saccharomyces cerevisiae</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to nonmarrow tissue have not been completely clarified. We describe here the technique of intravital microscopy applied to the mouse cremaster microcirculation for analysis of peripheral bone marrow stem cell migration in vivo. Intravital microscopy of the M. cremaster has been previously introduced in the field of inflammatory research for direct observation of leucocyte interaction with the vascular endothelium. Since sufficient peripheral stem and progenitor cell migration includes similar initial steps of rolling along and firm adhesion at the endothelial lining it is conceivable to apply the M. cremaster model for the observation and quantification of the interaction of intravasculary administered stem cells with the endothelium. As various chemical components can be selectively applied to the target tissue by simple superfusion techniques, it is possible to establish essential microenvironmental preconditions, for initial stem cell recruitment to take place in a living organism outside the bone marrow.

Intravital microscopy of the mouse M. cremaster microcirculation offers a unique and well-standardized in vivo model for the analysis of peripheral bone marrow stem cell migration.

Microscopia intravital de la microcirculación en el músculo de ratón Cremaster para el análisis de la migración de la célula madre periféricas

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to ...

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in the striated muscle fibers that appear as highly localized Ca2+ release events mediated by ryanodine receptor (RyR) Ca2+ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca2+ sparks could provide information on the intracellular Ca2+&#160;handling properties of healthy and diseased striated muscles. Although Ca2+ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.
Detailed here is an experimental protocol for measuring Ca2+ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca2+ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca2+ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca2+ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP3R) and RyR contributes to Ca2+ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca2+ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).

Described here is a method to directly measure calcium sparks, the elementary units of Ca2+ release from sarcoplasmic reticulum&#160;in intact skeletal muscle fibers. This method utilizes osmotic-stress-mediated triggering of Ca2+ release from ryanodine receptor in isolated muscle fibers. The dynamics and homeostatic capacity of intracellular Ca2+ signaling can be employed to assess muscle function in health and disease.

Evaluación de calcio en Sparks intactas las fibras musculares esqueléticas

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in ...

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

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with real time imaging, it becomes feasible to determine permeability changes in individual pulmonary microvessels. Herein we describe steps to isolate rat lungs and perfuse them with autologous blood. Then, we outline steps to infuse fluorophores or agents via a microcatheter into a small lung region. Using these procedures described, we determined permeability increases in rat lung microvessels in response to infusions of bacterial lipopolysaccharide. The data revealed that lipopolysaccharide increased fluid leak across both venular and capillary microvessel segments. Thus, this method makes it possible to compare permeability responses among vascular segments and thus, define any heterogeneity in the response. While commonly used methods to define lung permeability require postprocessing of lung tissue samples, the use of real time imaging obviates this requirement as evident from the present method. Thus, the isolated lung preparation combined with real time imaging offers several advantages over traditional methods to determine lung microvascular permeability, yet is a straightforward method to develop and implement.

The isolated blood-perfused lung preparation makes it feasible to visualize microvessel networks on the lung surface. Here we describe an approach to quantify permeability of single microvessels in isolated lungs using real time fluorescence imaging.

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 ...

Isolation of Murine Coronary Vascular Smooth Muscle Cells

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs from the coronary circulation. Hearts with intact aortic arches were removed and perfused via retrograde Langendorff with digestion solution containing 300 Units/ml of collagenase type II, 0.1 mg/ml soybean trypsin inhibitor and 1 M CaCl2. The perfusates were collected at 15 min intervals for 90 min, pelleted by centrifugation, resuspended in plating media, and plated on tissue culture dishes. VSMCs were characterized by presence of SM22&#945;, &#945;-SMA, and vimentin. One of the main advantages of using this technique is the ability to isolate VSMCs from the coronary circulation of mice. Although the small number of cells obtained can limit some of the applications for which the cells can be utilized, isolated coronary VSMCs can be used in a variety of well-established cell culture techniques and assays. Studies investigating VSMCs from genetically modified mice can provide further information about structure-function and signaling processes associated with vascular pathologies.

The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques.

Aislamiento de murino coronaria células musculares lisas vasculares

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

A Rapid Automated Protocol for Muscle Fiber Population Analysis in Rat Muscle Cross Sections Using Myosin Heavy Chain Immunohistochemistry

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle composition. Various time-consuming methods have traditionally been used to study fiber populations in many fields of research. However, recently developed immunohistochemical methods based on myosin heavy chain protein expression provide a quick alternative to identify multiple fiber types in a single section. Here, we present a rapid, reliable and reproducible protocol for improved staining quality, allowing automatic acquisition of whole cross sections and automatic quantification of fiber populations with ImageJ. For this purpose, embedded skeletal muscles are cut in cross sections, stained using myosin heavy chains antibodies with secondary fluorescent antibodies and DAPI for cell nuclei staining. Whole cross sections are then scanned automatically using a slide scanner to obtain high-resolution composite pictures of the entire specimen. Fiber population analyses are subsequently performed to quantify slow, intermediate and fast fibers using an automated macro for ImageJ. We have previously shown that this method can identify fiber populations reliably to a degree of &#177;4%. In addition, this method reduces inter-user variability and time per analyses significantly using the open source platform ImageJ.

Here, we present a protocol for rapid muscle fiber analyses, which allows improved staining quality, and thereby automatic acquisition and quantification of fiber populations using the freely available software ImageJ.

Un Protocolo Automatizado Rápido de Fibra Análisis Población en músculo de rata Secciones representativas Uso de la cadena pesada de miosina inmunohistoquímica

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle ...