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

Questo studio è in accordo con l&#39;Università Nazionale di Singapore Istituzionale cura degli animali e del Comitato uso (protocollo approvato senza. R15-0225). 1. Preparazione chirurgica del modello animale <ol><li> cannulazioni nave <ol><li> Anestetizzare a ratti maschi Sprague-Dawley (6 - 7 settimane) di pesatura (203 ± 20) g con ketamina (37,5 mg / ml) e xilazina (5 mg / ml) cocktail attraverso intraperitoneale (ip) iniezione (2 ml / kg) . Non ricoprire l&#39;ago o rimuoverlo dalla siringa dopo l&#39;iniezione. </li><li> Una volta che l&#39;animale è stato anestetizzato (confermata dalla punta pizzicare), posizionarlo su una piastra elettrica per mantenere la sua temperatura corporea a 37 ° C. radere delicatamente i capelli sulla scapole, collo dell&#39;utero anteriore, basso addome, zampa posteriore mediale e sacco scrotale. frenare delicatamente le gambe utilizzando nastri di carta adesiva. </li><li> Eseguire tutte le procedure chirurgiche con le forbici di microdissezione e pinze ad angolo, mentre la visualizzazione attraversouno stereomicroscopio. Mettere tutti gli strumenti chirurgici taglienti su un vassoio resistente alla perforazione per evitare lesioni durante l&#39;intervento chirurgico. </li><li> Scrub tutti i siti chirurgici 3 volte con alternanza di iodio e il 70% di alcool prima di eseguire l&#39;incisione. Lavare tutti i cateteri con 30 / soluzione di eparina-salina ml UI. </li><li> Fare A 1 - 1,5 centimetri incisione cutanea mediana sulla scapole usando un paio di forbici chirurgiche sopra la vena giugulare destra. Separare la fascia per via smussa per esporre la vena giugulare e cannulate con un tubo di polietilene (PE-50) riempita con eparina-salino con 5-0 punti di sutura in seta. Infondere anestesia supplementare quando necessario (1/3 a 1/2 della dose iniziale, per via endovenosa (iv)) durante tutto il corso della chirurgia e dell&#39;esperimento. </li><li> Eseguire tracheostomia per mantenere la pervietà delle vie aeree. Fare un 1 - cm un&#39;incisione 1.5 nella zona cervicale anteriore. Incannulare la trachea utilizzando un tubo di polietilene (PE-205) con 2-0 suture di seta per fissare il catetere in posizione. </li><li> Monitorare la pressione sanguignaattraverso incannulamento a livello dell&#39;arteria femorale. Fare un 1 - cm un&#39;incisione 1.5 sulla superficie mediale sinistro della zampa posteriore. Separare l&#39;arteria femorale per via smussa. Incannulare l&#39;arteria femorale con un tubo di polietilene (PE-10) riempito di eparina-salina utilizzando 5-0 suture di seta. </li></ol></li><li> Cremaster muscolare Preparazione e flusso di visualizzazione <ol><li> Inserire una sutura 5-0 seta attraverso l&#39;apice del sacco scrotale per estenderlo. Fare un&#39;incisione lungo la superficie ventrale del sacco scrotale. Regolarmente applicare soluzione calda isotonica (37 ° C, pH 7,4) al muscolo esposto. </li><li> Rimuovere circostante tessuti connettivi attentamente e accuratamente con un applicatore con punta di cotone. </li><li> Inserire una sutura 5-0 seta attraverso l&#39;apice del muscolo cremastere. Tagliare la sutura in due parti di uguale lunghezza e un nodo su ogni lato. Tagliare il muscolo tra i due nodi e si estendono su una piattaforma di plexiglas trasparente personalizzato tirando delicatamente la sutura. fissarel&#39;estremità della sutura sulla piattaforma con l&#39;aderenza blu. NOTA: la rimozione completa dei circostante tessuto connettivo è di fondamentale importanza per ottenere ottimale contrasto dell&#39;immagine. </li><li> Ripetere 1.2.3 passo fino al 5 a 6 fissazioni sono fatti. Rimuovere con attenzione il muscolo cremastere dall&#39;epididimo utilizzando alte temperature cauterizzazione. Superfuse soluzione isotonica calda al muscolo esposto per prevenire la disidratazione del tessuto. <ol><li> Circondano il muscolo cremastere con pezzi piegati di garza. Coprire il muscolo esposta con un film di polivinile. I pezzi di garza con il film formano un bacino poco profondo per tenere soluzione isotonica calda per l&#39;obiettivo dell&#39;acqua immersione microscopio (Figura 1A). </li></ol></li><li> Trasferire l&#39;animale sul palco animale di un microscopio intravitale (Figura 1C). Collegare l&#39;incannulamento arterioso ad un sistema di acquisizione dati fisiologici per il monitoraggio continuo della pressione (Figura 1E). </li><li> Mantenere la temperat muscolareure a 35 ° C con un elemento riscaldante allegata sotto la piattaforma animale (Figura 1B). Inserire una sonda di temperatura vicino al muscolo per fornire un feedback negativo al controllore potenza dell&#39;elemento riscaldante (Figura 1D). </li><li> Lasciare l&#39;animale sulla scena per 15 minuti per equilibrare con l&#39;ambiente. </li><li> Visualizza il flusso di sangue al microscopio intravitale con un obiettivo di acqua-immersion 40X e un condensatore nel tempo. </li><li> Scegliere un arteriole ramificati (&lt;60 micron) basata su una chiara focalizzazione e il contrasto tra il nucleo RBC, CFL e dalle pareti del recipiente, al fine di concentrarsi microscopio sul piano diametrale del vaso sanguigno. Ruotare la fotocamera montata sul microscopio per allineare la parete del serbatoio verticale. </li><li> Registrare il flusso di sangue utilizzando una videocamera ad alta velocità ad un frame rate di 3.000 / sec per 1 sec. Salvare il video registrato in formato AVI non compresso in scala di grigi a 8 bit per preservare la qualità delle immagini. NOTA:<html> Si raccomanda un frame rate minimo di registrazione di 3.000 fotogrammi / sec per garantire che la misura CFL può essere eseguita almeno una volta al RBC in condizioni di flusso arteriolare fisiologiche. </li><li> Utilizzare un filtro blu con trasmissione di picco alla lunghezza d&#39;onda di 394 nm e banda passante spettrale 310 - 510 nm per aumentare il contrasto tra i globuli rossi e plasma. NOTA: Assicurarsi che lo spettro di luce che passa attraverso il filtro blu dalla sorgente luminosa microscopica (lampada alogena 100 W) è di bassa intensità della luce per evitare qualsiasi potenziale danno tissutale. </li><li> Alla fine dell&#39;esperimento, eutanasia l&#39;animale con una overdose di pentobarbital sodico. </li></ol></li></ol> Analisi 2. Immagine <ol><li> Pre-elaborazione per la misura della larghezza CFL <ol><li> Aprire MATLAB ed eseguire il file &#39;CFL_pre.m&#39;. (Questo ed altri file MATLAB può essere trovato nelip &quot;&gt; Supplemental MATLAB Archive.) </li><li> Fare clic su &#39;Apri file&#39; per selezionare il file video da analizzare. </li><li> Regolare il dispositivo di scorrimento &#39;rotazione&#39; di allineare le pareti dei vasi in verticale. NOTA: Gli utenti possono visualizzare le linee della griglia assistenza per l&#39;allineamento nave selezionando il pulsante di opzione &#39;griglia su&#39;, e regolare il livello di zoom dell&#39;immagine facendo scorrere il cursore &#39;Zoom&#39;. </li><li> Fare clic sul pulsante &#39;Conferma Modifica&#39; per confermare l&#39;allineamento nave. </li><li> Fare clic sul pulsante &#39;Imposta ROI per ritagliare&#39; per definire la regione di interesse (ROI). L&#39;immagine allineata verrà visualizzato in una finestra pop-up. Regolare l&#39;obiettivo rettangolare sull&#39;immagine, e fare doppio clic per confermare il ROI. Ignorare questo passaggio se non è richiesto il ritaglio dell&#39;immagine. NOTA: includere solo una singola nave in ROI per analizzare la larghezza CFL dalla nave. Fare clic sul pulsante &#39;Reset immagine&#39; per ripristinare l&#39;immagine alla sua forma originale, se necessario. </li><li> Clicca il9; Estrarre &#39;pulsante per estrarre tutti i fotogrammi video modificati in immagini po&#39; mappa consecutivi (8-bit in scala di grigi &#39;immagini in formato bmp&#39;). Le immagini estratte possono essere trovate nella cartella con lo stesso nome del file video selezionato. </li></ol></li><li> La misura della larghezza CFL <ol><li> Aprire MATLAB ed eseguire il file &#39;CFL_measure.m&#39;. </li><li> Fare clic su &#39;Seleziona cartella&#39; per selezionare la cartella contenente le immagini estratte. </li><li> Fare clic sulla cartella contenente le immagini e cliccare su &#39;Seleziona cartella&#39;. Il primo fotogramma immagine nella cartella verrà caricato e mostrato nel pannello &#39;Scala di grigi immagine&#39;, insieme con il suo colore grigio istogramma intensità nel pannello &#39;immagine Istogramma&#39;. </li><li> Selezionare la cornice immagine desiderata dalla casella di riepilogo per effettuare l&#39;analisi, in caso contrario verrà selezionato il primo fotogramma dell&#39;immagine. </li><li> Fare clic su &#39;Trova nave Walls&#39; per identificare la parete del vaso interno nell&#39;immagine, che è determinata nel punto in cui illuce profilo di intensità transiti di punta dal buio alla luce più di due pixel. </li><li> Controllare &#39;filtro mediano&#39; applicare un filtro mediano l&#39;immagine per ridurre il rumore &#39;sale e pepe&#39;. </li><li> Controllare &#39;Contrasto automatico&#39; per regolare l&#39;intensità dell&#39;immagine digitale per migliorare il contrasto dell&#39;immagine. </li><li> Selezionare un algoritmo di soglia nella casella di riepilogo che determina automaticamente un valore soglia (τ) che divide i livelli di grigio in due classi - pixel bianchi con livelli di grigio sopra τ (CFL), e pixel neri con livelli di grigio inferiori τ (core RBC). NOTA: Come metodo alternativo, utilizzare soglia manuale se nessuno di algoritmi automatizzati-soglia fornisce un adeguato soglia immagine. Fare clic sul pulsante &#39;Manuale&#39; radio e regolare il cursore per definire il valore soglia manuale. </li><li> Per misurare la variazione spaziale delle larghezze CFL, inserire la risoluzione in pixel nella casella &#39;Pixel Resolution&#39; (la risoluzionecon questa configurazione sperimentale era 0,42 micron / pixel). </li><li> Fare clic sul pulsante &#39;Calcola&#39; per ottenere la variazione spaziale delle larghezze CFL. Fare clic su &#39;Esporta .csv&#39; per esportare i dati di larghezza CFL in forma di tabelle. </li><li> Per misurare la variazione temporale delle larghezze CFL ad una specifica linea di analisi lungo il vaso, cliccare sul pulsante di opzione &#39;temporale Variation&#39; e inserire le informazioni frame rate (il tasso di frame utilizzato in questa configurazione sperimentale era 3.000 fotogrammi / sec). </li><li> Inserisci il primo fotogramma e l&#39;ultimo fotogramma delle immagini per l&#39;analisi dei &#39;Frame Start&#39; e scatole &#39;ultimo fotogramma&#39;, rispettivamente. </li><li> Selezionare la posizione della linea di analisi lungo il vaso facendo scorrere il &#39;Analisi Line&#39; barra di scorrimento. Conferma la posizione della linea di analisi, che è illustrato sia sul &#39;immagine in scala di grigi&#39; e &#39;immagine binaria&#39;. </li><li> Fare clic su &#39;Calcola&#39; per ottenere la variazione temporale delle larghezze CFL. Click &#39;Export .csv&#39; per esportare i dati di larghezza CFL in forma di tabelle. </li></ol></li></ol>

<ol>
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The authors declare no competing financial interests.

La misura della larghezza CFL è essenziale per una migliore comprensione delle emodinamica del microcircolo. In particolare, la misurazione delle larghezze CFL è stata eseguita in mesenterica 6, spinotrapezius 24 e cerebrali 25 microcircolazione. La misura convenzionale in vivo larghezze CFL è stato limitato a stime mediante ispezione manuale dei fotogrammi video registrati. Le misurazioni manuali necessari la media di diversi fotogrammi video successivi prima di identificare...

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 style="width:253px;">Equipment</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>
		</tr>
		<tr height="21">
			<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 height="21" style="height:21px;width:251px;">Temperature controller</td>
			<td style="width:167px;">Shimaden</td>
			<td style="width:119px;">SR 1</td>
			<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>
			<td style="width:119px;">NDC:0338-0221</td>
			<td>Warm in 37 &#176;C water bath before use</td>
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			<td></td>
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			<td height="21" style="height:21px;width:251px;">Heparin (5000 IU/ml)</td>
			<td style="width:167px;">LEO</td>
			<td style="width:119px;"></td>
			<td></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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		<tr height="21">
			<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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		<tr height="21">
			<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>
			<td></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></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>
			<td></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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		<tr height="21">
			<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>
		</tr>
		<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>
		</tr>
		<tr height="21">
			<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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		<tr height="21">
			<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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		<tr height="21">
			<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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		<tr height="21">
			<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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		<tr height="21">
			<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>
		</tr>
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</table>

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 visualizzazione del CFL in vivo dipende in larga misura le preparazioni chirurgiche dell&#39;animale. eccessiva perdita di sangue o la durata intervento chirurgico esteso possono sottoporre l&#39;animale a urti e le aberrazioni del flusso sanguigno. Mantenimento della temperatura del tessuto utilizzando una piastra elettrica, nonché una piattaforma personalizzata durante l&#39;intervento chirurgico e esperimento è cruciale per mantenere le condizioni fisiologiche del ratto. Utilizzando una lampada alogena ...

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.

Visualizzazione e la quantificazione dello strato acellulare nelle arteriole del muscolo Cremaster Rat

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 Views.  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.

Quantificazione dei γH2AX Foci in risposta alle radiazioni ionizzanti

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

Ricerca

Biologia

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.

Purificazione di progenitori dal muscolo scheletrico

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.

La preparazione muscolare mouse Cremaster for Imaging intravitale del microcircolo

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.

Imaging del recettore dell&#39;estrogeno α in Rat Arteriole Pial utilizzando un microscopio digitale immunofluorescenza

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.

Visualizzazione e l&#39;analisi di molecole di mRNA mediante fluorescenza<em&gt; In Situ</em&gt; Ibridazione in<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 intravitale del microcircolo nel muscolo mouse Cremaster per l&#39;analisi di migrazione delle cellule staminali periferiche

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.

Valutazione del calcio Sparks in intatte fibre muscolari scheletriche

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.

Quantificare singolo microvasi Permeabilità in Isolata Blood-perfusione Rat Lung Preparazione

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.

L&#39;isolamento di Murine coronarica vascolari cellule muscolari lisce

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 rapido protocollo automatizzato per Fibra muscolare Analisi Popolazione nel ratto muscolari sezioni trasversali utilizzando una catena pesante della miosina immunoistochimica

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