The authors thank the Central Animal facility of the University Hospital Aachen for technical support. This work was supported by the German Research Foundation (DFG Ta434/2-1, DFG SFB/TRR 57) and by the Interdisciplinary Center for Clinical Research (IZKF) Aachen. This work was further supported by the Core Facility &#8221;Two-Photon Imaging&#8221;, a Core Facility of the Interdisciplinary Center for Clinical Research (IZKF) Aachen within the Faculty of Medicine at RWTH Aachen University.

NOTA: Gli esperimenti sono stati eseguiti in conformità con la normativa tedesca in materia di studi sugli animali in seguito alla &#39;Guida per la cura e l&#39;uso di animali da laboratorio &quot;(pubblicazione NIH, 8 ° edizione, 2011) e la direttiva 2010/63 / UE sulla protezione degli animali utilizzati a fini scientifici (Gazzetta ufficiale dell&#39;Unione europea, 2010). Permesso ufficiale è stata concessa dal governativa cura degli animali e uso ufficio (LANUV Nordrhein-Westfalen, Recklinghausen, Germania). NOTA: I passaggi che possono essere omessi per l&#39;imaging a breve termine (ad esempio, scattare colpi, 3-D pile o anche breve durata microscopia time-lapse) sono contrassegnati con un asterisco (*) per ridurre i tempi di preparazione e semplificare il protocollo chirurgico. Imaging può anche venire eseguito senza ampio controllo e monitoraggio circolazione se necessario, tuttavia, il tempo di sopravvivenza sarà notevolmente ridotto. 1. Setup Microscopio e Pre-chirurgia Preparazione (5-10 kmn) <ol><li> Sistema Accendere (microscopio, potere Lab, laser, rilievo di riscaldamento, camera climatica, web cam, dispositivo pompa a siringa, respiratore). </li><li> Collegare O 2 alimentazione isoflurano vapore e impostare il livello Isoflurano al 1,5 Vol% (v / v), la connessione a un piccolo respiratore animali. <ol><li> Per l&#39;imaging a breve termine, a mantenere l&#39;anestesia con isoflurano solo dopo l&#39;induzione dell&#39;anestesia iniziale ip (vedi punto 2.2). Quindi utilizzare 2 Vol% (v / v) isoflurano, e tenere il tempo di imaging di sotto di 2 ore per garantire la stabilità microcircolazione fegato. </li></ol></li><li> La respirazione a 120-140 cicli respiratori / min Set con un volume di 150-200 ml. </li><li> Impostare palco del mouse agarosio. Preparare 100 ml di 3% (w / v) agarosio, versando in un piatto (di circa 10 cm x 15 cm base) con un angolo di 40 °. </li><li> Impostare area di lavoro chirurgica raccogliere la strumentazione necessaria. Per prevenire l&#39;infezione microbica, disinfettare strumenti utilizzando una soluzione all&#39;1% di Sekusept Forte S (9,9% w / v), Glutaral 9,8% w / v, Formaldeide per 5 minuti prima dell&#39;esperimento (Figura 1A). </li><li> Ottenere rimanenti componenti: disinfettante della pelle (ad esempio, 10% di soluzione di povidone iodio), fegato (fase pile e bridge), soluzione di agarosio (3% (w / v) in PBS), pompa a siringa con 5% (w / v) soluzione di glucosio (G-5), pompa a siringa con soluzione anestetica I (ketamina 0,1 mg / ml, Xilazina 0,5 mg / ml, fentanil 5 mg / ml), tamponi di cotone, n-butil-2-Cyanoacrylat, e 1x PBS. Mettere piatto fase agarosio nella camera di incubazione per il preriscaldamento. </li></ol> 2. tracheotomia (10-15 min) <ol><li> Utilizzare C57BL / 6 topi da casa da riproduzione di almeno 25-28 g. Casa topi in condizioni specifiche esenti da organismi patogeni, in conformità con le linee guida FELASA, in un ambiente a temperatura e umidità controllata con 12 ore di luce / 12 hr ciclo scuro. Consentire animali l&#39;accesso gratuito alla dieta standard topi (Sniff standard roditore Diet) e acqua ad libitum. </li><li> Anestetizzare il mouse initial intraperitoneale (ip) iniezione di 250 microlitri della soluzione di anestetico II (ketamina 0,1 mg / ml, Xilazina 1 mg / ml, Buprenorfina 10 mcg / ml). <ol><li> * Per manutenzione durante l&#39;imaging intravitale, continuamente amministrare soluzione anestetica I di iniezione continua ip (ketamina 0,1 mg / ml, Xilazina 0,5 mg / ml, fentanil 5 mg / ml) utilizzando un dispositivo pompa a siringa con una portata di 0,2 ml / hr in combinazione con inalazione isoflurano 0,5 Vol% (v / v). </li><li> Controllare riflessi dopo 5 min (es pizzico zampe con le pinze), disinfettare la pelle per tracheotomia, iniziare la preparazione se il mouse è in stato di anestesia chirurgica tollerante. </li><li> Applicare occhio crema protettiva (ad esempio, Dexpantenolo 50 mg / g) per evitare di cornea si secchi (Figura 1B). </li></ol></li><li> Fissare il mouse sulla tabella di preparazione esporre lato ventrale con del nastro adesivo per le estremità; tirare troppo delicatamente collo, fissare in questa posizione, ad es., usando un elastico agganciato to gli incisivi. <ol><li> Disinfettare la pelle e la pelliccia con una soluzione di iodio povidone, applicando il disinfettante con un batuffolo di cotone. </li></ol></li><li> Eseguire il taglio della pelle iniziale (0,5-1 cm di lunghezza) direttamente sotto il mento (Figura 1C) <ol><li> Sezionare con cura il tessuto connettivo tra ghiandole salivari (Figura 1D). </li><li> Attenzione allo strappo aperto muscolare tubo circostante trachea (Figura 1E, F). </li></ol></li><li> Mettere filo chirurgico sotto la trachea per dopo tubo di ventilazione fissaggio (Figura 1G). <ol><li> Aprire trachea con micro-forbici tra gli anelli cartilaginei che svolgono una incisione a forma di T (Figura 1 H) </li></ol></li><li> Inserire tubo di ventilazione nella trachea attraverso l&#39;incisione (~ 0.5 cm). Per spingere il tubo in avanti, afferrare fine caudale con piccole pinze anatomiche e far avanzare delicatamente il tubo nella trachea (Figura 1I) </li><li> Fissare il tubo con filo chirurgico (es </em&gt;, 5-0) con filo inserito nel passo 2.5 un nodo intorno tubo all&#39;interno della trachea; effettuare secondo fissaggio craniale del tubo alla pelle per evitare accidentali depositioning (Figura 1J, K). </li><li> Taglio Seal con colla tissutale (ad esempio, n-butil-2-Cyanoacrylat) (Figura 1L). </li><li> Fissare il tubo alla testa con del nastro adesivo. </li></ol> 3. laparotomia (15-20 min) <ol><li> Posizionare il mouse sul tappetino di riscaldamento per evitare l&#39;ipotermia. Shave addome, rimuovere con attenzione i capelli dalla pelle. Disinfettare la pelle rasata e pelliccia utilizzando una soluzione di iodio povidone. </li><li> Eseguire una piccola pelle tagliata sotto lo sterno con le forbici chirurgiche (Figura 2a). Estendere il taglio lateralmente sotto le costole su entrambi i lati, cauterizzare tutti i vasi sanguigni visibili per prevenire le emorragie. </li><li> Eseguire accuratamente un piccolo taglio alla linea alba sotto lo sterno, aprendo il peritoneo (Figura 2B). Estendere il taglio di entrambi i lati con cauterizzione per prevenire le emorragie (Figura 2C, D). </li><li> Posizionare il mouse nel piatto fase agarosio (Figura 2E). Inserire pile di altezza adeguata su entrambi i lati del mouse (solitamente 12-14 coprioggetto) (Figura 2F). </li><li> Posizionare sensore di trigger respiratorio sotto parte superiore della schiena per sincronizzare il microscopio con la respirazione. Attivare unità di innesco (Figura 2G). </li><li> Mettere filo chirurgico (5-0) con lo sterno di ritrattare costole (Figura 2i). </li><li> Tagliare con cautela legamento che collega il fegato e il diaframma (Legamento falciforme), così come il fegato e il tratto gastrointestinale con le forbici chirurgiche curve. Tagliare il legamento falciforme verso l&#39;aorta (Figura 2J). </li></ol> 4. Configurazione di esempio (10-15 min) <ol><li> Posizionare il mouse sul lato destro con un angolo di 45 ° per un facile accesso al grande lobo epatico. </li><li> Aggiungere scena ponte sotto le costole, che copre il addominalecavità. Fabbricazione un ponte utilizzando un vetrino di serie con rivestimento del nastro adesivo per coprire spigoli vivi (Figura 2K). </li><li> Luogo grande lobo epatico sul palco. Scivolare delicatamente doppia sfera della sonda stilo o una spatola imbottita sotto il fegato e tenere la parte superiore dell&#39;organo con un tampone di cotone bagnato o tessuto bagnato. Sollevare lobo sul vetrino e applicare gentile trascinare. Lobe può essere piegato o piegata. Fornire attenzione a solo delicata manipolazione del fegato (Figura 2L). </li><li> Posizionare pali laterali di supporto, ad esempio, pile di piccoli slittamenti di copertura (20 mm x 20 mm), pari a circa la stessa altezza del lobo epatico accanto ad essa per sostenere vetrino. </li><li> Mettere grande vetrino (24 mm x 50 mm) sul lobo epatico. Assicurarsi che la copertura di slittamento è orientato più orizzontale possibile (Figura 2M). NOTA: Il vetrino deve essere in contatto con il tessuto senza schiacciarla. Verificare la presenza di segni visibili di compromissione del flusso sanguigno (tessuto bianco). Se MICROCIRClamento è perturbato, aggiungere ulteriore supporto pali. </li><li> * Inserire due cateteri intraperitoneali indipendenti (Figura 2N) per l&#39;anestesia a lungo termine e G-5 applicazione. Installare cateteri lateralmente nel basso addome accanto agli arti posteriori. NOTA: I cateteri intraperitoneale possono essere fatto da sé, collegando 27 aghi G con tubi in silicone flessibile (Figura 3). </li><li> * Fixate ago con un anello di 5-0 filo chirurgico alla pelle per evitare spostamenti accidentali. </li><li> * Collegare pompa a siringa con soluzione di anestesia I CATETERE 1, portata impostato a 0,2 ml / h; collegare pompa a siringa con G-5 a catetere 2, portata impostato a 0,1 ml / h. </li></ol> 5. Monitoraggio mouse <ol><li> * Elettrodi ECG posto in estremità anteriore e posteriori (figura 4a). </li><li> * Collegare il tubo di scadenza di CO 2 sensore di livello. </li><li> Aggiungi il sensore di temperatura esterna collegata al pad di calore (Figura 4C). </li></ol> 6. Integrazione e Tissue Fixation (5-10 min) <ol><li> Preparare 100 ml di 3% (w / v) agarosio in 1X PBS. Incorpora fegato quando la temperatura è a 41 ° C con una siringa da 5 ml e un ago da 18 G (Figura 5A). </li><li> Versare agarosio rimanente sul coprioggetto e intorno al mouse (Figura 5B, C). Attendere fino agarosio è completamente gelatinizzato. </li><li> Rimuovere agarosio in eccesso con la spatola Heidemann, la preparazione di un Viewfield abbastanza grande per eseguire la scansione del lobo epatico predisposto (Figura 5D, E). </li></ol> 7. Imaging <ol><li> Trasferire il mouse nella camera microscopio clima. </li><li> Aggiungere 50-100 ml di PBS 1x (preriscaldata a 37 ° C). </li><li> Coprire piatto del campione per evitare l&#39;evaporazione (figura 5F). </li><li> * Ridurre isoflurano inalazione al 0,5 Vol% (v / v). </li><li> * Avviare il software di monitoraggio, la registrazione ECG, frequenza cardiaca e CO 2 espiratorio. </li><li> Inizio di imaging: aprire lapersiane ser, identificano i campi di vista di interesse, definiscono limite superiore e inferiore per Z-stack, e avviare la registrazione lasso di tempo. Regolare Z-stack, se necessario, correggere Z-drift (ad es. A causa di cambiamenti nella pressione sanguigna o la variazione di temperatura, Figura (5G). NOTA: Al termine del periodo di imaging o se la circolazione o anestesia degli animali diventa instabile, sacrificare i topi da dislocazione cervicale (senza risveglio dall&#39;anestesia). </li></ol>

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V., 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=Two-photon+laser+scanning+microscopy+imaging+of+intact+spinal+cord+and+cerebral+cortex+reveals+requirement+for+CXCR6+and+neuroinflammation+in+immune+cell+infiltration+of+cortical+injury+sites.">Two-photon laser scanning microscopy imaging of intact spinal cord and cerebral cortex reveals requirement for CXCR6 and neuroinflammation in immune cell infiltration of cortical injury sites.</a> J Immunol Methods. 352 (1-2), 89-100 (2010).</li><li>Karlmark, K. R., 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=Hepatic+recruitment+of+the+inflammatory+Gr1++monocyte+subset+upon+liver+injury+promotes+hepatic+fibrosis.">Hepatic recruitment of the inflammatory Gr1+ monocyte subset upon liver injury promotes hepatic fibrosis.</a> Hepatology. 50 (1), 261-274 (2009).</li><li>Heymann, F., 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=Hepatic+macrophage+migration+and+differentiation+critical+for+liver+fibrosis+is+mediated+by+the+chemokine+receptor+C-C+motif+chemokine+receptor+8+in+mice.">Hepatic macrophage migration and differentiation critical for liver fibrosis is mediated by the chemokine receptor C-C motif chemokine receptor 8 in mice.</a> Hepatology. 55 (3), 898-909 (2012).</li><li>Ramachandran, 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=Differential+Ly-6C+expression+identifies+the+recruited+macrophage+phenotype,+which+orchestrates+the+regression+of+murine+liver+fibrosis.">Differential Ly-6C expression identifies the recruited macrophage phenotype, which orchestrates the regression of murine liver fibrosis.</a> Proc Natl Acad Sci U S A. 109 (46), E3186-E3195 (2012).</li><li>Moles, 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=A+TLR2/S100A9/CXCL-2+signaling+network+is+necessary+for+neutrophil+recruitment+in+acute+and+chronic+liver+injury+in+the+mouse.">A TLR2/S100A9/CXCL-2 signaling network is necessary for neutrophil recruitment in acute and chronic liver injury in the mouse.</a> J Hepatol. 60 (4), 782-791 (2014).</li><li>Hammerich, L., 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=Chemokine+receptor+CCR6-dependent+accumulation+of+&#947;&#948;+T+cells+in+injured+liver+restricts+hepatic+inflammation+and+fibrosis.">Chemokine receptor CCR6-dependent accumulation of &#947;&#948; T cells in injured liver restricts hepatic inflammation and fibrosis.</a> Hepatology. 59 (2), 630-642 (2014).</li><li>Syn, W. -. K., 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=NKT-associated+hedgehog+and+osteopontin+drive+fibrogenesis+in+non-alcoholic+fatty+liver+disease.">NKT-associated hedgehog and osteopontin drive fibrogenesis in non-alcoholic fatty liver disease.</a> Gut. 61 (9), 1323-1329 (2012).</li><li>McDonald, B., 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=Interaction+of+CD44+and+hyaluronan+is+the+dominant+mechanism+for+neutrophil+sequestration+in+inflamed+liver+sinusoids.">Interaction of CD44 and hyaluronan is the dominant mechanism for neutrophil sequestration in inflamed liver sinusoids.</a> J Exp Med. 205 (4), 915-927 (2008).</li><li>Egen, J. G., 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=Intravital+imaging+reveals+limited+antigen+presentation+and+T+cell+effector+function+in+mycobacterial+granulomas.">Intravital imaging reveals limited antigen presentation and T cell effector function in mycobacterial granulomas.</a> Immunity. 34 (5), 807-819 (2011).</li><li>Singer, G., Stokes, K. Y., Granger, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatic+microcirculation+in+murine+sepsis:+role+of+lymphocytes.">Hepatic microcirculation in murine sepsis: role of lymphocytes.</a> Pediatr Surg Int. 24 (1), 13-20 (2008).</li><li>Phillipson, M., Kubes, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neutrophil+in+vascular+inflammation.">The neutrophil in vascular inflammation.</a> Nat Med. 17 (11), 1381-1390 (2011).</li><li>Khandoga, A. 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The authors disclose no con&#64258;ict of interests.

Lo scopo del nostro studio è stato quello di sviluppare un metodo altamente standardizzato, stabile e riproducibile per l&#39;imaging intravitale TPLSM del fegato. Imaging intravitale in generale ha dato preziose informazioni sul comportamento cellulare in condizioni di vita reale seguenti homing e l&#39;interazione di diverse popolazioni di leucociti nello sviluppo, omeostasi e la malattia. Tuttavia, la posizione anatomica piuttosto impegnativa del fegato, a causa della quale respiratorio e movimento intestinale peris...

La visualizzazione delle cellule e funzioni cellulari di organi interi o persino interi organismi è stato di grande interesse per più di 50 anni, tra cui quasi tutte le parti del corpo 2. Pertanto, alcuni primi studi già impiegati l&#39;imaging intravitale del fegato 3,4. Tuttavia, alcune limitazioni esistono aggiornati riguardo a lungo termine ad alta risoluzione stabile di imaging di tessuto epatico. A causa della posizione anatomica del fegato in stretto contatto con il diaframma e il tratto gastrointestinale 5, il problema più comune per l&#39;imaging microscopico intravitale è movimento dovuto alla respirazione e, in misura minore, peristaltica del tratto intestinale 6. In confronto ad altri organi solidi, chirurgia epatica è particolarmente impegnativo. A causa della struttura microvascolare densa, manipolazione chirurgica può portare a massicce lesioni emorragiche, alterata microcircolazione 7 e anche l&#39;attivazione di residente icellule mmune come le cellule di Kupffer 8. Pertanto, fissaggio meccanico del tessuto come pubblicato altrove 6,9 rischia di interferire con imaging microscopia intravitale. In un fegato sano, 10-15% del volume totale del sangue risiede all&#39;interno del sistema vascolare del fegato, e l&#39;organo riceve circa il 25% della gittata cardiaca complessiva 10, rendendo l&#39;organo altamente sensibili alle variazioni di circolazione (ad esempio, variazioni di pressione sanguigna ). Di conseguenza, le interruzioni nel flusso epatico causa ad esempio, shear stress, spostamento, lesioni da uso eccessivo tessuto o circolazione centralizzata porterà ad alterazioni artificiali in leucociti comportamento migratorio, ridotta ossigenazione del fegato e quindi ulteriori danni al fegato, che colpisce fegato risposte immunitarie come pure come conservazione dell&#39;organo e la durata complessiva dell&#39;animale. I primi studi microscopici erano basati su mi epifluorescenza intravitalemicroscopia, ma molti vincoli tecnici, quali foto sbiancamento e la profondità di penetrazione bassa limitano l&#39;uso di questa tecnica per il lungo termine l&#39;imaging del fegato 4,11,12. Con lo sviluppo della microscopia multiphoton nel 1990, le limitazioni della foto sbianca o profondità di penetrazione erano principalmente risolti, come questo nuovo metodo era tecnicamente in grado di svolgere studi di imaging in quasi tutti gli organi sotto situazioni reali 13-15. Tuttavia, le principali sfide rimanenti rispetto all&#39;imaging fegato sono stati: i movimenti del respiro, autofluorescenza di tessuto epatico, che fissano inalterato il flusso di sangue nelle sinusoidi epatici, e di imaging particolarmente stabile per un periodo di diversi hr 16 più lunghi. Anche se diversi studi hanno affrontato la funzione e la migrazione dei vari leucociti nel fegato 17, ad esempio, NKT cellule 18-20, le cellule T 21,22, i macrofagi del fegato 23,24 o neutrofili 25, a lungo termine multiphoton mImaging icroscopy non era ancora stato stabilito con successo, un compito ancora più impegnativo in animali affetti da malattia epatica acuta o cronica a causa dei danni esistente e quindi maggiore suscettibilità a ulteriori danni 26. Tuttavia, il monitoraggio comportamento migratorio e la funzione cellulare dei leucociti nel fegato in tempo reale consente nuove intuizioni nel loro ruolo specifico nella omeostasi del fegato e malattie 27. Il CXCR6 recettore delle chemochine è espresso in diverse sottopopolazioni linfocitarie, tra cui natural killer (NK), le cellule NKT e alcune popolazioni di cellule T 18,28. Precedenti studi sui topi hanno indicato che CXCR6 e la sua CXCL16 ligando affine possono controllare il pattugliamento delle cellule NKT su sinusoidi del fegato durante l&#39;omeostasi. Di conseguenza, l&#39;uso di topi CXCR6.gfp (portando un knock-in della proteina fluorescente verde [GFP] nel locus CXCR6) è stata descritta per studiare la migrazione dei linfociti in vari organi come il cervello 29e anche il fegato 18,20, mostrando aumento infiltrazione di cellule CXCR6.gfp su infiammazione. Con il metodo fornito in questo studio è stato possibile seguire questi processi per un lungo periodo di tempo in condizioni stabilizzate. La procedura basata multiphoton intravitale permesso di imaging che era altamente riproducibile con il minimo turbamento dell&#39;animale e l&#39;organo; ottimizzata per il lungo termine animali sopravvivenza da un ampio monitoraggio seguito da vicino il controllo della respirazione e della circolazione; e altamente flessibile e facile da adottare anche ad altri organi parenchimatosi come il rene, milza.

<table border="0" cellpadding="0" cellspacing="0" width="1184">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Anesthetics</td>
			<td style="width:263px;"></td>
			<td style="width:219px;"></td>
			<td style="width:293px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Buprenorphine</td>
			<td style="width:263px;">Essex Pharma</td>
			<td style="width:219px;">997.00.00</td>
			<td>Analgeticum, 0.1 mg/kg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Fentanyl</td>
			<td style="width:263px;">Rotex Medica</td>
			<td style="width:219px;">charge: 30819</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Fluovac anesthesia system</td>
			<td style="width:263px;">Harvard Apparatus</td>
			<td style="width:219px;">34-1030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Glucose 5%</td>
			<td style="width:263px;">Braun</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ISOFLO (Isoflurane Vapor) vaporiser</td>
			<td style="width:263px;">Eickemeyer</td>
			<td style="width:219px;">4802885</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Isoflurane</td>
			<td style="width:263px;">Forene Abbott</td>
			<td style="width:219px;">B 506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Isotonic (0.9%) NaCl solution</td>
			<td style="width:263px;">DeltaSelect GmbH</td>
			<td style="width:219px;">PZN 00765145</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Ketamin 10%</td>
			<td style="width:263px;">ceva</td>
			<td style="width:219px;">Charge: 36217/09</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;"></td>
			<td style="width:263px;"></td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Xylazin 2%</td>
			<td style="width:263px;">medistar</td>
			<td style="width:219px;">Charge: 04-03-9338/23</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;"></td>
			<td style="width:263px;"></td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Consumable supplies</td>
			<td style="width:263px;"></td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">20ml Syringe</td>
			<td style="width:263px;">BD Plastipak</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">250ml Erlenmeyer flask</td>
			<td style="width:263px;">Schott Duran</td>
			<td>21 226 36</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">25mL Beaker 2x</td>
			<td style="width:263px;">Schott Duran</td>
			<td style="width:219px;">50-1150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">2ml syringe</td>
			<td style="width:263px;">BD Plastipak</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4-0 Vicryl suture</td>
			<td style="width:263px;">Ethicon</td>
			<td style="width:219px;">V7980</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Agarose</td>
			<td style="width:263px;">commercially available</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Bepanthen Eye and Nose ointment</td>
			<td style="width:263px;">Bayer Vital GmbH</td>
			<td style="width:219px;">6029009.00.00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Change-A-Tip Deluxe High-Temp Cautery Kit</td>
			<td style="width:263px;">Fine Science Tools Inc.</td>
			<td style="width:219px;">18010-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Cotton Gauze swabs</td>
			<td style="width:263px;">Fuhrmann GmbH</td>
			<td style="width:219px;">32014</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Cover Slip 24x50mm</td>
			<td style="width:263px;">ROTH</td>
			<td style="width:219px;">1871</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Durapore silk tape</td>
			<td style="width:263px;">3M</td>
			<td style="width:219px;">1538-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Feather disposable scalpel</td>
			<td style="width:263px;">Feather</td>
			<td style="width:219px;">02.001.30.011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Fine Bore Polythene Tubing 0,58mm ID</td>
			<td style="width:263px;">Smiths medical</td>
			<td style="width:219px;">800/100/200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Histoacryl</td>
			<td style="width:263px;">Braun</td>
			<td style="width:219px;">1050052</td>
			<td>5x 0,5ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Leukoplast</td>
			<td style="width:263px;">BSN Medical Inc.</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Microscope Slides</td>
			<td style="width:263px;">ROTH</td>
			<td style="width:219px;">1879</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Poly-Alcohol Haut&#8230;farblos Antisepticum</td>
			<td style="width:263px;">Antiseptica GmbH</td>
			<td style="width:219px;">72PAH200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Sterican needle 18 G x 1</td>
			<td style="width:263px;">B. Braun</td>
			<td style="width:219px;">304622</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Sterican needle 27 3/4 G x 1</td>
			<td style="width:263px;">B. Braun</td>
			<td style="width:219px;">4657705</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Tissue paper</td>
			<td style="width:263px;">commercially available</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;"></td>
			<td style="width:263px;"></td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Surgical Instruments</td>
			<td style="width:263px;"></td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amalgam burnisher 3PL</td>
			<td style="width:263px;">Gatz</td>
			<td style="width:219px;">0110?</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Blair retractors (4 pronged (blunt)) x2</td>
			<td style="width:263px;">Storz&#38;Klein</td>
			<td style="width:219px;">S-01134</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dumont No.7 forceps</td>
			<td style="width:263px;">Fine Science Tools Inc.</td>
			<td style="width:219px;">91197-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Graefe forceps curved x1</td>
			<td style="width:263px;">Fine Science Tools Inc.</td>
			<td style="width:219px;">11151-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Graefe forceps straight x2</td>
			<td style="width:263px;">Fine Science Tools Inc.</td>
			<td style="width:219px;">11050-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heidemann spatula HD2</td>
			<td style="width:263px;">Stoma</td>
			<td style="width:219px;">2030.00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Needle holder Mathieu</td>
			<td style="width:263px;">Fine Science Tools Inc.</td>
			<td style="width:219px;">12010-14</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Scissor</td>
			<td style="width:263px;">Fine Science Tools Inc.</td>
			<td style="width:219px;">14074-11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Semken forceps</td>
			<td style="width:263px;">Fine Science Tools Inc.</td>
			<td style="width:219px;">11008-13</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Small surgical scissors curved</td>
			<td style="width:263px;">Fine Science Tools Inc.</td>
			<td style="width:219px;">14029-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Small surgical scissors straight</td>
			<td style="width:263px;">Fine Science Tools Inc.</td>
			<td style="width:219px;">14028-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Standard pattern forceps</td>
			<td style="width:263px;">Fine Science Tools Inc.</td>
			<td style="width:219px;">11000-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vannas spring scissors</td>
			<td style="width:263px;">Fine Science Tools Inc.</td>
			<td style="width:219px;">15000-08</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;"></td>
			<td style="width:263px;"></td>
			<td style="width:219px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Equipment</td>
			<td style="width:263px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">ECG Trigger Unit</td>
			<td style="width:263px;">Rapid Biomedical</td>
			<td style="width:219px;">3000003686</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:411px;">MICROCAPSTAR End-Tidal Carbon Dioxide Analyzer</td>
			<td style="width:263px;">AD Instruments</td>
			<td style="width:219px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Minivent Typ 845</td>
			<td style="width:263px;">Harvard Apparatus</td>
			<td>73-0043</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Multiphoton microscope Trimscope I</td>
			<td style="width:263px;">LaVision</td>
			<td style="width:219px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Perfusor Compact</td>
			<td style="width:263px;">B. Braun</td>
			<td style="width:219px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:411px;">PowerLab 8/30 8 channel recorder</td>
			<td style="width:263px;">AD Instruments</td>
			<td style="width:219px;">PL3508</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Temperature controlled heating pad</td>
			<td style="width:263px;">Sygonix</td>
			<td style="width:219px;">26857617</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Temperature sensor</td>
			<td style="width:263px;">comercially available</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Temperature controlled System for Microscopes -Cube&#38;Box</td>
			<td style="width:263px;">Life Imaging Services</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
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long term intravital multiphoton microscopy imaging of immune cells in healthy and diseased liver using cxcr6.gfp reporter mice

<html> Fegato infiammazione come risposta al danno è un processo altamente dinamico che coinvolge l&#39;infiltrazione di distinti sottotipi di leucociti tra cui monociti, neutrofili, sottogruppi di cellule T, cellule B, natural killer (NK) e le cellule NKT. Microscopia intravitale del fegato per monitorare la migrazione delle cellule immunitarie è particolarmente difficile a causa degli elevati requisiti per quanto riguarda la preparazione del campione e la fissazione, risoluzione ottica e animale sopravvivenza a lungo termine. Tuttavia, la dinamica dei processi infiammatori così come studi di interazione cellulare potrebbe fornire informazioni fondamentali per comprendere meglio l&#39;iniziazione, la progressione e la regressione della malattia epatica infiammatoria. Pertanto, un metodo altamente sensibile ed affidabile è stato istituito per studiare migrazione e cellula-cellula-interazioni di diverse cellule immunitarie in fegato di topo per lunghi periodi (circa 6 ore) di intravitale due fotoni microscopia a scansione laser (TPLSM) in combinazione con terapia intensiva monitoraggio. ent &quot;&gt; Il metodo fornito comprende una preparazione dolce e fissazione stabile del fegato con il minimo turbamento dell&#39;organo; di imaging intravitale lungo termine utilizzando la microscopia multicolor multiphoton praticamente senza photobleaching o effetti fototossici per un periodo di tempo massimo di 6 ore, consentendo il monitoraggio di specifici sottoinsiemi di leucociti, e le condizioni di imaging stabile a causa monitoraggio estensivo di topo parametri vitali e la stabilizzazione della circolazione, la temperatura e lo scambio di gas. Per studiare la migrazione dei linfociti su infiammazione del fegato CXCR6.gfp knock-in topi sono stati sottoposti a esami del fegato intravitale in condizioni basali e dopo danno epatico acuto e cronico indotto da un&#39;iniezione intraperitoneale (s) di tetracloruro di carbonio (CCL 4). CXCR6 è un recettore per chemochine espressi sui linfociti, soprattutto su Natural Killer T (NKT) -, Natural Killer (NK) - e sottoinsiemi di linfociti T come le cellule T CD4 ma anche Associ mucoseATED invariante cellule (MAIT) T 1. Seguendo il modello migratorio e il posizionamento di CXCR6.gfp + cellule immunitarie permesso una visione dettagliata del loro comportamento alterato sul danno epatico e quindi il loro potenziale coinvolgimento nella progressione della malattia.

Per convalidare il nostro approccio TPLSM intravitale, abbiamo sottoposto CXCR6 GFP / + topi per l&#39;imaging intravitale TPLSM. I topi sono stati o non trattata come controlli di base o sottoposti a una singola iniezione intraperitoneale di tetracloruro di carbonio (CCL 4) per indurre un danno epatico acuto 20. Le sequenze video sono state scattate in un periodo di tempo di 2-5 ore, e le cellule sono state tracciate nel corso del tempo a causa della loro fl...

Watch this Scientific Journal Video about Long Term Intravital Multiphoton Microscopy Imaging of Immune Cells in Healthy and Diseased Liver Using CXCR6.Gfp Reporter Mice at JoVE.com

Long Term Intravital Multiphoton Microscopy Imaging of Immune Cells in Healthy and Diseased Liver Using CXCR6.Gfp Reporter Mice

Stable intravital high-resolution imaging of immune cells in the liver is challenging. Here we provide a highly sensitive and reliable method to study migration and cell-cell-interactions of immune cells in mouse liver over long periods (about 6 hours) by intravital multiphoton laser scanning microscopy in combination with intensive care monitoring.

Long Term intravitale Multiphoton Microscopia Imaging di cellule immunitarie in sani e malati di fegato Uso Mice CXCR6.Gfp Reporter

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, ...

long-term-intravital-multiphoton-microscopy-imaging-immune-cells

Research

JoVE Journal

Immunology and Infection

11.8K Views.  RWTH University-Hospital Aachen. Stable intravital high-resolution imaging of immune cells in the liver is challenging. Here we provide a highly sensitive and reliable method to study migration and cell-cell-interactions of immune cells in mouse liver over long periods (about 6 hours) by intravital multiphoton laser scanning microscopy in combination with intensive care monitoring.

Video: Long Term Intravital Multiphoton Microscopy Imaging of Immune Cells in Healthy and Diseased Liver Using CXCR6.Gfp Reporter Mice

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

Sette passi per Stellate Cellule

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

Ricerca

Immunologia e infezioni

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic tools and surgical procedures, TPM becomes a powerful technique to identify "niches" inside organs and to document cellular "behaviors" in live animals. While intravital imaging provides information that best resembles the real cellular behavior inside the organ, it is both more laborious and technically demanding in terms of required equipment/procedures than alternative ex vivo imaging acquisition. Thus, we describe a surgical procedure and novel "stereotactic" organ holder that allows us to follow the movements of Foxp3+ cells within the thymus.
Foxp3 is the master regulator for the generation of regulatory T cells (Tregs). Moreover, these cells can be classified according to their origin: ie. thymus-differentiated Tregs are called "naturally-occurring Tregs" (nTregs), as opposed to peripherally-converted Tregs (pTregs). Although significant amount of research has been reported in the literature concerning the phenotype and physiology of these T cells, very little is known about their in vivo interactions with other cells. This deficiency may be due to the absence of techniques that would permit such observations. The protocol described in this paper provides a remedy for this situation.
Our protocol consists of using nude mice that lack an endogenous thymus since they have a punctual mutation in the DNA sequence that compromises the differentiation of some epithelial cells, including thymic epithelial cells. Nude mice were gamma-irradiated and reconstituted with bone marrows (BM) from Foxp3-KIgfp/gfp mice. After BM recovery (6 weeks), each animal received embryonic thymus transplantation inside the kidney capsule. After thymus acceptance (6 weeks), the animals were anesthetized; the kidney containing the transplanted thymus was exposed, fixed in our organ holder, and kept under physiological conditions for in vivo imaging by TPM. We have been using this approach to study the influence of drugs in the generation of regulatory T cells.

We have developed novel laboratory tools and protocols for intravital imaging acquisition of the thymus. Our technique should help in the identification of &#x201C;niches&#x201D; within the thymus where T cell development occurs.

Imaging intravitale del Timo mouse utilizzando 2-Photon Microscopy

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic ...

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model itself. Early phases of inflammation and infection have been widely studied by using the Pseudomonas aeruginosa agar-beads mouse model, while only few reports have focused on the long-term chronic infection in vivo. The main challenge for long term chronic infection remains the low bacterial burden by P. aeruginosa and the low percentage of infected mice weeks after challenge, indicating that bacterial cells are progressively cleared by the host.
This paper presents a method for obtaining efficient long-term chronic infection in mice. This method is based on the embedding of the P. aeruginosa clinical strains in the agar-beads in vitro, followed by intratracheal instillation in C57Bl/6NCrl mice. Bilateral lung infection is associated with several measurable read-outs including weight loss, mortality, chronic infection, and inflammatory response. The P. aeruginosa RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chronic infection. P. aeruginosa colonization may persist in the lung for over three months. Murine lung pathology resembles that of CF patients with advanced chronic pulmonary disease.
This murine model most closely mimics the course of the human disease and can be used both for studies on the pathogenesis and for the evaluation of novel therapies.

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

We describe the agar-beads method to establish persistent long-term chronic Pseudomonas aeruginosa airway infection in the mouse model.

Long Term cronica<em&gt; Pseudomonas aeruginosa</em&gt; Infezione delle vie aeree nei topi

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model ...

Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick tissue samples and organs in health and disease. By controlling the scanning pattern in multi-photon microscopy and applying appropriate numerical algorithms, we developed a striped-illumination approach, which enabled us to achieve 3-fold better axial resolution and improved signal-to-noise ratio, i.e. contrast, in more than 100 &#181;m tissue depth within highly scattering tissue of lymphoid organs as compared to standard multi-photon microscopy. The acquisition speed as well as photobleaching and photodamage effects were similar to standard photo-multiplier-based technique, whereas the imaging depth was slightly lower due to the use of field detectors. By using the striped-illumination approach, we are able to observe the dynamics of immune complex deposits on secondary follicular dendritic cells &#8211; on the level of a few protein molecules in germinal centers.

High-resolution intravital imaging with enhanced contrast up to 120 &#181;m depth in lymph nodes of adult mice is achieved by spatially modulating the excitation pattern of a multi-focal two-photon microscope. In 100 &#181;m depth we measured resolutions of 487 nm (lateral) and 551 nm (axial), thus circumventing scattering and diffraction limits.

Altamente Risolto intravitale Striped-illuminazione Microscopia dei Centri Germinal

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick ...

Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

Intravital microscopy (IVM) is a powerful optical imaging technique that has made possible the visualization, monitoring and quantification of various biological events in real time and in live animals. This technology has greatly advanced our understanding of physiological processes and pathogen-mediated phenomena in specific organs.
In this study, IVM is applied to the mouse liver and protocols are designed to image in vivo the circulatory system of the liver and measure red blood cell (RBC) velocity in individual hepatic vessels. To visualize the different vessel subtypes that characterize the hepatic organ and perform blood flow speed measurements, C57Bl/6 mice are intravenously injected with a fluorescent plasma reagent that labels the liver-associated vasculature. IVM enables in vivo, real time, measurement of RBC velocity in a specific vessel of interest. Establishing this methodology will make it possible to investigate liver hemodynamics under physiological and pathological conditions. Ultimately, this imaging-based methodology will be important for studying the influence of L. donovani infection&#160;on hepatic hemodynamics.
This method can be applied to other infectious models and mouse organs and might be further extended to pre-clinical testing of a drug&#8217;s effect on inflammation by quantifying its effect on blood flow.

Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

This article reports on a detailed method for the dynamic measurement and quantification of blood flow velocity within individual blood vessels of the mouse liver vasculature using intravital microscopy imaging in combination with a specific methodology for image acquisition and analysis.

Intravitale Microscopia Imaging del Fegato seguente<em&gt; Leishmania</em&gt; Infezione: Valutazione della epatiche Emodinamica

Intravital microscopy (IVM) is a powerful optical imaging technique that has made possible the visualization, monitoring and quantification of various ...

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 ...

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target cells. Inside the host cell the effector proteins manipulate cellular functions to the benefit of the bacteria. To better understand the control of type III secretion during host cell interaction, sensitive and accurate assays to measure translocation are required. We here describe the application of an assay based on the fusion of a Yersinia enterocolitica effector protein fragment (Yersinia outer protein; YopE) with TEM-1 beta-lactamase for quantitative analysis of translocation. The assay relies on cleavage of a cell permeant FRET dye (CCF4/AM) by translocated beta-lactamase fusion. After cleavage of the cephalosporin core of CCF4 by the beta-lactamase, FRET from coumarin to fluorescein is disrupted and excitation of the coumarin moiety leads to blue fluorescence emission. Different applications of this method have been described in the literature highlighting its versatility. The method allows for analysis of translocation in vitro and also in in vivo, e.g., in a mouse model. Detection of the fluorescence signals can be performed using plate readers, FACS analysis or fluorescence microscopy. In the setup described here, in vitro translocation of effector fusions into HeLa cells by different Yersinia mutants is monitored by laser scanning microscopy. Recording intracellular conversion of the FRET reporter by the beta-lactamase effector fusion in real-time provides robust quantitative results. We here show exemplary data, demonstrating increased translocation by a Y. enterocolitica YopE mutant compared to the wild type strain.

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Effector translocation into host cells via a type III secretion system is a common virulence strategy among gram-negative bacteria. A beta-lactamase effector fusion based assay for quantitative analysis of translocation was applied. In Yersinia infected cells, conversion of a FRET reporter by the beta-lactamase is monitored using laser scanning microscopy.

Analisi di<em&gt; Yersinia enterocolitica</em&gt; Effector traslocazione in cellule ospiti Utilizzando beta-lattamasi Effector Fusions

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target ...

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 ...

Intravital Imaging of Neutrophil Priming Using IL-1&#946; Promoter-driven DsRed Reporter Mice

Neutrophils are the most abundant leukocytes in human blood circulation and are quickly recruited to inflammatory sites. Priming is a critical event that enhances the phagocytic functionality of neutrophils. Although extensive studies have unveiled the existence and importance of neutrophil priming during infection and injury, means of visualizing this process in vivo have been unavailable. The protocol provided enables monitoring of the dynamic process of neutrophil priming in living animals by combining three methodologies: 1) DsRed reporter signal &#8212; used as a measure of priming 2) in vivo neutrophil labeling &#8212; achieved by injection of fluorescence-conjugated anti-lymphocyte antigen 6G (Ly6G) monoclonal antibody (mAb) and 3) intravital confocal imaging. Several critical steps are involved in this protocol: oxazolone-induced mouse ear skin inflammation, appropriate sedation of animals, repeated injections of anti-Ly6G mAb, and prevention of focus drift during imaging. Although a few limitations have been observed, such as the limit of continuous imaging time (~ 8 hr) in one mouse and the leakage of fluorescein isothiocyanate-dextran from blood vessels in the inflammatory state, this protocol provides a fundamental framework for intravital imaging of primed neutrophil behavior and function, which can easily be expanded to examination of other immune cells in mouse inflammation models.

Intravital Imaging of Neutrophil Priming Using IL-1&amp;#946; Promoter-driven DsRed Reporter Mice

This current protocol employs fluorescent reporters, in vivo labeling, and intravital imaging techniques to enable monitoring of the dynamic process of neutrophil priming in living animals.

Imaging intravitale di neutrofili adescamento Utilizzando IL-1b Promoter-driven DsRed topi reporter

Neutrophils are the most abundant leukocytes in human blood circulation and are quickly recruited to inflammatory sites. Priming is a critical event that ...

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by fluorescence or bioluminescence. We utilize BlaC, an enzyme expressed constitutively by all M. tuberculosis strains. REF allows rapid quantification of bacteria in lungs of infected mice. The same group of mice can be imaged at many time points, greatly reducing costs, enumerating bacteria more quickly, allowing novel observations in host-pathogen interactions, and increasing statistical power, since more animals per group are readily maintained. REF is extremely sensitive due to the catalytic nature of the BlaC enzymatic reporter and specific due to the custom flourescence resonance energy transfer (FRET) or fluorogenic substrates used. REF does not require recombinant strains, ensuring normal host-pathogen interactions. We describe the imaging of M. tuberculosis infection using a FRET substrate with maximal emission at 800 nm. The wavelength of the substrate allows sensitive deep tissue imaging in mammals. We will outline aerosol infection of mice with M. tuberculosis, anesthesia of mice, administration of the REF substrate, and optical imaging. This method has been successfully applied to evaluating host-pathogen interactions and efficacy of antibiotics targeting M. tuberculosis.

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

We describe the optical imaging of mice infected with Mycobacterium tuberculosis (M. tuberculosis) using reporter enzyme fluorescence (REF). This protocol facilitates the sensitive and specific detection of M. tuberculosis in pre-clinical animal models for pathogenesis, therapeutics and vaccine research.

Tubercolosi del micobatterio in topi con fluorescenza enzima Reporter di imaging

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by ...