L&#39;intero protocollo dopo l&#39;isolamento delle cellule richiede circa 2 ore. 1. Microsurgical Preparazione <ol><li> Eseguire isolamento delle cellule staminali del midollo osseo del donatore e marcatura fluorescente della sottopopolazione isolato secondo protocolli standardizzati 9,12 permettere alle cellule di riposare durante il tempo viene eseguita l&#39;operazione animale. Per l&#39;etichettatura fluorescente ti consigliamo di CFDA (Carboxyfluorescein diacetate succinimidyl estere). </li><li> Anestetizzare un topo maschio pesa 20-25 g con ketamina (75 mg / kg) e xilazina (2,5 mg / kg). Nel corso degli interventi chirurgici e protocolli sperimentali, anestesia deve essere mantenuta dai integratori (10% di iniezione iniziale, ip) come necessario (di solito ogni 30-60 min). </li><li> Shave delicatamente la faccia anteriore dello scroto destra così come la coscia destra e l&#39;inguine. Raccogliere tutti perdono i capelli con un batuffolo di cotone inumidito. Posizionare il lato ventrale del mouse su un palco di visualizzazione plexiglass e fissare la tassat con telo elastico. Lo stadio è su ordine, costituito da una piastra di base e una seconda piastra sul bordo caudale della piastra di base per l&#39;elevazione di entrambe le gambe e scroto. Posizionare la fase su una piastra elettrica per mantenere la temperatura corporea a 37 ° C. Dopo la fissazione sul palco, mettere l&#39;animale sotto un microscopio di funzionamento ed effettuare le seguenti fasi operative utilizzando 10 - 16 volte di ingrandimento. </li><li> Rendere l&#39;incisione iniziale con le forbici pelle alla coscia destra, solo anteriore dei vasi femorali dal ginocchio fino all&#39;inguine. </li><li> Identificare l&#39;arteria femorale e seguirla prossimale, mobilitando l&#39;arteria da tessuti molli circostanti. Identificare e tagliare un grosso ramo al testicolo sinistro con termocauterio. Successivamente inserire un soggiorno di sutura superficiale al testicolo sinistro. Lieve trazione faciliterà ulteriormente mobilitazione prossimale dell&#39;arteria femorale. </li><li> Dopo il completamento della mobilitazione prossimale, identificare e tagliare un grosso ramo esecuzione medially alla coscia da termocauterio. Successivamente legare l&#39;arteria femorale nella parte distale della coscia destra. Trazione delicata sulla legatura sarà di aiuto ulteriore preparazione. </li><li> Girare l&#39;attenzione ora l&#39;arteria femorale prossimale di nuovo e mettere una sutura intorno all&#39;arteria quanto prossimale possibile. Preparare un nodo, ma non legarlo giù ancora. Applicare una leggera trazione per la sutura e creare un attorcigliamenti arteriosa. </li><li> Dopo aver stabilito il piegamento, incise l&#39;arteria viene accuratamente usando un microscissor. Fate attenzione a non danneggiare la vena femorale. </li><li> Inserire un microcatetere preparata (diametro interno 0,28 millimetri) attraverso l&#39;incisione. Il catetere deve essere anidro (cloruro di sodio 0,9%, NaCl) e collegato ad una siringa da 1 ml prima dell&#39;inserimento. </li><li> Dopo successo inserzione del catetere, allentare attentamente il piegamento per agevolare il passaggio del catetere. Flusso di sangue arterioso dovrebbe essere immediatamente visibile all&#39;interno del catetere. Far avanzare delicatamente il catetere pochi millimetris oltre il piegamento precedente. Successivamente, lega giù la legatura predisposto per il fissaggio del catetere. </li><li> Lavare delicatamente il catetere (0,9% NaCl) e aspirare a confermare la posizione corretta. Usare un pezzo di nastro adesivo per fissare ulteriormente il catetere per la fase di esercizio. </li><li> Dopo la fissazione adeguata, mettere il catetere da parte per le seguenti fasi di preparazione del muscolo cremastere. Applicare lavaggio regolare e l&#39;aspirazione del catetere ogni 5-10 minuti, al fine di prevenire la formazione di coaguli. </li><li> Girare l&#39;attenzione ora allo scroto destra e fare un&#39;incisione tagliando la pelle e la fascia sopra l&#39;aspetto ventrale dello scroto destra. Fare attenzione a non toccare il tessuto sottostante con qualsiasi strumento. </li><li> Estendere l&#39;incisione fino alla piega inguinale e caudalmente all&#39;estremità distale dello scroto. </li><li> Tessuto accuratamente separato sottostante morbido e connettivo sopra il sacco Cremaster, senza toccarlo. Identificare l&#39;estremità distale del Cremaster, e inserire un soggiorno sutura qui per slightly estendere il muscolo. Resecare ora restante del tessuto molle sotto il sacco muscolo tirando delicatamente il muscolo caudale e anteriormente. </li><li> Dopo la separazione completa da tessuto connettivo, aprire il sacco cremastere in un punto sul bordo craniale con le forbici. Estendere l&#39;incisione da questa apertura fino all&#39;estremità distale del muscolo. </li><li> Sigillare la piccola imbarcazione che collega il testicolo e cremastere utilizzando termocauterio, successivamente tagliare i restanti collegamenti tra il muscolo e il testicolo con le forbici. </li><li> Mettere da parte il testicolo, fuori dal campo microscopia. </li><li> Cremastere aperto ora adagiata sul palco. Mettere quattro 6-0 suture Prolene ai bordi del tessuto e fissarli con drappeggio elastico, al fine di diffondere il tessuto del muscolo cremastere. Da ora in poi inumidire continuamente il tessuto con soluzione salina tamponata con fosfato (PBS) usando una siringa da 1 ml (Figura 1). </li><li> Lasciare ora la preparazione riposare per 15 min. Successivamente iniettare0,05-0,1 ml di 1% rodamina-destrano attraverso l&#39;arteria femorale sinistra per migliorare il contrasto del microcircolo. Se si prevedono eventuali chemochine o agenti infiammatori per l&#39;applicazione, si dovrebbe ora topica inumidire il muscolo con il rispettivo reagente con una siringa da 1 ml. Nel caso di un esperimento di controllo, continuare inumidendo con PBS. </li></ol> 2. Intravitale Microscopia <ol><li> Mantenere il mouse fissato sulla fase di esercizio e muoversi sotto un microscopio a fluorescenza modificato per epi-illuminazione e collegato ad un CCD (dispositivo ad accoppiamento di carica) videocamera. Fate attenzione a non dislocare il catetere arteria femorale. </li><li> Condurre esperimenti utilizzando un obiettivo immersione in acqua. </li><li> Dopo aver regolato l&#39;obiettivo per la visualizzazione sufficiente del microcircolo Cremaster e prima iniezione di cellule, stocasticamente definire sei venule postcapillari per una successiva analisi di ferma adesione delle cellule staminali. </li><li> Somministrare cellule staminali fluorescenti marcato,disciolto in 200 microlitri di PBS, a 0.4 x 10 6 cellule per iniezione, con un totale di cinque iniezioni consecutive utilizzando il catetere femorale e una siringa da 1 ml. Agitare con precauzione il campione cellulare prima della prima iniezione. </li><li> Cellule staminali Record rotolamento immediatamente dopo l&#39;iniezione delle cellule, definendo così &quot;rolling&quot; come una riduzione superiore al 50% della velocità cella lungo il rivestimento endoteliale in combinazione con tipico cellulare &quot;bastone e rilasciare&quot; movimenti. Effettuare il conteggio delle cellule staminali di laminazione e di tutte le cellule staminali che passano una distanza venulare predefinita per 1 min di osservazione dopo ogni singola iniezione. La quantità di rotolamento cellule staminali è espressa come percentuale di tutte le cellule che passano. Non contare le cellule espositrici occasionali fenomeni tethering brevi come cellule di rotolamento. </li><li> Dopo l&#39;ultima iniezione cella determinare il numero di cellule staminali saldamente aderenti contando e indica la quantità in relazione alla superficie endoteliale calcolato di the sei venule predefiniti (diametro x lunghezza x π) come cellule aderenti / mm 2. Adesione Studio è considerato quando non il movimento delle cellule è rilevabile per 30 sec. </li><li> Dopo il completamento della microscopia intravitale rimuovere delicatamente il muscolo cremastere e memorizzarlo in corrispondenza successiva analisi (immunoistochimica o biologia molecolare). Sacrifica l&#39;animale da dislocazione cervicale. L&#39;analisi delle registrazioni microscopia intravitale per la quantificazione del gambo rotolamento cellulare e ferma adesione endoteliale, nonché emodinamica microvascolari, come la velocità del sangue rosso cella (mm / sec), la lunghezza della nave (micron), diametro del vaso (micron), parete tasso quota (sec -1); deve essere eseguita in linea da un investigatore cieco al rispettivo trattamento tessutale sperimentale utilizzando un software di elaborazione delle immagini. </li></ol>

<ol>
	<li>Madlambayan, G. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=marrow+stem+and+progenitor+cell+contribution+to+neovasculogenesis+is+dependent+on+model+system+with+SDF-1+as+a+permissive+trigger.">marrow stem and progenitor cell contribution to neovasculogenesis is dependent on model system with SDF-1 as a permissive trigger.</a> Blood. 114, 4310-4319 (2009).</li><li>Lapidot, T., Dar, A., Kollet, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+do+stem+cells+find+their+way+home?.">How do stem cells find their way home?.</a> Blood. 106, 1901-1910 (2005).</li><li>Lehr, H. A., Vollmar, B., Vajkoczy, P., Menger, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+fluorescence+microscopy+for+the+study+of+leukocyte+interaction+with+platelets+and+endothelial+cells.">Intravital fluorescence microscopy for the study of leukocyte interaction with platelets and endothelial cells.</a> Methods Enzymol. 300, 462-481 (1999).</li><li>Gavins, F. N., Chatterjee, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+microscopy+for+the+study+of+mouse+microcirculation+in+anti-inflammatory+drug+research:+focus+on+the+mesentery+and+cremaster+preparations.">Intravital microscopy for the study of mouse microcirculation in anti-inflammatory drug research: focus on the mesentery and cremaster preparations.</a> J. Pharmacol. Toxicol. Methods. 49, 1-14 (2004).</li><li>Leister, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+peritoneal+cavity+chamber+for+intravital+microscopy+of+the+liver+under+conditions+of+pneumoperitoneum.">A peritoneal cavity chamber for intravital microscopy of the liver under conditions of pneumoperitoneum.</a> Surg. Endosc. 17, 939-942 (2003).</li><li>Sriramarao, P., Anderson, W., Wolitzky, B. A., Broide, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+bone+marrow-derived+mast+cells+roll+on+P-selectin+under+conditions+of+flow+in+vivo.">Mouse bone marrow-derived mast cells roll on P-selectin under conditions of flow in vivo.</a> Lab Invest. 74, 634-643 (1996).</li><li>Baez, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+open+cremaster+muscle+preparation+for+the+study+of+blood+vessels+by+in+vivo+microscopy.">An open cremaster muscle preparation for the study of blood vessels by in vivo microscopy.</a> Microvasc. Res. 5, 384-394 (1973).</li><li>Bagher, P., Segal, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mouse+cremaster+muscle+preparation+for+intravital+imaging+of+the+microcirculation.">The mouse cremaster muscle preparation for intravital imaging of the microcirculation.</a> J. Vis. Exp. , e2874 (2011).</li><li>Kaminski, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+NOS+is+required+for+SDF-1alpha/CXCR4-mediated+peripheral+endothelial+adhesion+of+c-kit++bone+marrow+stem+cells.">Endothelial NOS is required for SDF-1alpha/CXCR4-mediated peripheral endothelial adhesion of c-kit+ bone marrow stem cells.</a> Lab Invest. 88, 58-69 (2008).</li><li>Furlani, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HMGB-1+induces+c-kit++cell+microvascular+rolling+and+adhesion+via+both+toll-like+receptor-2+and+toll-like+receptor-4+of+endothelial+cells.">HMGB-1 induces c-kit+ cell microvascular rolling and adhesion via both toll-like receptor-2 and toll-like receptor-4 of endothelial cells.</a> J. Cell Mol. Med. 16, 1094-1105 (2012).</li><li>Furlani, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+the+intravascular+administration+of+mesenchymal+stem+cells+safe?+Mesenchymal+stem+cells+and+intravital+microscopy.">Is the intravascular administration of mesenchymal stem cells safe? Mesenchymal stem cells and intravital microscopy.</a> Microvasc. Res. 77, 370-376 (2009).</li><li>Donndorf, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysing+migratory+properties+of+human+CD+133(+)+stem+cells+in+vivo+after+intraoperative+sternal+bone+marrow+isolation.">Analysing migratory properties of human CD 133(+) stem cells in vivo after intraoperative sternal bone marrow isolation.</a> Cell Transplant. , (2012).</li></ol>

Tutti gli esperimenti sugli animali, condotti per questo manoscritto sono stati approvati dalla cura degli animali e l&#39;uso comitato locale. Gli autori dichiarano di non avere interessi finanziari in competizione.

<html> Microscopia a fluorescenza intravital permette la visualizzazione diretta e l&#39;analisi quantitativa delle interazioni cellula-endotelio staminali. Il modello cremastere muscolare è particolarmente utile, poiché l&#39;esposizione microchirurgico e le tecniche di visualizzazione intravitale sono stati ben stabiliti nel corso della ricerca sulle interazioni leucociti-endotelio e vari componenti chimici può essere applicato selettivamente al tessuto bersaglio semplice tecnica superfusione. A causa dell&#39;assen...

Lo scopo del presente articolo è quello di descrivere la tecnica della microscopia intravitale (IVM) applicato sul cremastere microcircolazione del mouse per l&#39;osservazione diretta e l&#39;analisi del midollo osseo migrazione di cellule staminali periferiche. Rigenerazione dei tessuti e organi Il concetto attuale di cellule staminali basato comporta la homing delle cellule staminali del midollo osseo al tessuto danneggiato 1. Un passo fondamentale per la migrazione di cellule staminali successo include staminali interazione delle cellule con l&#39;endotelio locale all&#39;interno dell&#39;organo feriti seguita dalla migrazione transendoteliale e, infine, organo attecchimento 2. Analisi intravital di queste cellule staminali - interazioni cellule endoteliali costituisce un parametro ideale per la quantificazione di una risposta rigenerativa basata sulle cellule staminali in differenti impostazioni fisiopatologiche in vivo. Inoltre, sembra concepibile, che, in futuro, analisi intravital della capacità migratoria di specifico p cellule staminaliopulations all&#39;interno di ambienti microcircolazione standardizzati, potrebbero essere applicati prima avanzando queste popolazioni a ulteriori test clinici. Microscopia intravital è stato inizialmente sviluppato per l&#39;osservazione e la quantificazione delle interazioni cellula-endoteliale dei leucociti in vivo nel campo della ricerca infiammatorio 3. Le prime registrazioni di successo di studi di microscopia intravitale sono stati riportati da Cohnheim già nel 19 ° secolo, studiando lingue e mesenteri rana sotto un microscopio ottico 4. Dal suo primo uso IVM ha subito un enorme sviluppo tecnico e oggi alcuni componenti essenziali costituiscono prerequisiti per eseguire IVM quantitativa: (i) preparazioni di tessuto che consentono l&#39;accesso ottico, (ii) sonde molecolari che possono essere rilevati da un microscopio, (iii ) un microscopio collegato ad un sistema di rilevamento e (iv) sistemi di analisi computerizzati in grado di estrarre i parametri di interesse dati di immagineset 4. Una varietà di preparazioni di tessuto è stato introdotto per gli studi IVM compresi i mesenteri e il fegato di topo e nel ratto 5, le camere plica dorsale di topo 6 e il criceto, l&#39;orecchio di coniglio e il sacchetto criceto guancia per citarne alcuni. Tuttavia, nel seguito ci concentreremo sul muscolo cremastere topo, che rappresenta un tessuto ideale per le osservazioni intravitale, come la preparazione e la visualizzazione sono possibili da una procedura chirurgica ben standardizzata, e in generale non si verifichino problemi di artefatti da movimento. La preparazione cremastere muscolare aperta si è svolta per la prima volta nel 1970 da Baez e colleghi 7. Originariamente descritto per i ratti, è stato adottato con successo anche al mouse 8. Dopo gli studi precedenti si erano concentrati principalmente sulle interazioni leucociti con la parete del vaso, il nostro gruppo ha recentemente introdotto il mouse preparazione cremastere muscolo come valutaziostrumento BLE per visualizzazione diretta e l&#39;analisi quantitativa di staminali interazioni cellula-endotelio in un microambiente definito 9. Varie sottopopolazioni di cellule staminali sono state studiate utilizzando questo modello, compresi murino c-kit + cellule staminali del midollo osseo e di cellule staminali mesenchimali, nonché CD umano cellule staminali del midollo osseo 133 + 10-12. Dopo aver isolato cellule da donatore di midollo osseo e marcatura fluorescente per la visualizzazione, le cellule staminali vengono selettivamente applicati nella microcircolazione cremastere utilizzando una iniezione arteriosa attraverso l&#39;arteria femorale, evitando così qualsiasi intrappolamento cella all&#39;interno organi remoti. Inoltre, il modello cremastere muscolare è particolarmente utile in quanto varie chemochine potenzialmente mediano la migrazione delle cellule staminali locale entro le rispettive impostazioni, possono essere applicati localmente al tessuto bersaglio di semplice tecnica superfusione.

<table><tbody><tr><td>Carboxy-fluorescein diacetate succinimidylester (CFDA)</td><td>Invitrogen, Carlsbad, CA, USA</td><td>C1157</td><td></td></tr><tr><td>Rhodamine 6 G (1%)</td><td>Sigma-Aldrich, Munich, Germany</td><td>83697</td><td></td></tr><tr><td>Ketamin (Ketanest)</td><td>Pfizer, Berlin, Germany</td><td>not available</td><td></td></tr><tr><td>Xylacin (Rompun)</td><td>Bayer, Leverkusen, Germany</td><td>not available</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate - buffered saline (PBS)</td><td>PAN Biotech, Aidenbach, Germany</td><td>P04-36500</td><td></td></tr></tbody></table>

intravital microscopy of the microcirculation in the mouse cremaster muscle for the analysis of peripheral stem cell migration

Nell&#39;era della intravascolari protocolli di applicazione di cellule nel contesto della terapia cellulare rigenerativa, i meccanismi alla base della migrazione di cellule staminali per nonmarrow tessuto non sono stati completamente chiariti. Descriviamo qui la tecnica della microscopia intravitale applicata al cremastere microcircolazione del mouse per l&#39;analisi del midollo osseo periferico migrazione delle cellule staminali in vivo. Microscopia intravitale del cremastere M. è stato precedentemente introdotto nel campo della ricerca infiammatorio per l&#39;osservazione diretta dell&#39;interazione dei leucociti con l&#39;endotelio vascolare. Da staminali periferiche sufficiente e la migrazione delle cellule progenitrici include analoghe fasi iniziali di laminazione lungo e ferma adesione alla endoteliali che rivestono è concepibile applicare il modello cremastere M. per l&#39;osservazione e la quantificazione dell&#39;interazione delle cellule staminali somministrate intravasculary con l&#39;endotelio. Come vari componenti chimici possono essere applicati selettivamente al bersaglio trilascio da semplici tecniche di superfusione, è possibile stabilire condizioni essenziali del microambiente, per l&#39;assunzione iniziale di cellule staminali che si terrà in un organismo vivente al di fuori del midollo osseo.

In generale, l&#39;interazione di direttamente iniettato cellule staminali o progenitrici con l&#39;endotelio vascolare all&#39;interno della microcircolazione cremastere muscolare è un evento raro e avviene esclusivamente in venule postcapillari (diametro: 30-80 micron). A causa fluorescenza etichettatura rotolamento e cellule staminali saldamente aderenti possono essere chiaramente quantificati in separazione da leucociti circolanti endogeni nelle venule osservati (Figura 2). <p class="jove_conte...

Watch this Scientific Journal Video about Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration at JoVE.com

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

Microscopia intravitale del microcircolo cremastere topo M. offre un unico e ben standardizzato-modello in vivo per l&#39;analisi del midollo osseo migrazione di cellule staminali periferiche.

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 ...

intravital-microscopy-microcirculation-mouse-cremaster-muscle-for

Research

JoVE Journal

Biology

15.2K Views.  University Rostock.  Microscopia intravitale del microcircolo cremastere topo M. offre un unico e ben standardizzato-modello in vivo per l'analisi del midollo osseo migrazione di cellule staminali periferiche. 

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

In vivo Micro-circulation Measurement in Skeletal Muscle by Intra-vital Microscopy

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of imaging had invented. While many macroscopic parameters of blood flow reflect flow velocity, changes in blood flow velocity and red blood cell (RBC) flux does not hold linear relationship in the microscopic observations. There are reports of discrepancy between RBC velocity and RBC flux, RBC flux and plasma flow volume, and of spatial and temporal heterogeneity of flow regulation in the peripheral tissues in microscopic observations, a scientific basis for the requirement of more detailed studies in microcirculatory regulation using intravital microscopy. METHODS: We modified Jeff Lichtman's method of in vivo microscopic observation of mouse sternomastoid muscles. Mice are anesthetized,
ventilated, and injected with PKH26L-fluorescently labeled RBCs for microscopic observation.RESULT &amp; CONCLUSIONS: Fluorescently labeled RBCs are detected and distinguished well by a wide-field microscope. Muscle contraction evoked by electrical stimulation induced increase in RBC flux. Quantification of other parameters including RBC velocity and capillary density were feasible. Mice tolerated well the surgery, injection of stained RBCs, microscopic observation, and electrical stimulation. No muscle or blood vessel damage was observed, suggesting that our method is relatively less invasive and suited for long-term observations.

A new versatile method for observation of microcirculation is presented. It is considered suitable for long-term observation, and for combination with pharmacophysiological or molecular biological interventions.

In vivo micro-circolazione di misura nel muscolo scheletrico da Intra-vitale Microscopia

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of ...

Ricerca

Biologia

Intravital Microscopy of the Mouse Brain Microcirculation using a Closed Cranial Window

This experimental model was designed to assess the mouse pial microcirculation during acute and chronic, physiological and pathophysiological hemodynamic, inflammatory and metabolic conditions, using in vivo fluorescence microscopy. A closed cranial window is placed over the left parieto-occipital cortex of the mice. Local microcirculation is recorded in real time through the window using epi and fluorescence illumination, and measurements of vessels diameters and red blood cell (RBC) velocities are performed. RBC velocity is measured using real-time cross-correlation and/or fluorescent-labeled erythrocytes. Leukocyte and platelet adherence to pial vessels and assessment of perfusion and vascular leakage are made with the help of fluorescence-labeled markers such as Albumin-FITC and anti-CD45-TxR antibodies. Microcirculation can be repeatedly video-recorded over several days. We used for the first time the close window brain intravital microscopy to study the pial microcirculation to follow dynamic changes during the course of Plasmodium berghei ANKA infection in mice and show that expression of CM is associated with microcirculatory dysfunctions characterized by vasoconstriction, profound decrease in blood flow and eventually vascular collapse.

Intravital microscopy to follow temporal and spatial hemodynamic and inflammatory events in the pial microcirculation.

Microscopia intravitale della microcircolazione cervello di topo usando una finestra chiusa del cranio

This experimental model was designed to assess the mouse pial microcirculation during acute and chronic, physiological and pathophysiological hemodynamic, ...

Neuroscienze

Tracking Neutrophil Intraluminal Crawling, Transendothelial Migration and Chemotaxis in Tissue by Intravital Video Microscopy

The recruitment of circulating leukocytes from blood stream to the inflamed tissue is a crucial and complex process of inflammation1,2. In the postcapillary venules of inflamed tissue, leukocytes initially tether and roll on the luminal surface of venular wall. Rolling leukocytes arrest on endothelium and undergo firm adhesion in response to chemokine or other chemoattractants on the venular surface. Many adherent leukocytes relocate from the initial site of adhesion to the junctional extravasation site in endothelium, a process termed intraluminal crawling3. Following crawling, leukocytes move across endothelium (transmigration) and migrate in extravascular tissue toward the source of chemoattractant (chemotaxis)4. Intravital microscopy is a powerful tool for visualizing leukocyte-endothelial cell interactions in vivo and revealing cellular and molecular mechanisms of leukocyte recruitment2,5. In this report, we provide a comprehensive description of using brightfield intravital microscopy to visualize and determine the detailed processes of neutrophil recruitment in mouse cremaster muscle in response to the gradient of a neutrophil chemoattractant. To induce neutrophil recruitment, a small piece of agarose gel (~1-mm3 size) containing neutrophil chemoattractant MIP-2 (CXCL2, a CXC chemokine) or WKYMVm (Trp-Lys-Tyr-Val-D-Met, a synthetic analog of bacterial peptide) is placed on the muscle tissue adjacent to the observed postcapillary venule. With time-lapsed video photography and computer software ImageJ, neutrophil intraluminal crawling on endothelium, neutrophil transendothelial migration and the migration and chemotaxis in tissue are visualized and tracked. This protocol allows reliable and quantitative analysis of many neutrophil recruitment parameters such as intraluminal crawling velocity, transmigration time, detachment time, migration velocity, chemotaxis velocity and chemotaxis index in tissue. We demonstrate that using this protocol, these neutrophil recruitment parameters can be stably determined and the single cell locomotion conveniently tracked in vivo.

We describe a protocol of brightfield intravital microscopy for measuring dynamic neutrophil-endothelial cell interactions during neutrophil recruitment in response to the source of a neutrophil chemoattractant in vivo. Neutrophil intraluminal crawling, transendothelial migration and chemotaxis in mouse cremaster muscle tissue are visualized with time-lapsed video photography and tracked with ImageJ.

Monitoraggio Crawling neutrofili intraluminale, migrazione e chemiotassi transendoteliale in tessuto al microscopio video intravitale

The recruitment of circulating leukocytes from blood stream to the inflamed tissue is a crucial and complex process of inflammation1,2. In the ...

Immunologia e infezioni

Real-time Digital Imaging of Leukocyte-endothelial Interaction in Ischemia-reperfusion Injury (IRI) of the Rat Cremaster Muscle

Ischemia-reperfusion injury (IRI) has been implicated in a large array of pathological conditions such as cerebral stroke, myocardial infarction, intestinal ischemia as well as following transplant and cardiovascular surgery.1 Reperfusion of previously ischemic tissue, while essential for the prevention of irreversible tissue injury, elicits excessive inflammation of the affected tissue. Adjacent to the production of reactive oxygen species, activation of the complement system and increased microvascular permeability, the activation of leukocytes is one of the principle actors in the pathological cascade of inflammatory tissue damage during reperfusion.2, 3 Leukocyte activation is a multistep process consisting of rolling, firm adhesion and transmigration and is mediated by a complex interaction between adhesion molecules in response to chemoattractants such as complement factors, chemokines, or platelet-activating factor.4
While leukocyte rolling in postcapillary venules is predominantly mediated by the interaction of selectins5 with their counter ligands, firm adhesion of leukocytes to the endothelium is selectin-controlled via binding to intercellular adhesion molecules (ICAM) and vascular cellular adhesion molecules (VCAM).6, 7 
Gold standard for the in vivo observation of leukocyte-endothelial interaction is the technique of intravital microscopy, first described in 1968.8
Though various models of IRI (ischemia-reperfusion injury) have been described for various organs, 9-12 only few are suitable for direct visualization of leukocyte recruitment in the microvascular bed on a high level of image quality.8 
We here promote the digital intravital epifluorescence microscopy of the postcapillary venule in the cremasteric microcirculation of the rat 13 as a convenient method to qualitatively and quantitatively analyze leukocyte recruitment for IRI-research in striated muscle tissue and provide a detailed manual for accomplishing the technique. We further illustrate common pitfalls and provide useful tips which should enable the reader to truly appreciate, and safely perform the method.
In a step by step protocol we depict how to get started with respiration controlled anesthesia under sufficient monitoring to keep the animal firmly anesthetized for longer periods of time. We then describe the cremasteric preparation as a thin flat sheet for outstanding optical resolution and provide a protocol for leukocyte imaging in IRI that has been well established in our laboratories.

Real-time Digital Imaging of Leukocyte-endothelial Interaction in Ischemia-reperfusion Injury IRI of the Rat Cremaster Muscle

Digital intravital epifluorescence microscopy of postcapillary venules in the cremasteric microcirculation is a convenient method to gain insights into leukocyte-endothelial interaction in vivo in ischemia-reperfusion injury (IRI) of striated muscle tissue. We here provide a detailed protocol to safely perform the technique and discuss its applications and limitations.

In tempo reale Imaging digitale di leucociti-endotelio in ischemia-riperfusione (IRI) del muscolo Cremaster Rat

Ischemia-reperfusion injury (IRI) has been implicated in a large array of pathological conditions such as cerebral stroke, myocardial infarction, ...

Medicina

Real-time Imaging of Heterotypic Platelet-neutrophil Interactions on the Activated Endothelium During Vascular Inflammation and Thrombus Formation in Live Mice

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 During thrombus formation at the site of arteriolar injury, platelets adherent to the activated endothelium and subendothelial matrix proteins support neutrophil rolling and adhesion.3 Conversely, under venular inflammatory conditions, neutrophils adherent to the activated endothelium can support adhesion and accumulation of circulating platelets. Heterotypic platelet-neutrophil aggregation requires sequential processes by the specific receptor-counter receptor interactions between cells.4 It is known that activated endothelial cells release adhesion molecules such as von Willebrand factor, thereby initiating platelet adhesion and accumulation under high shear conditions.5 Also, activated endothelial cells support neutrophil rolling and adhesion by expressing selectins and intercellular adhesion molecule-1 (ICAM-1), respectively, under low shear conditions.4 Platelet P-selectin interacts with neutrophils through P-selectin glycoprotein ligand-1 (PSGL-1), thereby inducing activation of neutrophil &beta;2 integrins and firm adhesion between two cell types. Despite the advances in in vitro experiments in which heterotypic platelet-neutrophil interactions are determined in whole blood or isolated cells,6,7 those studies cannot manipulate oxidant stress conditions during vascular disease. In this report, using fluorescently-labeled, specific antibodies against a mouse platelet and neutrophil marker, we describe a detailed intravital microscopic protocol to monitor heterotypic interactions of platelets and neutrophils on the activated endothelium during TNF-&alpha;-induced inflammation or following laser-induced injury in cremaster muscle microvessels of live mice.

Here we report an experimental technique of fluorescence intravital microscopy to visualize heterotypic platelet-neutrophil interactions on the activated endothelium during vascular inflammation and thrombus formation in live mice. This microscopic technology will be valuable to study the molecular mechanism of vascular disease and to test pharmacologic agents under pathophysiological conditions.

In tempo reale di immagine degli eterotipica piastrine-neutrofili interazioni sull&#39;endotelio Attivato durante l&#39;infiammazione vascolare e la formazione di trombi in topi vivi

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 ...

Intravital Microscopy of Leukocyte-endothelial and Platelet-leukocyte Interactions in Mesenterial Veins in Mice

Intravital microscopy is a method that can be used to investigate different processes in different regions and vessels in living animals. In this protocol, we describe intravital microscopy of mesentery veins. This can be performed in a short period of time with reproducible results showing leukocyte-endothelial interactions in vivo. We describe an inflammatory setting after LPS challenge of the endothelium. But in this model one can apply many different types of inflammatory conditions, like bacterial, chemical or biological and investigate the administration of drugs and their direct effects on the living animal and its impact on leukocyte recruitment. This protocol has been applied successfully to a number of different treatments of mice and their effects on inflammatory response in vessels. Herein, we describe the visualization of leukocytes and platelets by fluorescently labeling these with rhodamine 6G. Additionally, any specific imaging can be performed using targeted fluorescently labeled molecules.

This simplified application of intravital microscopy of mesentery veins in mice can be used in different models of inflammation to observe leukocyte-endothelial and platelet-leukocyte interactions.

Interazioni intravitale Microscopia di leucociti-endoteliali e piastrine-leucociti in Mesenterial Vene Mice

Intravital microscopy is a method that can be used to investigate different processes in different regions and vessels in living animals. In this ...

Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in vivo is crucial for understanding the molecular basis of leukocyte transendothelial migration and interaction with vascular endothelium. One powerful approach involves intravital microscopy on transgenic mice expressing fluorescent proteins in cells of interest.
Here we present a protocol for imaging monocytes and neutrophils in the CX3CR1gfp/wt mouse i.v. injected with orange dye-labeled neutrophils with an inverted confocal microscope. Time-lapse movies gathered from 30 min&#160;to several hours of imaging allow the analysis of leukocyte behavior in mesenteric veins under both steady state and inflammatory conditions. We also describe the steps to locally induce blood vessel inflammation with TLR2/TLR1 agonist Pam3SK4 and monitor the subsequent recruitment of neutrophils and monocytes.
The presented technique can also be used to monitor other populations of leukocytes and investigate molecules implicated in leukocyte recruitment or trafficking using other stimuli or transgenic mice.

We detail a protocol to monitor the behavior of neutrophils and monocytes in mesenteric veins under steady state and inflammatory conditions using intravital confocal microscopy on anaesthetized mice.

Imaging neutrofili e monociti in mesenterica Veins intravitale Microscopia su topi anestetizzati in tempo reale

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in ...

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 ...

Microiontophoresis and Micromanipulation for Intravital Fluorescence Imaging of the Microcirculation

Microiontophoresis entails passage of current through a micropipette tip to deliver a solute at a designated site within an experimental preparation. Microiontophoresis can simulate synaptic transmission1 by delivering neurotransmitters and neuropeptides onto neurons reproducibly2. Negligible volume (fluid) displacement avoids mechanical disturbance to the experimental preparation. Adapting these techniques to the microcirculation3 has enabled mechanisms of vasodilation and vasoconstriction to be studied at the microscopic level in vivo4,5. A key advantage of such localized delivery is enabling vasomotor responses to be studied at defined sites within a microvascular network without evoking systemic or reflexive changes in blood pressure and tissue blood flow, thereby revealing intrinsic properties of microvessels.
A limitation of microiontophoresis is that the precise concentration of agent delivered to the site of interest is difficult to ascertain6. Nevertheless, its release from the micropipette tip is proportional to the intensity and duration of the ejection current2,7, such that reproducible stimulus-response relationships can be readily determined under defined experimental conditions (described below). Additional factors affecting microiontophoretic delivery include solute concentration and its ionization in solution. The internal diameter of the micropipette tip should be &tilde; 1 &mu;m or less to minimize diffusional 'leak', which can be counteracted with a retaining current. Thus an outward (positive) current is used to eject a cation and a negative current used to retain it within the micropipette.
Fabrication of micropipettes is facilitated with sophisticated electronic pullers8. Micropipettes are pulled from glass capillary tubes containing a filament that 'wicks' solution into the tip of the micropipette when filled from the back end ("backfilled"). This is done by inserting a microcapillary tube connected to a syringe containing the solution of interest and ejecting the solution into the lumen of the micropipette. Micromanipulators enable desired placement of micropipettes within the experimental preparation. Micromanipulators mounted on a movable base can be positioned around the preparation according to the topography of microvascular networks (developed below). 
The present protocol demonstrates microiontophoresis of acetylcholine (ACh+ Cl-) onto an arteriole of the mouse cremaster muscle preparation (See associated protocol: JoVE ID#2874) to produce endothelium-dependent vasodilation. Stimulus delivery is synchronized with digitized image acquisition using an electronic trigger. The use of Cx40BAC-GCaMP2 transgenic mice9 enables visualization of intracellular calcium responses underlying vasodilation in arteriolar endothelial cells in the living microcirculation.

Microiontophoresis entails movement of ions from a micropipette in response to a difference in electrical potential between the inside and outside of the micropipette. Biologically active molecules are thereby delivered in proportion to electrical current. We illustrate acetylcholine microiontophoresis in conjunction with micromanipulation to study endothelium-dependent vasodilation in the microcirculation.

Microiontophoresis e Micromanipolazione per intravitale Imaging di fluorescenza del microcircolo

Microiontophoresis entails passage of current through a micropipette tip to deliver a solute at a designated site within an experimental  preparation. ...

Leukocyte Infiltration of Cremaster Muscle in Mice Assessed by Intravital Microscopy

Intravital microscopy (IVM) is widely used to monitor physiological and pathophysiological processes within the leukocyte recruitment cascade in vivo. The current protocol represents a practical and reproducible method to visualize the leukocyte endothelium interaction leading to leukocyte recruitment in skeletal muscle derived tissue within the intact organism of the mouse. The model is applicable to all fields of research that focus on granulocyte activation and their role in disease.
We provide a step by step protocol to guide through the method and to highlight potential pitfalls and technical difficulties. The protocol covers the following aspects: experimental settings and required material, anesthesia of the mouse, dissection of the cremaster muscle as well as tracheal and carotid cannulation, IVM recordings and offline analysis. Data formats like adherent leukocytes, rolling flux (RF) and rolling flux fraction (RFF) are explained in detail and appropriate applications are discussed. Representative results from dystrophin deficient mdx mice are provided in the results section.
IVM is a powerful tool to assess leukocyte recruitment in an in vivo setting; however, delineating for example endothelial and leukocyte function may require a combination with ex vivo setups like flow chamber experiments. Furthermore, the genetic background of animals of interest may greatly influence baseline recruitment, requiring individual fine tuning of the protocol provided. Despite its limitations, IVM may serve as a platform to readily translate in vitro findings into a living vertebrate organism.

Here, we show how to perform intravital microscopy on post-capillary venules of the mouse cremaster muscle. Commonly applied to different models of inflammation and sepsis, particularly those induced by chemokines and cytokines, we highlight its relevance in the study of muscolopathies involving exaggerated muscular leukocyte infiltration.

Infiltrazione leucocita del muscolo Cremaster nei topi valutata dalla microscopia intravitale

Intravital microscopy (IVM) is widely used to monitor physiological and pathophysiological processes within the leukocyte recruitment cascade in vivo. The ...

Microscopia intravitale del microcircolo nel muscolo mouse Cremaster per l'analisi di migrazione delle cellule staminali periferiche

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