Questa ricerca è stata sostenuta dal INSERM, l&#39;Università di Aix-Marseille e un premio di sviluppo della carriera da HFSPO ottenuto dalla CL Forestier.

Dichiarazione etica: Tutti gli studi sugli animali sono stati eseguiti in conformità con le linee guida e protocolli che sono stati approvati dalla cura e l&#39;uso degli animali Comitato istituzionale del Aix-Marseille Université, Francia. Femminile C57Bl / 6 topi a 8-10 settimane di vita sono stati in commercio ottenuti e trattati secondo le regole di Décret N ° 8 87-848, 19 Ottobre, 1987, Parigi. Tutti gli esperimenti usando L. parassiti donovani LD1S sono state condotte in conformità alla normativa biosicurezza della legislazione francese e dell&#39;Unione europea. 1. L&#39;infezione Mouse con L. donovani promastigote Parassiti <ol><li> Preparare il terreno di coltura per la manutenzione in vitro di L. donovani parassiti promastigote, LD1S (MHOM / SD / 62 / 1S-CL2D). <ol><li> Utilizzare M199 media. Supplemento M199 mezzo con siero bovino fetale al 10% (calore-inattivati ​​a 56 ° C per 30 min),, 1 glu mM HEPES 25 mM pH 6.9, 12 mM NaHCO 3tamine, RPMI 16040 miscela di vitamine, 10 mM di acido folico, adenosina 100 mM, emina 7,6 mM, 50 U / ml di penicillina e 50 mg / ml di streptomicina. </li></ol></li><li> Culture LD1S parassiti in 10 ml di mezzo completo M199 a 26 ° C in un pallone ventilata a 25 cm2. </li><li> Utilizzando successive fasi di centrifugazione differenziale, preparare una popolazione di parassiti promastigote che si arricchiscono in forme metacyclic. <ol><li> Raccolto un mid-log cultura fase di parassita. Conta su un hemocytomer. Risospendere 5 x 10 5 parassiti / ml in 10 ml di mezzo completo M199. Crescere la coltura parassita in condizioni anaerobiche fino a raggiungere la fase stazionaria tardiva (in circa 5-6 giorni). </li><li> Spin cultura LD1S a 1.200 xg per 10 min a 26 ° C a pellet parassiti non metacyclic, recuperare il surnatante e girare nuovamente a 2500 xga 26 ° C per 10 min. Nota: Questo pellet finale è altamente arricchito nei parassiti promastigote metacyclic. </li><li> Risospendere il pellet in 100 ml di PBS. Rimuovere una piccola aliquota per il fissaggio con il 25% glutaraldeide 1/100 v: v e contare i parassiti con un emocitometro. </li></ol></li><li> Diluire la popolazione parassitaria arricchito-metacyclic a 1 x 10 7/100 ml in PBS. Iniettare la sospensione parassita nella vena della coda di un mouse anestetizzato (vedi sotto) usando una siringa da 1 ml con un ago 30 G. Per gli animali di controllo, iniettare 100 microlitri di PBS nella vena della coda. </li></ol> 2. Procedure chirurgiche <ol><li> Disinfettare lo spazio di lavoro con uno spray disinfettante biocida e tutti gli strumenti chirurgici con il 70% di etanolo per 30 minuti. </li><li> Preparare una soluzione di anestetico, secondo il peso dell&#39;animale, contenente 125 mg / kg di ketamina e 12,5 mg / kg di xilazina diluito in PBS. </li><li> Pesare il mouse e intraperitoneale iniettare la dose appropriata di anestetico utilizzando una siringa da 1 ml con un ago 30 G. </li><li> Tenere il mouse caldo e controllare che il mouse è completamente anestheammortizzato pizzicando il rilievo del piede prima di procedere. Re-iniettare una mezza dose della soluzione anestetico nel topo ogni 60 min. </li><li> Shave addome del mouse e pulirlo con Vetedine. Fai una piccola incisione nella pelle e la muscolatura sotto la gabbia toracica sul lato sinistro dell&#39;addome. </li><li> Lay una garza umida sull&#39;addome appena sotto l&#39;area aperta. Esporre il fegato e posizionarlo sul garza. Posare un pezzo di garza umida del fegato. </li><li> Inserire un 24 x 60 mm 2 sino a 150 micron di spessore coprioggetto, cianoacrilato-legato ad un telaio metallico, sul fegato per la visualizzazione al microscopio. </li><li> Proteggere gli occhi del mouse con una garza umida. </li><li> Dopo la sessione di imaging, eutanasia l&#39;animale anestetizzato per dislocazione cervicale. </li></ol> 3. intravitale Microscopia Imaging del Fegato Architettura <ol><li> Eseguire gli esperimenti di microscopia intravitale su un microscopio confocale invertito dotato dicontrollato hermostatic camera, un apocromatico 63X obiettivo glicerolo olio di immersione (NA 1,4), argon (488 nm) e HeNe (543 nm, 633 nm) di laser e di un diodo blu (405 nm). </li><li> Posizionare il mouse sul palco del microscopio con il vetrino che copre il volto di fegato in giù sull&#39;obiettivo. Impostare la temperatura della camera a 29 ° C. Regolare il focus usando l&#39;autofluorescenza di fegato per consentire la visualizzazione delle sinusoidi. </li><li> Per visualizzare la vascolarizzazione, preparare una soluzione di 500 mg BSA-Alexa 647 diluito in 100 ml di PBS, per ogni mouse. Iniettare la soluzione per via endovenosa nella vena della coda del topo anestetizzato utilizzando una siringa da 1 ml con un ago 30 G. </li><li> Per visualizzare i nuclei delle cellule del fegato, preparare una soluzione di Hoechst 33342 in PBS. Iniettare questa soluzione al 8 mg / kg di topo per via intraperitoneale con una siringa da 1 ml con un ago G 30. </li><li> Utilizzare la modalità sequenziale normale, l&#39;obiettivo ad immersione 63X e lo scanner a 400 Hz per l&#39;immagine del epaticanuclei delle cellule e la vascolarizzazione del fegato. Accendere il 405 nm diodo blu per Hoechst l&#39;eccitazione e il laser 633 nm per BSA-Alexa 647 eccitazione. Accendere il nm laser Argon 488 e definire una larga fascia di acquisizione per visualizzare il autofluorescenza fegato. </li></ol> 4. intravitale Microscopia Imaging del Fegato del Sangue di velocità del flusso di misura <ol><li> Aprire il software LAS del microscopio (LAS-AF spettatore Versione 3.1.0 costruire 8587). Selezionare la modalità scanner di risonanza quando si apre il software microscopio. </li><li> Fare clic sulla scheda di configurazione per impostare l&#39;hardware. Clicca sul laser e selezionare il laser HeNe 633. Fare clic su Impostazioni e selezionare la risoluzione 12 bit. </li><li> Fare clic sul menu di acquisizione. Nella finestra delle impostazioni pathway fascio, selezionare l&#39;obiettivo 63X e impostare la potenza del laser 633 al 100%. Attivare il guadagno del segnale e fuori parametri impostati selezionando Alexa-633 dal menu PM1-3 discesa. </li><li> Nella scheda acquisiscono, selezionare l&#39;acquisizione &#39;XYT&#39;modalità. Impostare la larghezza formato a 1.024 x 1.024. Selezionare la velocità a 8.000 Hz. Selezionare il foro stenopeico di 3 unità Airy. Selezionare un fattore di zoom pari a 3. Non selezionare la modalità bidirezionale. </li></ol> 5. Analisi quantitativa di RBC Velocity Utilizzando le XYT Immagini (Metodo 1) <ol><li> Clicca sull&#39;icona &#39;Live&#39; di visualizzare il flusso di sangue in una modalità live. Scegliere l&#39;area di interesse e selezionare un vaso sanguigno specifico per l&#39;analisi. Impostare il vaso di interesse in posizione orizzontale, regolando la &#39;X&#39;, &#39;Y&#39; e scansione di rotazione campo coordinate sul pannello di controllo USB. </li><li> Arrestare la modalità &#39;live&#39; una volta che la nave di interesse è fissato. Cambiare il set larghezza formato 1.024 x 256 nella finestra, media riga = 1, fotogramma media = 1 Impostazione modalità di acquisizione, selezionare &quot;stack&quot; 150. Fare clic su &#39;Start&#39; per acquisire le immagini. </li><li> Per l&#39;analisi quantitativa della velocità RBC utilizzando le immagini &quot;XYT&quot; aprire il software ImageJ. Selezionare &#39;File&#39;scheda in ImageJ, fare clic su &quot;Apri&quot; e poi scegliere il file di interesse. </li><li> Vai al menu &quot;plugin&quot; in ImageJ, selezionare Bio-formati LOCI e le opzioni di importazione. In questa finestra, selezionare lo stack di visualizzazione dei parametri come &#39;HyperStack&#39;. Nell&#39;opzione ordine pila, selezionare l&#39;opzione: &quot;xyzct&quot;. Nota: Si apre una finestra con tutte le serie di acquisizioni. </li><li> Clicca su &#39;Plugin&#39; nel menu ImageJ e selezionare l&#39;opzione MTrackJ. Clicca sull&#39;icona &#39;add&#39; sulla piccola finestra e fare clic sul RBC per tenere traccia. Clicca sulla stessa RBC in ogni fotogramma seguente e cliccare sull&#39;icona &#39;misura&#39; per misurare la velocità. Nota: Il file generato può essere salvato in formato .EXL. </li><li> Traccia di un minimo di cinque globuli rossi per nave e analizzare un minimo di tre recipienti individuali della stessa dimensione. </li></ol> 6. Analisi quantitativa di RBC Velocity Utilizzando le xt Linea Immagini (metodo 2) <ol><li> Clicca su &#39;Live&#39; di visualizzare il sangue fbasso in modalità live. Selezionare un vaso sanguigno specifico per l&#39;analisi. Impostare il vaso di interesse in posizione orizzontale. Arrestare la modalità &#39;live&#39; una volta che la nave di interesse è fissato. </li><li> Selezionare la modalità di scansione &#39;xt&#39; e impostare il lume centrale della nave selezionata lungo la linea di scansione. Imposta larghezza formato a 1.024 x 512, velocità = 8,000Hz, linea di media = 32, tempo = 512. Fare clic su &#39;Start&#39; e acquisire le immagini della linea xt. </li><li> Generare striature di analisi quantitativa della velocità RBC aprendo il file .lif e aprendo l&#39;immagine linea xt di interesse. Nel software LAS-AF, selezionare &quot;esperimenti&quot;, aprire il .lif e selezionare l&#39;immagine. Nota: Si aprirà automaticamente una chimografo. </li><li> Misurare il tempo (t, ottenuto sull&#39;asse Y) necessaria per una striatura particella (mostrando riflessione scuro) a percorrere una determinata distanza (d, ottenuto sull&#39;asse X) su questa chimografo. Selezionate lo strumento &quot;disegnare la linea&quot; e tracciare una linea orizzontale per la striscia. Nota la distanza inmicron e il tempo in secondi generati nella scheda risultati nella parte inferiore dell&#39;immagine. </li><li> Calcola la velocità come V = distanza / tempo utilizzando i valori ottenuti dal chimografo. </li><li> Quantificare un minimo di cinque particelle striature immagine xt e per un minimo di tre recipienti individuali della stessa dimensione. </li></ol>

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+screening+test+using+Leishmania+promastigotes+stably+expressing+mCherry+protein.">In vitro screening test using Leishmania promastigotes stably expressing mCherry protein.</a> Antimicrob. Agents Chemother. 58, 1825-1828 (2014).</li><li>Ferrer, M., Martin-Jaular, L., Calvo, M., del Portillo, H. A. <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+of+the+spleen:+quantitative+analysis+of+parasite+mobility+and+blood.">Intravital microscopy of the spleen: quantitative analysis of parasite mobility and blood.</a> J. Vis. Exp. , (2012).</li><li>Kamoun, W. 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The authors have nothing to disclose.

Il recente sviluppo della microscopia intravitale del fegato del topo apre nuove possibilità per la ricerca di risposta fisiologica alle infezioni in vivo e in tempo reale 5,9,10. Organo flusso di sangue è un parametro fisiologico fondamentale che viene spesso alterata in molte malattie. Tuttavia, lo stato di emodinamica del fegato in condizioni fisiologiche e infettive rimane una zona poco esplorata. In questo studio, metodi basati IVM, che in precedenza sono stati adattati per lo studio della mil...

Emodinamica organo-specifiche sono importanti caratteristiche fisiologiche di qualsiasi organo dei mammiferi. Anomalie nel flusso di sangue possono essere la conseguenza di infiammazione e un segno di disfunzione d&#39;organo 1. Così, il flusso di sangue organizzazione, la struttura e la funzione appaiono parametri critici per l&#39;analisi in condizioni fisiologiche e patologiche. Le tecniche che sono comunemente impiegate per analizzare il flusso di sangue in un organo specifico contengono diverse limitazioni, tra cui il limite di risoluzione della tecnica stessa (es Doppler del flusso di sangue), la capacità di misurazione del flusso sanguigno assoluta solo (volume di sangue per unità che serve un organo) (ad esempio Optical Coherence Tomography) e la misurazione delle variazioni medie di velocità in un grande ed eterogenea popolazione di vasi sanguigni 2,3. Il sistema circolatorio del fegato collega diversi sottotipi vaso eterogenee nella loro dimensioni, struttura e funzione. Inquesto studio, la microscopia intravitale (IVM) tecnologia di imaging è applicata per valutare l&#39;emodinamica fegato in vivo, in tempo reale, ad alta risoluzione e in parallelo per scoprire le caratteristiche dei singoli vasi sanguigni che compongono l&#39;organo epatica. Il recente sviluppo di questa potente tecnica di imaging ottico consente al ricercatore di raccogliere dati dinamici sugli animali che vivono ad una risoluzione spaziale e temporale elevata. Consentendo la visualizzazione diretta e il monitoraggio in tempo reale dei processi biologici specifici e rapidi in vivo, IVM offre un&#39;opportunità unica al ricercatore di vasi sanguigni immagine individuale, e misurare e quantificare la velocità di singole cellule rosse del sangue (globuli rossi) all&#39;interno di un appositamente selezionati nave epatica. In questo studio, abbiamo implementato la tecnica IVM nel fegato mouse per studiare l&#39;influenza di infezione del mouse da parte del parassita Leishmania epatotropico sull&#39;emodinamica fegato. L. donovaniè l&#39;agente responsabile leishmaniosi viscerale, una malattia grave caratterizzata da risposte infiammatorie acute-on-cronica e una patologia che è presente in diversi organi, compreso la milza e nel fegato. In un modello sperimentale murino di leishmaniosi viscerale, l&#39;infezione del fegato è auto-risolvere, mentre l&#39;infezione della milza è progressiva 4. Questi risultati di infezione Leishmania rispetto ai singoli organi non sono ancora completamente compreso. Indagine su fegato e della milza emodinamica in condizioni patologiche getterà nuova luce sulle interazioni ospite-parassita e patogenesi della malattia. Il nostro sistema di modello sperimentale si basa sull&#39;esposizione di imaging e il fegato di un topo anestetizzato che ha ricevuto l&#39;iniezione endovenosa di coloranti fluorescenti specifici per l&#39;etichettatura del intravasculature epatica. Il fegato è un organo favorevole per la microscopia intra-vitale. Dopo aver eseguito un piccolo incision nell&#39;addome, il fegato è delicatamente esternalizzato e collocato su una garza bagnata, poi su un vetrino con l&#39;obiettivo di ridurre eventuali artefatti da movimento a causa di battito cardiaco e la respirazione. Il fegato è poi collocato all&#39;interno della vista di una lente microscopio. Rispetto al nodo milza e linfa che richiedono l&#39;uso di due fotoni microscopia per studi IVM, il vantaggio del fegato risiede nella sua architettura 3D omogeneo / anatomia che consente l&#39;uso di un microscopio confocale convenzionale, con una profondità massima penetrazione circa 50 micron, per la microscopia intravitale immagini 5-8. Questo studio descrive due metodi di imaging indipendenti per la misurazione quantitativa di RBC velocità e la velocità del flusso sanguigno nei singoli vasi sanguigni. Nel primo metodo, flusso ematico del fegato viene eseguita utilizzando una modalità bi-dimensionale xy nel tempo. I dati XYT risultanti vengono analizzati utilizzando il plugin MtrackJ nel software ImageJ gratuito, che consente la tracciabilità dei individUAL globuli rossi nel corso del tempo. Nel secondo metodo, un singolo vaso sanguigno è selezionato e il corrispondente flusso di sangue viene analizzato tramite la scansione di riga modalità veloce acquisizione del microscopio confocale a scansione laser. Il vaso di interesse viene scansionato ad alta frequenza lungo il suo asse centrale attraverso una linea assiale. La velocità del flusso di sangue viene poi quantificato in base alla differenza di contrasto tra senza etichetta eritrociti scuro e plasma fluorescente. Le intensità di fluorescenza di eritrociti e plasma acquisiti lungo la linea di scansione vengono rilevate in tempo per ottenere striature, i cui angoli sono proporzionali alle velocità di un individuo RBC. L&#39;obiettivo di questo articolo è di fornire un metodo semplice e riproducibile per l&#39;imaging e misurando la velocità del flusso di sangue all&#39;interno dei singoli vasi sanguigni del fegato e di mettere a disposizione gli strumenti di base per l&#39;esecuzione di successo di chirurgia mouse IVM e analisi quantitative della velocità di singoli globuli rossi. Til suo approccio permetterà ai ricercatori di acquisire nuovi elementi in velocità del sangue in condizioni patologiche.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Hoechst 33342</td>
			<td>Sigma Aldrich</td>
			<td>B2261</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA-Alexa 647</td>
			<td>lifetechnologies</td>
			<td>A34785</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextran-FITC 500 mol wt</td>
			<td>SIGMA</td>
			<td>46947</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>PanPharma</td>
			<td>20434</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Bayer</td>
			<td>KP07KEU</td>
			<td></td>
		</tr>
		<tr>
			<td>Vetedine</td>
			<td>Pharma Animal</td>
			<td>6869029</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyanoacrylate liquid</td>
			<td>Cyanolit</td>
			<td>5833300005</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslip frame: Membrane slide for microdissection part N&#176;: 5013</td>
			<td>Molecular machines</td>
			<td>50103</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslip DiaPath 24x60 ep: 1.6 mm</td>
			<td>DiaPath</td>
			<td>61061</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal laser scanning microscope</td>
			<td>Leica&#160;</td>
			<td>TCS-SP5</td>
			<td></td>
		</tr>
		<tr>
			<td>LAS-AF viewer&#160;</td>
			<td>Leica software</td>
			<td>Version 3.1.0 buid 8587</td>
			<td></td>
		</tr>
	</tbody>
</table>

intravital microscopy imaging of the liver following leishmania infection: an assessment of hepatic hemodynamics

Microscopia intravitale (IVM) è un potente tecnica di imaging ottico che ha reso possibile la visualizzazione, il monitoraggio e la quantificazione di vari eventi biologici in tempo reale e di animali vivi. Questa tecnologia ha notevolmente avanzato la nostra comprensione dei processi fisiologici e fenomeni patogeni mediata in organi specifici. In questo studio, IVM viene applicato al fegato mouse e protocolli sono progettati per immagini in vivo il sistema circolatorio del fegato e misurare globuli rossi (RBC) Velocità in singoli vasi epatici. Per visualizzare i diversi sottotipi dei vasi che caratterizzano l&#39;organo epatica ed eseguire misure di velocità del flusso sanguigno, topi C57BL / 6 vengono iniettate per via endovenosa con un reagente fluorescente plasma che contrassegna la vascolarizzazione epatica associata. IVM consente in vivo, in tempo reale, la misurazione della velocità RBC in un recipiente specifico di interesse. Stabilire questa metodologia renderà possibileindagare l&#39;emodinamica del fegato in condizioni fisiologiche e patologiche. In definitiva, questa metodologia di imaging basata sarà importante per studiare l&#39;influenza di L. infezione donovani sull&#39;emodinamica epatici. Questo metodo può essere applicato ad altri modelli infettive e organi di topo e potrebbe essere estesa ai test preclinici di effetto di un farmaco su infiammazione quantificando il suo effetto sul flusso ematico.

L&#39;organizzazione architettonico specifico delle sinusoidi del fegato può essere visualizzato basa sulla proprietà autofluorescenti di questo organo (figura 1, pannello B e C, verde), l&#39;iniezione intraperitoneale di Hoechst per l&#39;etichettatura di epatociti nuclei (Figura 1B, blu) e l&#39;iniezione endovenosa di BSA fluorescente per la colorazione del sistema circolatorio epatica (Figura 1C, rosso). Il fegato è composto da vari differenti sottotipi imbarcaz...

Watch this Scientific Journal Video about Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics at JoVE.com

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-imaging-liver-following-leishmania-infection-an

Research

JoVE Journal

Immunology and Infection

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

11.4K Views.  University of Aix-Marseille. 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.

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

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most common forms of disease are visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). Mouse models of leishmaniasis are widely used, but quantification of parasite burdens during murine disease requires mice to be euthanized at various times after infection. Parasite loads are then measured either by microscopy, limiting dilution assay, or qPCR amplification of parasite DNA. The in vivo imaging system (IVIS) has an integrated software package that allows the detection of a bioluminescent signal associated with cells in living organisms. Both to minimize animal usage and to follow infection longitudinally in individuals, in vivo models for imaging Leishmania spp. causing VL or CL were established. Parasites were engineered to express luciferase, and these were introduced into mice either intradermally or intravenously. Quantitative measurements of the luciferase driving bioluminescence of the transgenic Leishmania parasites within the mouse were made using IVIS. Individual mice can be imaged multiple times during longitudinal studies, allowing us to assess the inter-animal variation in the initial experimental parasite inocula, and to assess the multiplication of parasites in mouse tissues. Parasites are detected with high sensitivity in cutaneous locations. Although it is very likely that the signal (photons/second/parasite) is lower in deeper visceral organs than the skin, but quantitative comparisons of signals in superficial versus deep sites have not been done. It is possible that parasite numbers between body sites cannot be directly compared, although parasite loads in the same tissues can be compared between mice. Examples of one visceralizing species (L. infantum chagasi) and one species causing cutaneous leishmaniasis (L. mexicana) are shown. The IVIS procedure can be used for monitoring and analyzing small animal models of a wide variety of Leishmania species causing the different forms of human leishmaniasis.

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

An in vivo imaging system is used to generate quantitative measurements of murine infection with the Trypanosomatid protozoan Leishmania. This is a non-invasive and non-lethal method for detecting parasites expressing luciferase within many tissues throughout the course of chronic Leishmania spp. infection.

Imaging in vivo del transgenico parassiti Leishmania in un host live

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most ...

Ricerca

Immunologia e infezioni

Intravital Microscopy of the Inguinal Lymph Node

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune response and therefore, the development of effector cells. As such, LNs are key to fighting invading pathogens and maintaining health. The choice of LN to study is dictated by accessibility and the desired model; the inguinal lymph node is well situated and easily supports studies of biologically relevant models of skin and genital mucosal infection.
The inguinal LN, like all LNs, has an extensive microvascular network supplying it with blood. In general, this microvascular network includes the main feed arteriole of the LN that subsequently branches and feeds high endothelial venules (HEVs). HEVs are specialized for facilitating the trafficking of immune cells into the LN during both homeostasis and infection. How HEVs regulate trafficking into the LN under both of these circumstances is an area of intense exploration. The LN feed arteriole, has direct upstream influence on the HEVs and is the main supply of nutrients and cell rich blood into the LN. Furthermore, changes in the feed arteriole are implicated in facilitating induction of adaptive immune response. The LN microvasculature has obvious importance in maintaining an optimal blood supply to the LN and regulating immune cell influx into the LN, which are crucial elements in proper LN function and subsequently immune response. 
The ability to study the LN microvasculature in vivo is key to elucidating how the immune system and the microvasculature interact and influence one another within the LN. Here, we present a method for in vivo imaging of the inguinal lymph node. We focus on imaging of the microvasculature of the LN, paying particular attention to methods that ensure the study of healthy vessels, the ability to maintain imaging of viable vessels over a number of hours, and quantification of vessel magnitude. Methods for perfusion of the microvasculature with vasoactive drugs as well as the potential to trace and quantify cellular traffic are also presented. 
Intravital microscopy of the inguinal LN allows direct evaluation of microvascular functionality and real-time interface of the direct interface between immune cells, the LN, and the microcirculation. This technique potential to be combined with many immunological techniques and fluorescent cell labelling as well as manipulated to study vasculature of other LNs.

A technique for performing intravital microscopy of the inguinal lymph node (LN) is outlined. Such technique allows for real-time, in vivo study of the lymph node microvasculature and structure both during homeostasis and infection. This technique can be adapted to cell trafficking studies and to other lymph node sites.

Microscopia intravitale del linfonodo inguinale

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune ...

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 ...

Intravital Microscopy of the Spleen: Quantitative Analysis of Parasite Mobility and Blood Flow

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. Thus, in vivo imaging of malaria parasites during pre-erythrocytic stages have revealed the active entrance of parasites into skin lymph nodes 3, the complete development of the parasite in the skin 4, and the formation of a hepatocyte-derived merosome to assure migration and release of merozoites into the blood stream 5. Moreover, the development of individual parasites in erythrocytes has been recently documented using 4D imaging and challenged our current view on protein export in malaria 6. Thus, intravital imaging has radically changed our view on key events in Plasmodium development. Unfortunately, studies of the dynamic passage of malaria parasites through the spleen, a major lymphoid organ exquisitely adapted to clear infected red blood cells are lacking due to technical constraints.
Using the murine model of malaria Plasmodium yoelii in Balb/c mice, we have implemented intravital imaging of the spleen and reported a differential remodeling of it and adherence of parasitized red blood cells (pRBCs) to barrier cells of fibroblastic origin in the red pulp during infection with the non-lethal parasite line P.yoelii 17X as opposed to infections with the P.yoelii 17XL lethal parasite line 7. To reach these conclusions, a specific methodology using ImageJ free software was developed to enable characterization of the fast three-dimensional movement of single-pRBCs. Results obtained with this protocol allow determining velocity, directionality and residence time of parasites in the spleen, all parameters addressing adherence in vivo. In addition, we report the methodology for blood flow quantification using intravital microscopy and the use of different colouring agents to gain insight into the complex microcirculatory structure of the spleen. 
Ethics statement
All the animal studies were performed at the animal facilities of University of Barcelona in accordance with guidelines and protocols approved by the Ethics Committee for Animal Experimentation of the University of Barcelona CEEA-UB (Protocol No DMAH: 5429). Female Balb/c mice of 6-8 weeks of age were obtained from Charles River Laboratories.

We show the method for performing intravital microscopy of the spleen using GFP transgenic malaria parasites and the quantification of parasite mobility and blood flow within this organ.

Microscopia intravitale della Milza: analisi quantitativa della mobilità Parasite e del flusso sanguigno

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. ...

Intravital Imaging of the Mouse Popliteal Lymph Node

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign antigens from peripheral tissues to prime the adaptive immune response. Juxtaposed between innate and adaptive immune responses, the LN is an ideal site to study immune cell interactions1,2. Lymphocytes (T cells, B cells and NK cells), dendritic cells (DCs), and macrophages comprise the bulk of bone marrow-derived cellular elements of the LN. These cells are strategically positioned in the LN to allow efficient surveillance of self antigens and potential foreign antigens3-5. The process by which lymphocytes successfully encounter cognate antigens is a subject of intense investigation in recent years, and involves an integration of molecular contacts including antigen receptors, adhesion molecules, chemokines, and stromal structures such as the fibro-reticular network2,6-12. 
Prior to the development of high-resolution real-time fluorescent in vivo imaging, investigators relied on static imaging, which only offers answers regarding morphology, position, and architecture. While these questions are fundamental in our understanding of immune cell behavior, the limitations intrinsic with this technique does not permit analysis to decipher lymphocyte trafficking and environmental clues that affect dynamic cell behavior. Recently, the development of intravital two-photon laser scanning microscopy (2P-LSM) has allowed investigators to view the dynamic movements and interactions of individual cells within live LNs in situ12-16. In particular, we and others have applied this technique to image cellular behavior and interactions within the popliteal LN, where its compact, dense nature offers the advantage of multiplex data acquisition over a large tissue area with diverse tissue sub-structures11,17-18. It is important to note that this technique offers added benefits over explanted tissue imaging techniques, which require disruption of blood, lymph flow, and ultimately the cellular dynamics of the system. Additionally, explanted tissues have a very limited window of time in which the tissue remains viable for imaging after explant. With proper hydration and monitoring of the animal's environmental conditions, the imaging time can be significantly extended with this intravital technique. Here, we present a detailed method of preparing mouse popliteal LN for the purpose of performing intravital imaging.

Recent advances in 2-photon microscopy have enabled real-time in situ imaging of live tissues in animal models, thereby enhancing our ability to investigate cellular behavior in both physiologic and pathologic conditions. Here, we outline the preparations required to perform intravital imaging of the mouse popliteal lymph node.

Imaging intravitale del Nodo poplitea Lymph mouse

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign ...

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 ...

A Flow Adhesion Assay to Study Leucocyte Recruitment to Human Hepatic Sinusoidal Endothelium Under Conditions of Shear Stress

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the development of fibrosis and progression to cirrhosis. Understanding the molecular mechanisms that mediate leucocyte recruitment to the liver could identify important therapeutic targets for liver disease. The key interaction during leucocyte recruitment is that of inflammatory cells with endothelium under conditions of shear stress. Recruitment to the liver occurs within the low shear channels of the hepatic sinusoids which are lined by hepatic sinusoidal endothelial cells (HSEC). The conditions within the hepatic sinusoids can be recapitulated by perfusing leucocytes through channels lined by human HSEC monolayers at specific flow rates. In these conditions leucocytes undergo a brief tethering step followed by activation and firm adhesion, followed by a crawling step and subsequent transmigration across the endothelial layer. Using phase contrast microscopy, each step of this &#39;adhesion cascade&#39; can be visualized and recorded followed by offline analysis. Endothelial cells or leucocytes can be pretreated with inhibitors to determine the role of specific molecules during this process.

Leucocyte recruitment to the liver occurs within the specialized channels of the hepatic sinusoids which are lined by unique hepatic sinusoidal endothelial cells. Phase contrast microscopy of leucocyte recruitment across human hepatic sinusoidal endothelium under conditions of physiological shear stress can facilitate the elucidation of the molecular mechanisms which underlie this process.

Un flusso di Adesione saggio per studiare Leucocyte reclutamento a epatici umani sinusoidali Endotelio in condizioni di stress Shear

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the ...

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

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, neutrophils, T cell subsets, B cells, natural killer (NK) and NKT cells. Intravital microscopy of the liver for monitoring immune cell migration is particularly challenging due to the high requirements regarding sample preparation and fixation, optical resolution and long-term animal survival. Yet, the dynamics of inflammatory processes as well as cellular interaction studies could provide critical information to better understand the initiation, progression and regression of inflammatory liver disease. Therefore, a highly sensitive and reliable method was established to study migration and cell-cell-interactions of different immune cells in mouse liver over long periods (about 6 hr) by intravital two-photon laser scanning microscopy (TPLSM) in combination with intensive care monitoring.
The method provided includes a gentle preparation and stable fixation of the liver with minimal perturbation of the organ; long term intravital imaging using multicolor multiphoton microscopy with virtually no photobleaching or phototoxic effects over a time period of up to 6 hr, allowing tracking of specific leukocyte subsets; and stable imaging conditions due to extensive monitoring of mouse vital parameters and stabilization of circulation, temperature and gas exchange.
To investigate lymphocyte migration upon liver inflammation CXCR6.gfp knock-in mice were subjected to intravital liver imaging under baseline conditions and after acute and chronic liver damage induced by intraperitoneal injection(s) of carbon tetrachloride (CCl4).
CXCR6 is a chemokine receptor expressed on lymphocytes, mainly on Natural Killer T (NKT)-, Natural Killer (NK)- and subsets of T lymphocytes such as CD4 T cells but also mucosal associated invariant (MAIT) T cells1. Following the migratory pattern and positioning of CXCR6.gfp+ immune cells allowed a detailed insight into their altered behavior upon liver injury and therefore their potential involvement in disease progression.

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, ...

Two-photon Intravital Imaging of Leukocytes During the Immune Response in Lipopolysaccharide-treated Mouse Liver

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are extremely high, no direct treatment or organ-related mechanism has been examined in detail in real time. The liver is the key organ that manages toxins and infections in the human body. Herein, we aimed to perform intravital imaging of mouse liver after induction of endotoxemia in order to track the motility of immune cells, such as neutrophils and liver capsular macrophages (LCMs). Accordingly, we designed a novel surgical method for exposure of the liver with minimally invasive surgery. Mice were intraperitoneally injected with lipopolysaccharide (LPS), a common endotoxin. Using our novel surgical approach for exposure and intravital imaging of the mouse liver, we found that neutrophil recruitment in LPS-treated LysM-green fluorescent protein (GFP) mouse liver was increased compared with that in phosphate-buffered saline-treated liver. After LPS treatment, the number of neutrophils increased significantly with time. Additionally, using CX3Cr1-GFP mice, we successfully visualized liver resident macrophages called LCMs. Therefore, to investigate the efficacy of new reagents to control immune mobility in vivo, determining the motility and morphology of neutrophils and LCMs in the liver may allow us to identify therapeutic effect in organ failure and tissue damage caused by leukocytes activation in sepsis.

We established a novel surgical protocol for two-photon imaging of live mice liver with minimal invasion. With this technique, we identified the detailed structure of the liver during lipopolysaccharide-induced endotoxemia. We anticipate that this method may be utilized to determine the effectiveness of various reagents treatment to hepatic leukocyte migration.

Due-fotone videomicroscopia Imaging dei leucociti durante la risposta immunitaria nel fegato Lipopolysaccharide-trattati del topo

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are ...

Imaging Cell Interaction in Tracheal Mucosa During Influenza Virus Infection Using Two-photon Intravital Microscopy

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. Two-photon intravital microscopy (2P-IVM) allows the observation of cell interactions in deep tissue in living animals, while minimizing the photobleaching generated during image acquisition. To date, different models for 2P-IVM of lymphoid and non-lymphoid organs have been described. However, imaging of respiratory organs remains a challenge due to the movement associated with the breathing cycle of the animal.
Here, we describe a protocol to visualize in vivo immune cell interactions in the trachea of mice infected with influenza virus using 2P-IVM. To this purpose, we developed a custom imaging platform, which included the surgical exposure and intubation of the trachea, followed by the acquisition of dynamic images of neutrophils and dendritic cells (DC) in the mucosal epithelium. Additionally, we detailed the steps needed to perform influenza intranasal infection and flow cytometric analysis of immune cells in the trachea. Finally, we analyzed neutrophil and DC motility as well as their interactions during the course of a movie. This protocol allows for the generation of stable and bright 4D images necessary for the assessment of cell-cell interactions in the trachea.

In this study, we present a protocol to perform two-photon intravital imaging and cell interaction analysis in the murine tracheal mucosa after infection with influenza virus. This protocol will be relevant for researchers studying immune cell dynamics during respiratory infections.

La formazione immagine interazione cellula nella Mucosa trachea durante l'infezione da Virus influenzale usando la microscopia Intravital due-fotone

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. ...