Questo lavoro è stato sostenuto in parte da sovvenzioni dal National Institutes of Health (P30 HL101302 e RO1 HL109439 a JC) e dell&#39;American Heart Association (SDG 5.270.005 a JC). A. Barazia è stato sostenuto da una borsa di formazione T32HL007829 NIH.

1. Preparazione di Microscopio intravitale (Figura 1A) <ol><li> Preparare tampone superfusione (125 mM NaCl, 4,5 mM KCl, 2,5 mM CaCl 2, 1 mM MgCl 2, e 17 mM NaHCO 3, pH 7,4). </li><li> Attivare un bagnomaria circolatorio per mantenere la temperatura del tampone e termo-controllata coperta a 37 ° C. Aerare tampone con gas di azoto (5% di CO2 equilibrato con azoto). </li><li> Accendere il sistema di microscopio (Sutter Lambda DG-4 ad alta velocità di lunghezza d&#39;onda cambia, postazione computer, microscopio Olympus BX61W, MPC-200 multi-manipolatore, ROE-200 fase, macchina fotografica ad alta velocità, e intensificatore). </li><li> Per il modello di infiammazione vascolare, usare uno specchio dicroico 100%. Di indurre la formazione di trombi da lesioni laser, sostituire uno specchio 100% con uno specchio 50/50% ed attivare un sistema di ablazione laser Micropoint. </li></ol> 2. Preparazione di Muscle Cremaster per microscopia intravitale (Figura 1B) <ol><li> Anestetizzare untopo maschio (6-8 settimane di vita, C57BL / 6) mediante iniezione ip di ketamina (125 mg / kg di peso corporeo (BW)) e xilazina (12,5 mg / kg di peso corporeo). L&#39;Università di cura degli animali e del Comitato Istituzionale Illinois Usa ha approvato tutte le cure degli animali e procedure sperimentali. Per un modello di infiammazione vascolare, iniettare murino TNF-α (0,5 mg in 250 microlitri salina) intrascrotally in un mouse 3 ore prima di imaging (2-2,5 ore prima dell&#39;intervento). </li><li> Posizionare il mouse su una coperta termoregolata a 37 ° C su un vassoio intravitale microscopio. </li><li> Tirare verso l&#39;alto la pelle sulla superficie del collo con pinze e incidere in senso orizzontale la linea mediana della pelle del collo fino a 1 cm con le forbici. </li><li> Aprire delicatamente il muscolo cervicale con le forbici smussate e rimuovere il muscolo che circonda la trachea. </li><li> Incannulare una PE90 tubo nella trachea per eliminare difficoltà di respirazione. </li><li> Rimuovere delicatamente il lato sinistro del muscolo cervicale e isolare la vena giugulare dai tessuti circostanti. </li><li> C annutardi un tubo PE10 nella vena giugulare sinistra per l&#39;infusione di anticorpi e anestetici aggiuntivi. </li><li> Estrarre delicatamente e incidere la pelle dello scroto in senso orizzontale. Per mantenere l&#39;integrità del tessuto durante l&#39;esperimento, tampone preriscaldato stato superfuse sul campo chirurgico. </li><li> Premere sul basso addome con una pinza ed estrarre delicatamente un testicolo con l&#39;altra pinza. </li><li> Rimuovere i tessuti circostanti connettivo di tutto il testicolo. </li><li> Incidere il muscolo cremastere verticale e appiattire il muscolo su un vetrino di vetro su un vassoio microscopio intravitale da appuntare la periferia. </li><li> Lasciare 10-20 minuti per il muscolo si stabilizzi prima della raccolta dei dati. Arteriole muscolari Cremaster (per la formazione del trombo) e venule (per infiammazione vascolare) con un diametro di 30-45 micron sono stati scelti per il nostro studio. </li><li> Per mantenere condizioni anestetiche, infondere 30-50 ml dello stesso anestetico circa ogni 30 min attraverso il cannulus giugulare. </li></ol> &lt;p class = &quot;jove_title&quot;&gt; 3. Microscopia intravitale per TNF-α indotta infiammazione vascolare <ol><li> Dopo il posizionamento del mouse sul microscopio intravitale, aperto SlideBook software 5.0 e fare clic su &quot;Finestra di messa a fuoco&quot; nella barra dei menu per impostare l&#39;ottica adeguati e oggettivi (60X). </li><li> Infondere Dylight 488-coniugato anti-topo ratto CD42c (0,1 mcg / g BW in 100 microlitri soluzione salina) e Alexa Fluor 647-coniugati ratto anti-topo Gr-1 anticorpi (0,05 mg / g BW in 100 microlitri soluzione salina) attraverso un cannulus giugulare . </li><li> Vai alla barra dei menu, e fare clic su &quot;Cattura immagine&quot; nella barra degli strumenti. </li><li> Un nuovo menu pop-up; In &quot;Image&quot;, selezionare &quot;Bin Factor&quot; come 1x1 e selezionare &quot;640&quot; sulla larghezza e &quot;460&quot; in altezza. </li><li> In &quot;Imposta Filtro&quot;, contrassegnare la casella di controllo per i canali che si desidera esporre e impostare il tempo di esposizione per ogni. Ad esempio, canale aperto: 10 msec, FITC canale: 50 msec, e Cy5 canale: 50 msec. </li><li> Selezionare &quot;Time-Lapse Capture&quot; per regolare il numero di punti di tempo e l&#39;intervallo. For il modello di infiammazione, il numero di punti temporali è impostata 1000-1500 con un intervallo di 200 msec (Tempo totale: 5 min). </li><li> Fare clic su &quot;Test&quot; per vedere un singolo piano dell&#39;immagine al tempo di esposizione specificato per quel canale. Regolare il tempo di esposizione e / o la luce del microscopio fino a quando l&#39;istogramma mostra una distribuzione adeguata intensità (per multi-canale di acquisizione, ripetere questo processo per ogni canale). </li><li> Una volta che tutti i parametri sono impostati (canali, tempi di esposizione, e così via) in &quot;Cattura immagine&quot;, selezionare &quot;Start&quot; per avviare l&#39;acquisizione. </li><li> Monitorare piastrine di laminazione e aderente e neutrofili per 5 minuti nella metà superiore di un venule cremastere infiammata nel modello di infiammazione vascolare. Da otto a dieci venule sono registrati in un topo. </li><li> Le immagini sono state scattate con un ingrandimento totale di 60X (1,0 obiettivo NA acqua ad immersione), dando una dimensione della finestra di 157 micron x 118 micron. </li><li> Al termine dell&#39;esperimento, i topi sono stati sacrificati tramite cervicale dislocation. </li></ol> 4. Analisi dei dati per il modello Infiammazione venulare <ol><li> Aprire SlideBook software 5.0 e fare clic su &quot;Cartella&quot; nella barra dei menu per aprire un file. </li><li> Fare clic sul menu a discesa e selezionate &quot;Apri, FITC, Cy5, ecc&quot; </li><li> Fare clic su &quot;Renormalize (barre rosse e verdi)&quot; e selezionare il canale. Trascinare le barre rosse e verdi a sinistra oa destra per ottimizzare o meno il segnale di fluorescenza. </li><li> Fare clic su &quot;Applica&quot; e &quot;OK&quot; per aggiornare l&#39;immagine. </li><li> Fare clic su &quot;Pulsante Miniatura&quot; per selezionare la visualizzazione corrente come predefinito. </li><li> Gioca immagine Timelapse catturata, e contare neutrofili laminazione e aderente durante il periodo di 5 minuti. </li><li> Per analizzare piastrine formazione di trombi, andare a &quot;Mask&quot; e selezionare &quot;Crea&quot;. </li><li> Il nome di una maschera e marchio &quot;Nell&#39;immagine corrente&quot;. </li><li> Fare clic su &quot;icona grande matita&quot; in &quot;Tool Marquee&quot; nella vista principale. </li><li> Il colore di una regione al di fuori della nave nella vista principale per impostare una maschera di sfondo. </li><li> Vai to &quot;Maschera&quot;, fare clic su &quot;Copia This Plane&quot;, fare clic su &quot;Copia in maschera timepoint corrente&quot; e selezionare &quot;Tutti i riferimenti temporali&quot;, e fare clic su &quot;OK&quot;. </li><li> Per calcolare segnale di fondo, andare su &quot;Statistiche&quot; e fare clic su &quot;Statistiche Mask&quot;. </li><li> Fare clic su &quot;Current Time lapse 2D o 4D Image (s)&quot; in Scope, e selezionare &quot;intera maschera&quot; in ambito Mask. In Funzioni, selezionare &quot;Tempo trascorso (hh: mm: ms)&quot; sotto data di cattura, selezionare &quot;Area (pixel)&quot; sotto morfometria, selezionare &quot;Potenza massima&quot; sotto intensità, e fare clic su &quot;Esporta&quot; (Questo metodo ci permette di statistica calcolo automatico intensità di fluorescenza (segnale di fondo)). </li><li> Aprire il file di testo in MS Office Excel e calcolare il valore medio del segnale di fondo per tutto il periodo di registrazione. Questo è l&#39;intensità di fluorescenza di sfondo. </li><li> Per determinare l&#39;intensità del trombo, fare clic su &quot;icona grande matita&quot; di nuovo in &quot;strumento Marquee&quot; nella vista principale. </li><li> Colore all&#39;interno della nave nella vista principale per impostare il segnale delle piastrine. </li><li> Vai su &quot;Mask&quot;, fare clic su &quot;Copia This Plane&quot;, fare clic su &quot;Copia in maschera timepoint corrente&quot;, selezionare &quot;tutte le visite&quot;, e fare clic su &quot;OK&quot;. </li><li> Vai su &quot;Statistiche&quot; e fare clic su &quot;Statistiche Mask&quot;. </li><li> Fare clic su &quot;Current Time lapse 2D o 4D Image (s)&quot; in Scope, e selezionare &quot;intera maschera&quot; in ambito Mask. In Funzioni, selezionare &quot;Tempo trascorso (hh: mm: ms)&quot; sotto data di cattura, selezionare &quot;Area (pixel)&quot; sotto morfometria, selezionare &quot;Potenza Sum&quot; sotto intensità, e fare clic su &quot;Esporta&quot; (Questo metodo ci permette di statistica calcolare l&#39;intensità di fluorescenza all&#39;interno del recipiente). </li><li> Aprire il file di testo in Microsoft Office Excel. Segnale di fluorescenza di piastrine viene calcolato sottraendo il valore medio di intensità Area sfondo x (pixel) dalla somma di intensità della parte interna del vaso. Questo è l&#39;intensità di fluorescenza del trombo di piastrine. </li><li> Ripetere questo in 30 diversi venule in 3-5 diversi tipi di mouse. Quindi, calcola il valore medio della intensità di fluorescenza delle piastrine. </li><li> Aanalizzare il rotolamento dei neutrofili e l&#39;adesione, l&#39;afflusso di rotolamento dei neutrofili è stata determinata oltre 5 min in ciascuna venule e presentati come numero di cellule al minuto. Il numero di neutrofili aderenti che sono stati fermi per&gt; 30 sec e lentamente strisciando (&lt;10 micron più di 30 secondi), ma non rotolare, sono stati contati e presentati come numero di cellule per 5 min. </li></ol> 5. Microscopia intravitale per Laser-indotta trombosi arteriolare 5-1 Calibrazione di ablazione laser <ol><li> Inserire una diapositiva a specchio sul palco microscopio, applicare una piccola goccia di acqua distillata fino alla parte superiore della diapositiva, e regolare l&#39;intensità e mettere a fuoco con l&#39;oculare. </li><li> Aprire &quot;Finestra di messa a fuoco&quot; e selezionare la scheda &quot;FRAP&quot; nel software SlideBook 5.0. </li><li> Impostare la potenza del laser a 40-50, e impostare entrambe le &quot;ripetizioni Fare doppio clic su&quot; e &quot;dimensione Doppio click&quot; a 8. Segnare la casella &quot;Guida&quot; per far apparire una serie di mirini. </li><li> Selezionare l&#39;icona &quot;Freccia&quot; dalla barra delle applicazioni on parte superiore dello schermo. </li><li> Nella sezione &quot;Allineamento FRAP&quot; scheda, fare clic su &quot;Fuoco prossimo&quot;. Un piccolo punto dovrebbe apparire vicino all&#39;angolo in basso a sinistra sul monitor. Una volta che il punto viene visualizzata, fare doppio clic sul centro di esso. Una volta cliccato, un altro punto dovrebbe apparire in alto a destra del primo spot, fare doppio clic su di esso. Ripetere questa operazione per 16 punti (16 ° posto apparirà nell&#39;angolo in basso a destra.) Fare clic su &quot;Salva&quot; quando completo per aggiornare i parametri di calibrazione. </li><li> Per verificare la precisione della calibrazione, fare clic sul pulsante, un &quot;Centro&quot; piccolo punto dovrebbe apparire al centro dello schermo. In caso contrario, ripetere la calibrazione fino a quando la precisione desiderata. </li></ol> 5-2 laser-indotta la formazione di trombi arteriolare <ol><li> Inserire un mouse anestetizzato (vedere la procedura di 2-2 a 2-12) sul palco microscopio. </li><li> Sotto la finestra &quot;Capture&quot;, selezionare un protocollo o di crearne uno nuovo. Le impostazioni sono le seguenti: CY5 (70 esposizione msec), Open (10 mseesposizione c), e FITC (50 esposizione msec). L&#39;immagine è catturata più di 20 minuti (circa 2.400 punti di tempo con un intervallo di tempo di 500 msec). </li><li> Infondere Dylight 488-coniugato anti-topo CD42c e Alexa Fluor 647-coniugato anti-topo Gr-1 anticorpi come descritto sopra. </li><li> Ottimizzare i parametri microscopio tra intensità di fluorescenza e di immagine in campo chiaro come descritto in 3-3 per 3-7. </li><li> Fare clic sul pulsante &quot;Test&quot; per garantire la giusta intensità campo chiaro o segnale anticorpo, arteriola posizionamento e messa a fuoco. </li><li> Fare clic su &quot;Start&quot; per avviare il processo di cattura dell&#39;immagine. </li><li> Prima di sparare il laser ablazione, accertarsi che l&#39;icona del puntatore del mouse è stato selezionato e che la parete del vaso è a fuoco chiaro. </li><li> Per sparare il laser, fare doppio clic su un punto 2-3 micron internamente dalla parete del serbatoio. Lesioni delle cellule endoteliali arteriolare provoca un cambiamento visivo forma che dovrebbe essere evidente nell&#39;immagine chiaro, seguito da accumulo piastrinica. Monitor tromboformazione per 5 min dopo la lesione laser. </li><li> Cinque minuti dopo la lesione laser (la dimensione del trombo dovrebbe essere stabile) mettere in pausa e annullare la cattura. Successivamente, avviare una cattura con 2400 punti di tempo con un intervallo di 500 msec per registrare piastrine aderenti e neutrofili laminazione / aderente per 20 min. Da tre a cinque arteriole sono registrati in un mouse. </li><li> Al termine dell&#39;esperimento, i topi sono stati sacrificati mediante dislocazione cervicale. </li></ol> 6. Analisi dei dati per il modello Trombosi arteriolare <ol><li> Andare oltre 4,1 passi attraverso 4.5. </li><li> Per ottenere l&#39;intensità di fluorescenza di fondo durante le piastrine formazione di trombi, andare oltre i passaggi attraverso 4,7 4.14. </li><li> Vai su &quot;Mask&quot;, fare clic su &quot;Segmento&quot;, selezionare FITC nel canale, e inserire il valore medio del segnale di fondo su Low. Fare clic su &quot;Applica&quot; e &quot;OK&quot;. </li><li> Per analizzare l&#39;intensità di fluorescenza del trombo di piastrine (Dylight 488-coniugato anticorpo anti-CD42c), </strong&gt; andare oltre 4,18 passaggi attraverso 4.19. Per calcolare il segnale anticorpo, &quot;Intensity Somma&quot; viene sottratto dal segnale di fondo (&quot;Maximum Intensity&quot; x &quot;Area (pixel)&quot;). Questo è l&#39;intensità di fluorescenza del trombo di piastrine. </li><li> Per quantificare i neutrofili di laminazione e aderente, giocare il timelapse e contare il numero di celle che visibilmente rotolano nei trombi piastrinici oltre 20 min. Rotolamento è definita come una diminuzione della velocità neutrofili mentre interagisce con il trombo di piastrine per almeno 2 sec. Neutrofili aderenti si intende qualsiasi neutrofili che rimangono attaccati ai trombi piastrinici per almeno 2 min. Il numero di neutrofili laminazione e aderente è stato presentato come il numero di cellule per 20 min. Velocità di laminazione è stata calcolata usando un sistema di tracciamento di particelle. </li></ol>

<ol>
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Qui si descrive un protocollo dettagliato per tempo reale microscopia a fluorescenza intravitale di visualizzare eterotipiche piastrine-neutrofili interazioni sull&#39;endotelio vascolare attivato durante l&#39;infiammazione e trombosi. In precedenza, simili approcci fluorescenza microscopiche sono stati segnalati per studiare il meccanismo molecolare della formazione di trombi e infiammazione vascolare. 8,12 Poiché il heterotypic interazione cellula-cellula potrebbe essere importante per vaso-occlusione al ...

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher Scientific</td>
			<td>7647-14-5</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>7447-40-7</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>CaCl2 2H2O</td>
			<td>Sigma-Aldrich</td>
			<td>10035-04-8</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MgCl2 6H2O</td>
			<td>Fisher Scientific</td>
			<td>7791-18-6</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Fisher Scientific</td>
			<td>144-55-8</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>0.9% NaCl Saline</td>
			<td>Hospira</td>
			<td>0409-4888-10</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Hospira</td>
			<td>0409-2051-05</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Lloyd</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Intramedic Tubing (PE 90)</td>
			<td>BD Diagnostics</td>
			<td>427421</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Intramedic Tubing (PE 10)</td>
			<td>BD Diagnostics</td>
			<td>427401</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Murine TNF-&alpha;</td>
			<td>R&amp;D Systems</td>
			<td>410-MT</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dylight 488- labeled rat anti-mouse CD42b antibody</td>
			<td>Emfret Analytics</td>
			<td>X488</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647-conjugated anti-mouse Ly-6G/Ly-6C (Gr-1) Antibody</td>
			<td>BioLegend</td>
			<td>108418</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NESLAB EX water bath/circulator</td>
			<td>Thermo-Scientific</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Olympus BX61W microscope</td>
			<td>Olympus</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>TH4-100 Power</td>
			<td>Olympus</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Lambda DG-4</td>
			<td>Sutter</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MPC-200 multi-manipulator</td>
			<td>Sutter</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>ROE-200 stage controller</td>
			<td>Sutter</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>C9300 high-speed camera</td>
			<td>Hamamatsu</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Intensifier</td>
			<td>Video Scope International</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ablation Laser</td>
			<td>Photonic Instruments, Inc.</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SlideBook 5.0</td>
			<td>Intelligent Imaging Innovations</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

real-time imaging of heterotypic platelet-neutrophil interactions on the activated endothelium during vascular inflammation and thrombus formation in live mice

Interazione delle piastrine attivate e leucociti neutrofili (principalmente) sulla trombosi attivato media endotelio e infiammazione vascolare. 1,2 Durante la formazione del trombo nel sito di lesione arteriolare, piastrine aderenti all&#39;endotelio attivato e proteine ​​della matrice subendoteliali supportare rotolamento dei neutrofili e adesione. 3 Viceversa, in venulare condizioni infiammatorie, aderenti neutrofili all&#39;endotelio attivato può sostenere l&#39;adesione e l&#39;accumulo di piastrine circolanti. Eterotipica piastrine-neutrofili aggregazione richiede processi sequenziali dagli specifici recettori-counter interazioni tra cellule recettore. 4 È noto che cellule endoteliali attivate rilasciare molecole di adesione come fattore di von Willebrand, avviando adesione piastrinica e accumulo condizioni di alto shear. 5 Inoltre, cellule endoteliali attivate supportare rotolamento dei neutrofili e adesione da selectine esprimono unmolecola-1 di adesione intercellulare d (ICAM-1), rispettivamente, in condizioni di basso attrito. 4 piastrine P-selectina interagisce con neutrofili attraverso P-selectina glicoproteina ligando-1 (PSGL-1), inducendo così l&#39;attivazione dei neutrofili β2 integrine e azienda adesione tra due tipi di cellule. Nonostante i progressi in esperimenti in vitro in cui sono determinati eterotipiche piastrine-neutrofili interazioni in sangue intero o cellule isolate, 6,7 quegli studi non può manipolare durante condizioni di stress ossidativo malattia vascolare. In questo rapporto, con marcati in fluorescenza, anticorpi specifici contro un mouse piastrinica e dei neutrofili marcatore, si descrive un dettagliato protocollo intravitale microscopica per monitorare le interazioni eterotipiche di piastrine e dei neutrofili sul endotelio attivato durante il TNF-α indotta da infiammazione o dopo laser-indotta lesioni in Cremaster microvasi muscolari di topi vivi.

Con un&#39;analisi dettagliata microscopia intravitale, eterotipiche piastrine-neutrofili interazioni sul endotelio attivato sono state visualizzate mediante infusione di anticorpi marcati con fluorescenza contro una piastrina (CD42c) o neutrofili marcatore (Gr-1) in topi vivi. In un modello di TNF-α indotta infiammazione venulare, neutrofili più laminazione sono stabilmente aderito all&#39;endotelio presumibilmente dall&#39;interazione attivati ​​β2 integrine con ICAM-1 durante il pe...

Watch this Scientific Journal Video about Real-time Imaging of Heterotypic Platelet-neutrophil Interactions on the Activated Endothelium During Vascular Inflammation and Thrombus Formation in Live Mic

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

Qui riportiamo una tecnica sperimentale di fluorescenza microscopia intravitale di visualizzare eterotipiche piastrine-neutrofili interazioni sul endotelio attivato durante l&#39;infiammazione vascolare e la formazione di trombi nei topi vivi. Questa tecnologia microscopica sarà prezioso per studiare il meccanismo molecolare della malattia vascolare e per testare agenti farmacologici in condizioni fisiopatologiche.

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

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

real-time-imaging-heterotypic-platelet-neutrophil-interactions-on

Research

JoVE Journal

Immunology and Infection

15.6K Views.  University of Illinois at Chicago. Qui riportiamo una tecnica sperimentale di fluorescenza microscopia intravitale di visualizzare eterotipiche piastrine-neutrofili interazioni sul endotelio attivato durante l'infiammazione vascolare e la formazione di trombi nei topi vivi. Questa tecnologia microscopica sarà prezioso per studiare il meccanismo molecolare della malattia vascolare e per testare agenti farmacologici in condizioni fisiopatologiche.

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

Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with &beta;-arrestin

G-protein coupled receptors (GPCRs) belong to the seven transmembrane protein family and mediate the transduction of extracellular signals to intracellular responses. GPCRs control diverse biological functions such as chemotaxis, intracellular calcium release, gene regulation in a ligand dependent manner via heterotrimeric G-proteins1-2. Ligand binding induces a series of conformational changes leading to activation of heterotrimeric G-proteins that modulate levels of second messengers such as cyclic adenosine monophosphate (cAMP), inositol triphosphate (IP3) and diacyl glycerol (DG). Concomitant with activation of the receptor ligand binding also initiates a series of events to attenuate the receptor signaling via desensitization, sequestration and/or internalization. The desensitization process of GPCRs occurs via receptor phosphorylation by G-protein receptor kinases (GRKs) and subsequent binding of &beta;-arrestins3. &beta;-arrestins are cytosolic proteins and translocate to membrane upon GPCR activation, binding to phosphorylated receptors (most cases) there by facilitating receptor internalization 4-6.
Leukotriene B4 (LTB4) is a pro-inflammatory lipid molecule derived from arachidonic acid pathway and mediates its actions via GPCRs, LTB4 receptor 1 (BLT1; a high affinity receptor) and LTB4 receptor 2 (BLT2; a low affinity receptor)7-9. The LTB4-BLT1 pathway has been shown to be critical in several inflammatory diseases including, asthma, arthritis and atherosclerosis10-17. The current paper describes the methodologies developed to monitor LTB4-induced leukocyte migration and the interactions of BLT1 with &beta;-arrestin and , receptor translocation in live cells using microscopy imaging techniques18-19.
Bone marrow derived dendritic cells from C57BL/6 mice were isolated and cultured as previously described 20-21. These cells were tested in live cell imaging methods to demonstrate LTB4 induced cell migration. The human BLT1 was tagged with red fluorescent protein (BLT1-RFP) at C-terminus and &beta;-arrestin1 tagged with green fluorescent protein (&beta;-arr-GFP) and transfected the both plasmids into Rat Basophilic Leukomia (RBL-2H3) cell lines18-19. The kinetics of interaction between these proteins and localization were monitored using live cell video microscopy. The methodologies in the current paper describe the use of microscopic techniques to investigate the functional responses of G-protein coupled receptors in live cells. The current paper also describes the use of Metamorph software to quantify the fluorescence intensities to determine the kinetics of receptor and cytosolic protein interactions.

Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with &amp;#946;-arrestin

This paper describes the methodology to determine the chemotactic response of leukocytes to specific ligands and identify interactions between the cell surface receptors and cytosolic proteins using live cell imaging techniques.

In tempo reale immagini di leucotriene B 4 Migrazione cellulare mediata e BLT1 Interazioni con β-arrestina

G-protein coupled receptors (GPCRs) belong to the seven transmembrane protein family and mediate the transduction of extracellular signals to ...

Ricerca

Immunologia e infezioni

Imaging Leukocyte Adhesion to the Vascular Endothelium at High Intraluminal Pressure

Worldwide, hypertension is reported to be in approximately a quarter of the population and is the leading biomedical risk factor for mortality worldwide. In the vasculature hypertension is associated with endothelial dysfunction and increased inflammation leading to atherosclerosis and various disease states such as chronic kidney disease2, stroke3 and heart failure4. An initial step in vascular inflammation leading to atherogenesis is the adhesion cascade which involves the rolling, tethering, adherence and subsequent transmigration of leukocytes through the endothelium. Recruitment and accumulation of leukocytes to the endothelium is mediated by an upregulation of adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1), intracellular cell adhesion molecule-1 (ICAM-1) and E-selectin as well as increases in cytokine and chemokine release and an upregulation of reactive oxygen species5. In vitro methods such as static adhesion assays help to determine mechanisms involved in cell-to-cell adhesion as well as the analysis of cell adhesion molecules. Methods employed in previous in vitro studies have demonstrated that acute increases in pressure on the endothelium can lead to monocyte adhesion, an upregulation of adhesion molecules and inflammatory markers6 however, similar to many in vitro assays, these findings have not been performed in real time under physiological flow conditions, nor with whole blood. Therefore, in vivo assays are increasingly utilised in animal models to demonstrate vascular inflammation and plaque development. Intravital microscopy is now widely used to assess leukocyte adhesion, rolling, migration and transmigration7-9. When combining the effects of pressure on leukocyte to endothelial adhesion the in vivo studies are less extensive. One such study examines the real time effects of flow and shear on arterial growth and remodelling but inflammatory markers were only assessed via immunohistochemistry10. Here we present a model for recording leukocyte adhesion in real time in intact pressurised blood vessels using whole blood perfusion. The methodology is a modification of an ex vivo vessel chamber perfusion model9 which enables real-time analysis of leukocyte -endothelial adhesive interactions in intact vessels. Our modification enables the manipulation of the intraluminal pressure up to 200 mmHg allowing for study not only under physiological flow conditions but also pressure conditions. While pressure myography systems have been previously demonstrated to observe vessel wall and lumen diameter11 as well as vessel contraction this is the first time demonstrating leukocyte-endothelial interactions in real time. Here we demonstrate the technique using carotid arteries harvested from rats and cannulated to a custom-made flow chamber coupled to a fluorescent microscope. The vessel chamber is equipped with a large bottom coverglass allowing a large diameter objective lens with short working distance to image the vessel. Furthermore, selected agonist and/or antagonists can be utilized to further investigate the mechanisms controlling cell adhesion. Advantages of this method over intravital microscopy include no involvement of invasive surgery and therefore a higher throughput can be obtained. This method also enables the use of localised inhibitor treatment to the desired vessel whereas intravital only enables systemic inhibitor treatment.

This is a method to visualise leukocyte adhesion to the endothelium in harvested pressurised vessels. The technique enables studying vascular adhesion under shear flow with differing intraluminal pressures up to 200 mmHg thus mimic-ing the pathophysiological conditions of high blood pressure.

Imaging adesione dei leucociti all&#39;endotelio vascolare ad alta pressione intraluminale

Worldwide, hypertension is reported to be in approximately a quarter of the population and is the leading biomedical risk factor for mortality worldwide. ...

Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many diseases associated with neuroinflammation such as multiple sclerosis (MS)2. Microglia in a normal CNS express macrophage marker CD11b and exhibit a resting phenotype by expressing low levels of activation markers such as CD45. During pathological events in the CNS, microglia become activated as determined by upregulation of CD45 and other markers3. The factors that affect microglia phenotype and functions in the CNS are not well studied. MicroRNAs (miRNAs) are a growing family of conserved molecules (~22 nucleotides long) that are involved in many normal physiological processes such as cell growth and differentiation4 and pathologies such as inflammation5. MiRNAs downregulate the expression of certain target genes by binding complementary sequences of their mRNAs and play an important role in the activation of innate immune cells including macrophages6 and microglia7. In order to investigate miRNA-mediated pathways that define the microglial phenotype, biological function, and to distinguish microglia from other types of macrophages, it is important to quantitatively assess the expression of particular microRNAs in distinct subsets of CNS-resident microglia. Common methods for measuring the expression of miRNAs in the CNS include quantitative PCR from whole neuronal tissue and in situ hybridization. However, quantitative PCR from whole tissue homogenate does not allow the assessment of the expression of miRNA in microglia, which represent only 5-15% of the cells of neuronal tissue. Hybridization in situ allows the assessment of the expression of microRNA in specific cell types in the tissue sections, but this method is not entirely quantitative. In this report we describe a quantitative and sensitive method for the detection of miRNA by real-time PCR in microglia isolated from normal CNS or during neuroinflammation using experimental autoimmune encephalomyelitis (EAE), a mouse model for MS. The described method will be useful to measure the level of expression of microRNAs in microglia in normal CNS or during neuroinflammation associated with various pathologies including MS, stroke, traumatic injury, Alzheimer's disease and brain tumors.

Microglia are resident macrophages that provide the first line of defense and immune surveillance of the central nervous system. MicroRNAs are regulatory molecules that play an important role in many physiological processes including activation and differentiation of macrophages. In this article, we describe the method for measurement of microRNAs in microglia.

Rilevamento dei microRNA nella microglia da Real-time PCR in CNS normale e durante neuroinfiammazione

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many ...

Real-time Live Imaging of T-cell Signaling Complex Formation

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating the activation and responses of multiple immune cells 3,4. T-cell activation is dependent on the recognition of specific antigens displayed by antigen presenting cells (APCs). The T-cell antigen receptor (TCR) is specific to each T-cell clone and determines antigen specificity 5. The binding of the TCR to the antigen induces the phosphorylation of components of the TCR complex. In order to promote T-cell activation, this signal must be transduced from the membrane to the cytoplasm and the nucleus, initiating various crucial responses such as recruitment of signaling proteins to the TCR;APC site (the immune synapse), their molecular activation, cytoskeletal rearrangement, elevation of intracellular calcium concentration, and changes in gene expression 6,7. The correct initiation and termination of activating signals is crucial for appropriate T-cell responses. The activity of signaling proteins is dependent on the formation and termination of protein-protein interactions, post translational modifications such as protein phosphorylation, formation of protein complexes, protein ubiquitylation and the recruitment of proteins to various cellular sites 8. Understanding the inner workings of the T-cell activation process is crucial for both immunological research and clinical applications.
Various assays have been developed in order to investigate protein-protein interactions; however, biochemical assays, such as the widely used co-immunoprecipitation method, do not allow protein location to be discerned, thus precluding the observation of valuable insights into the dynamics of cellular mechanisms. Additionally, these bulk assays usually combine proteins from many different cells that might be at different stages of the investigated cellular process. This can have a detrimental effect on temporal resolution. The use of real-time imaging of live cells allows both the spatial tracking of proteins and the ability to temporally distinguish between signaling events, thus shedding light on the dynamics of the process 9,10. We present a method of real-time imaging of signaling-complex formation during T-cell activation. Primary T-cells or T-cell lines, such as Jurkat, are transfected with plasmids encoding for proteins of interest fused to monomeric fluorescent proteins, preventing non-physiological oligomerization 11. Live T cells are dropped over a coverslip pre-coated with T-cell activating antibody 8,9, which binds to the CD3/TCR complex, inducing T-cell activation while overcoming the need for specific activating antigens. Activated cells are constantly imaged with the use of confocal microscopy. Imaging data are analyzed to yield quantitative results, such as the colocalization coefficient of the signaling proteins.

We describe a live-cell imaging method that provides insight into protein dynamics during the T-cell activation process. We demonstrate the combined usage of the T-cell spreading assay, confocal microscopy and imaging analysis to yield quantitative results to follow signaling complex formation throughout T-cell activation.

Tempo reale Imaging in diretta di cellule T Signaling formazione del complesso

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating ...

Real-time Imaging of Endothelial Cell-cell Junctions During Neutrophil Transmigration Under Physiological Flow

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known as leukocyte transendothelial migration. Two routes for leukocytes to cross the endothelial monolayer have been described: the paracellular route, i.e., through the cell-cell junctions and the transcellular route, i.e., through the endothelial cell body. However, it has been technically difficult to discriminate between the para- and transcellular route. We developed a simple in vitro assay to study the distribution of endogenous VE-cadherin and PECAM-1 during neutrophil transendothelial migration under physiological flow conditions. Prior to neutrophil perfusion, endothelial cells were briefly treated with fluorescently-labeled antibodies against VE-cadherin and PECAM-1. These antibodies did not interfere with the function of both proteins, as was determined by electrical cell-substrate impedance sensing and FRAP measurements. Using this assay, we were able to follow the distribution of endogenous VE-cadherin and PECAM-1 during transendothelial migration under flow conditions and discriminate between the para- and transcellular migration routes of the leukocytes across the endothelium.

Leukocytes cross the endothelial monolayer using the paracellular or the transcellular route. We developed a simple assay to follow the distribution of endogenous junctional VE-cadherin and PECAM-1 during leukocyte transendothelial migration under physiological flow to discriminate between the two transmigration routes.

In tempo reale Imaging del endoteliali giunzioni cellula-cellula Durante neutrofili Trasmigrazione Sotto fisiologico flusso

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known ...

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways activated during cardiovascular pathologies are, however, limited by the lack of robust and physiologically relevant methods. In order to model pathological vascular events we have developed an in vitro assay for studying the interaction between endothelium and whole blood. The assay consists of primary human endothelial cells, which are placed in contact with human whole blood. The method utilizes native blood with no or very little anticoagulant, enabling study of delicate interactions between molecular and cellular components present in a blood vessel.
We investigated functionality of the assay by comparing activation of coagulation by different blood volumes incubated with or without human umbilical vein endothelial cells (HUVEC). Whereas a larger blood volume contributed to an increase in the formation of thrombin antithrombin (TAT) complexes, presence of HUVEC resulted in reduced activation of coagulation. Furthermore, we applied image analysis of leukocyte attachment to HUVEC stimulated with tumor necrosis factor (TNF&#945;) and found the presence of CD16+ cells to be significantly higher on TNF&#945; stimulated cells as compared to unstimulated cells after blood contact. In conclusion, the assay may be applied to study vascular pathologies, where interactions between the endothelium and the blood compartment are perturbed.

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

The accessibility of reliable models to investigate vascular blood interactions in humans is lacking. We present an in vitro model of cultured primary human endothelial cells combined with human whole blood to investigate cellular interactions both in the blood (ELISA) and the vascular compartment (microscopy).

A Novel<em&gt; In vitro</em&gt; Modello per lo studio delle interazioni tra l&#39;uomo di sangue intero e l&#39;endotelio

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways ...

Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

Zebrafish (Danio rerio) embryos are increasingly used as an infection model to study the function of the vertebrate innate immune system in host-pathogen interactions.&#160;The ease of obtaining large numbers of embryos, their accessibility due to external development, their optical transparency as well as the availability of a wide panoply of genetic/immunological tools and transgenic reporter line collections, contribute to the versatility of this model.&#160;In this respect, the present manuscript describes the use of zebrafish as an&#160;in vivo&#160;model system to investigate the chronology of Mycobacterium abscessus infection. This human pathogen can exist either as smooth (S) or rough (R) variants, depending on cell wall composition, and their respective virulence can be imaged and compared in zebrafish embryos and larvae. Micro-injection of either S or R fluorescent variants directly in the blood circulation via the caudal vein, leads to chronic or acute/lethal infections, respectively. This biological system allows high resolution visualization and analysis of the role of mycobacterial cording in promoting abscess formation. In addition, the use of fluorescent bacteria along with transgenic zebrafish lines harbouring fluorescent macrophages produces a unique opportunity for multi-color imaging of the host-pathogen interactions. This article describes detailed protocols for the preparation of homogenous M. abscessus inoculum and for intravenous injection of zebrafish embryos for subsequent fluorescence imaging of the interaction with macrophages. These techniques open the avenue to future investigations involving mutants defective in cord formation and are dedicated to understand how this impacts on M. abscessus pathogenicity in a whole vertebrate.

Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

Optically transparent zebrafish embryos are widely used to study and visualize in real time the interactions between pathogenic microorganisms and the innate immune cells. Micro-injection of Mycobacterium abscessus, combined with fluorescence imaging, is used to scrutinize essential pathogenic features such as cord formation in zebrafish embryos.

Decifrare e Imaging Patogenesi e Cording di<em&gt; Mycobacterium abscessus</em&gt; In Zebrafish embrioni

Zebrafish (Danio rerio) embryos are increasingly used as an infection model to study the function of the vertebrate innate immune system in host-pathogen ...

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 ...

Metabolic Characterization of Polarized M1 and M2 Bone Marrow-derived Macrophages Using Real-time Extracellular Flux Analysis

Specific metabolic pathways are increasingly being recognized as critical hallmarks of macrophage subsets. While LPS-induced classically activated M1 or M(LPS) macrophages are pro-inflammatory, IL-4 induces alternative macrophage activation and these so-called M2 or M(IL-4) support resolution of inflammation and wound healing. Recent evidence shows the crucial role of metabolic reprogramming in the regulation of M1 and M2 macrophage polarization. 
In this manuscript, an extracellular flux analyzer is applied to assess the metabolic characteristics of naive, M1 and M2 polarized mouse bone marrow-derived macrophages. This instrument uses pH and oxygen sensors to measure the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), which can be related to glycolytic and mitochondrial oxidative metabolism. As such, both glycolysis and mitochondrial oxidative metabolism can be measured in real-time in one single assay.
Using this technique, we demonstrate here that inflammatory M1 macrophages display enhanced glycolytic metabolism and reduced mitochondrial activity. Conversely, anti-inflammatory M2 macrophages show high mitochondrial oxidative phosphorylation (OXPHOS) and are characterized by an enhanced spare respiratory capacity (SRC). 
The presented functional assay serves as a framework to investigate how particular cytokines, pharmacological compounds, gene knock outs or other interventions affect the macrophage&#8217;s metabolic phenotype and inflammatory status.

Metabolic reprogramming is a characteristic and prerequisite for M1 and M2 macrophage polarization. This manuscript describes an assay for the measurement of fundamental parameters of glycolysis and mitochondrial function in mouse bone marrow-derived macrophages. This tool can be applied to investigate how particular factors affect the macrophage&#8217;s metabolism and phenotype.

Caratterizzazione metabolica di Polarized M1 e M2 macrofagi derivate dal midollo osseo Utilizzando analisi in tempo reale extracellulare Flux

Specific metabolic pathways are increasingly being recognized as critical hallmarks of macrophage subsets. While LPS-induced classically activated M1 or ...

In Vitro Microfluidic Disease Model to Study Whole Blood-Endothelial Interactions and Blood Clot Dynamics in Real-Time

The formation of blood clots involves complex interactions between endothelial cells, their underlying matrix, various blood cells, and proteins. The endothelium is the primary source of many of the major hemostatic molecules that control platelet aggregation, coagulation, and fibrinolysis. Although the mechanism of thrombosis has been investigated for decades, in vitro studies mainly focus on situations of vascular damage where the subendothelial matrix gets exposed, or on interactions between cells with single blood components. Our method allows studying interactions between whole blood and an intact, confluent vascular cell network.
By utilizing primary human endothelial cells, this protocol provides the unique opportunity to study the influence of endothelial cells on thrombus dynamics and gives&#160;valuable insights into the pathophysiology of thrombotic disease. The use of custom-made microfluidic flow channels allows application of disease-specific vascular geometries and model specific morphological vascular changes. The development of a thrombus is recorded in real-time and quantitatively characterized by platelet adhesion and fibrin deposition. The effect of endothelial function in altered thrombus dynamics is determined by postanalysis through immunofluorescence staining of specific molecules.
The representative results describe the experimental setup, data collection, and data analysis. Depending on the research question, parameters for every section can be adjusted including cell type, shear rates, channel geometry, drug therapy, and postanalysis procedures. The protocol is validated by quantifying thrombus formation on the pulmonary artery endothelium of patients with chronic thromboembolic disease.

We present an in vitro vascular disease model to investigate whole blood interactions with patient-derived endothelium. This system allows the study of thrombogenic properties of primary endothelial cells under various circumstances. The method is especially suited to evaluate in situ thrombogenicity and anticoagulation therapy during different phases of coagulation.

Modello di malattia microfluidico in vitro per studiare le interazioni sangue-endoteliali intere e le dinamiche del coagulo di sangue in tempo reale

The formation of blood clots involves complex interactions between endothelial cells, their underlying matrix, various blood cells, and proteins. The ...