Questo studio è stato sostenuto in parte dal Programma OIS del Governo del Victoria, il National Health and Medical Research Council of Australia borse di programmi e progetti (JPF Chin-spolverare) e borsa di studio post-laurea (D Michell).

1. Isolando le arterie carotidee <ol><li> Euthanase10 settimana di vita ratti Sprague Dawley via di CO 2 / O 2 asfissia. </li><li> Accise arterie comuni a sinistra ea destra della carotide con aorta e il cuore garantendo allungamento minimo dei vasi. </li><li> Nel buffer di ghiaccio freddo Krebs separare le arterie della carotide e dell&#39;aorta dal cuore e di eseguire la dissezione chiudere. </li><li> Mantenere i vasi isolati in Krebs sul ghiaccio prima del montaggio. </li></ol> Ca. tempo = 45 minuti 2. Priming la nave da camera <ol><li> Al connettori prossimale e distale del buffer nave camera filo Krebs mantenuta a pH fisiologico infondendo CarboGen gas (95% O 2, 5% CO 2) attraverso il buffer a 37 ° C. </li><li> Garantire tubi e cannule sono lavata completamente e sono allineati. </li><li> Flush del buffer Kreb attraverso il P1 (prossimale) e P2 (distale) trasduttori. Chiudere i rubinetti per assicurarsi che non bolle d&#39;aria nel trasduttori. </li><li> Collegare i trasduttori ai connettori corrispondenti e ancora di più a livello di buffer di Krebs garantendo l&#39;assenza di bolle d&#39;aria. Chiudere i rubinetti alla camera. </li></ol> Ca. tempo = 15 min 3. Pressurizzazione della camera di nave <ol><li> Accendere attrezzature a pressione: servo di pressione, controllo della pressione, pompa peristaltica (Figura 1). </li><li> Con la pompa peristaltica sulla pressione e il servo di pressione in automatico tampone Krebs correre attraverso il tubo a 20 mmHg (quadrante a 1), garantendo l&#39;assenza di bolle d&#39;aria. </li><li> Collegare il tubo al trasduttore P2 chiuso. Pressione diventerà stabile. </li><li> Aprire il rubinetto P2 alla camera nave per scovare eventuali bolle poi chiudere. </li><li> Riempire il bagno con tampone Krebs (5 - 7 mL). </li></ol> Ca. tempo = 10 minuti 4. Montaggio della nave <ol><li> Sotto un microscopio a dissezione legami poliestere posto nero su ogni cannula (Figura 2). </li><li> Spostare cannule e titolari cannula a parte e montare nave ¼ (arco aortico fine) sulla cannula P1. Garantire a non strappare la nave. </li><li> Utilizzando una siringa piena di Krebs tampone delicatamente lavare il sangue in eccesso della nave tramite trasduttore P1. Chiudere P1 a camera. </li><li> Nave sicuro alla cannula P1 con la cravatta di poliestere. </li><li> Spostare cannule / titolari cannula più vicino per il montaggio su la cannula P2. </li><li> Monte fine distale del vaso (~ ¼ della lunghezza) sulla cannula distale. Fissare con cravatta poliestere. </li></ol> Ca. tempo = 20 minuti 5. Pressurizzazione della nave <ol><li> Regolare i titolari cannula per garantire che non piegare o tirare in vaso. </li><li> Con P1 e P2 chiuso alla camera di cambiare il servo di pressione da automatico a manuale. Controllare il servo di pressione non è costante riduzione della pressione entro 10 secondi. Se c&#39;è, si sta verificando una perdita e le connessioni e trasduttori devono essere fissati al suolo. </li><li> Regolare il servo pressione a automatica P2 quindi aprire alla camera. Al microscopio osservare la nave si dilatano quando il rubinetto è aperto alla camera. </li><li> Accendere il servo pressione manuale. Anche in questo caso verificare la costante riduzione della pressione entro 10 secondi. Se c&#39;è una perdita allora il sistema non è a tenuta di pressione e non ci sarà probabilmente un buco nel serbatoio. </li><li> Tornare alla automatica P1 aperto alla camera. L&#39;impostazione manuale verificare che la pressione rimane stabile. </li><li> Se nessuna perdita, in linea manuale aumentare l&#39;impostazione di 2 (40 mmHg). </li><li> Regolare in automatico e osservare la nave sotto il microscopio. Se necessario, regolare il titolare cannula per assicurarsi che non piegare in vessel.Check per perdite su impostazione manuale. </li><li> Ripetere i passaggi 5,6-5,7 aumentando il selettore per 3 (60 mmHg), 4 (80 mmHg) e così via fino a quando la pressione desiderata. </li></ol> Ca. tempo = 15 - 30 minuti 6. Incubando la nave sotto pressione <ol><li> Collegare il set di controllo della temperatura a 37 ° C. </li><li> Con una seconda pompa peristaltica profumato tampone Krebs (1 ml / min) nel bagno della camera nave. </li><li> Collegare aspiratore alla camera garantire solo lo strato superiore del bagno è stato rimosso. </li><li> Incubare nave pressurizzata a 37 ° C per 1 ora con la pressione impostato su Automatico. </li><li> Controllare la pressione non perde (il passaggio a manuale) periodicamente. </li><li> Gli effetti dei vari interventi farmacologici possono osservare semplicemente aggiungendo il composto al bagno durante l&#39;incubazione. </li></ol> Ca. tempo = 1 ora 7. Perfusione del vaso pressurizzato con sangue intero <ol><li> Durante l&#39;incubazione ottenere almeno 7,5 ml di sangue intero / nave raccolti in 40 eparina U / ml di sangue. Incubare in un falco da 50 ml a 37 ° C. </li><li> 10 minuti prima della fine del tsi etichetta sangue incubazione con VybrantDil (1:1000) per 10 minuti a 37 ° C al buio. </li><li> Dopo 10 minuti, raccogliere il sangue in una siringa di compensazione eventuali bolle e attaccare su una pompa a siringa con una giacca di calore a 37 ° C. </li><li> Chiudere trasduttore P1 alla camera / navi e collegare la siringa e tubo di scarico. </li><li> Sangue purga a 1000 ml / min attraverso il tubo di scarico per rimuovere eventuali bolle. </li><li> Aprire P1 alla camera. Il servo di pressione si regola automaticamente per mantenere la pressione desiderata. </li><li> Perfusione di sangue a 100 l / min. </li><li> Utilizzando un microscopio a fluorescenza accoppiato ad una fotocamera digitale registrare due campi della nave perfusi per 15 secondi a 1, 3, 5, 7,5 e 10 minuti. </li><li> Perfusione Messaggio integrità e la funzione endoteliale può essere valutata mediante tecniche farmacologiche come miografo molecola di espressione e di adesione può essere determinato mediante immunoistochimica per validare ulteriormente la risposta infiammatoria. </li></ol> Ca. tempo = 15 min 8. Rappresentante dei risultati: Un diagramma schematico della configurazione camera di pressione è mostrata in Figura 1. Con una fotocamera digitale accoppiato ad un microscopio a fluorescenza risultati possono essere visualizzati immediatamente tramite registrazioni video in diretta. Immagini video rappresentativi sono visto in Figura 3, dove sono considerati leucociti per essere aderente al endotelio se è rimasto fermo per 10 secondi. Con le registrazioni video sulle cellule ciclo continuo aderito può essere contato come un campo medio per. Mentre sia basso (Figura 3A) e alta (Figura 3B) pressione causerà una certa quantità di adesione un aumento significativo l&#39;adesione dei leucociti con la pressione più alta intraluminale è visto e questo è anche dimostrato quantitativamente (Figura 4). <img alt="Figura 1" src="/files/ftp_upload/3221/3221fig1.jpg" /> Figura 1. Pressurizzato ex vivo camera schematica nave. Una nave cannulata collegato ad una prossimale (P1) e di un trasduttore distale (P2), trasduttori di pressione del sangue che permette di essere manipolati all&#39;interno della nave. Perfusione è attraverso il trasduttore di P1 e la pressione viene mantenuta tramite trasduttore P2. <img alt="Figura 2" src="/files/ftp_upload/3221/3221fig2.jpg" /> Figura 2. Cannuala con legami. Cravatte in poliestere nero sono collegati a ciascun cannula. <img alt="Figura 3" src="/files/ftp_upload/3221/3221fig3.jpg" /> Figura 3. Immagini video rappresentativo. Adesione cellulare dinamico (freccia rossa) con fluorescenza a 80 mmHg (A) e 120 mmHg (B) dopo 10 minuti di perfusione. <img alt="Figura 4" src="/files/ftp_upload/3221/3221fig4.jpg" /> Figura 4. Adesione dei leucociti in Sprague Dawley arterie carotidi dopo 1 ore di incubazione a basse (80 mmHg) e ad alta pressione (120 mmHg). *** P &lt;0,001, come analizzato da 2-way ANOVA a misure ripetute utilizzando il test post hoc di Bonferroni.

<ol>
	<li>Lopez, A. D., Mathers, C. D., Ezzati, M., Jamison, D. T., Murray, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+and+regional+burden+of+disease+and+risk+factors.">Global and regional burden of disease and risk factors.</a> Lancet. 367, 1747-1757 (2001).</li><li>Szczech, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+kidney+injury+and+cardiovascular+outcomes+in+acute+severe+hypertension.">Acute kidney injury and cardiovascular outcomes in acute severe hypertension.</a> Circulation. 121, 2183-2191 .</li><li>Rodriguez-Garcia, J. L., Botia, E., de La Sierra, A., Villanueva, M. A., Gonzalez-Spinola, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Significance+of+elevated+blood+pressure+and+its+management+on+the+short-term+outcome+of+patients+with+acute+ischemic+stroke.">Significance of elevated blood pressure and its management on the short-term outcome of patients with acute ischemic stroke.</a> Am J Hypertens. 18, 379-384 (2005).</li><li>Levy, D., Larson, M. G., Vasan, R. S., Kannel, W. B., Ho, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+progression+from+hypertension+to+congestive+heart+failure.">The progression from hypertension to congestive heart failure.</a> JAMA. 275, 1557-1562 (1996).</li><li>Ley, K., Laudanna, C., Cybulsky, M. I., Nourshargh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Getting+to+the+site+of+inflammation:+the+leukocyte+adhesion+cascade+updated.">Getting to the site of inflammation: the leukocyte adhesion cascade updated.</a> Nat Rev Immunol. 7, 678-689 (2007).</li><li>Riou, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+pressure+promotes+monocyte+adhesion+to+the+vascular+wall.">High pressure promotes monocyte adhesion to the vascular wall.</a> Circ Res. 100, 1226-1233 (2007).</li><li>Ley, K., Gaehtgens, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial,+not+hemodynamic,+differences+are+responsible+for+preferential+leukocyte+rolling+in+rat+mesenteric+venules.+Circ+Res.">Endothelial, not hemodynamic, differences are responsible for preferential leukocyte rolling in rat mesenteric venules. Circ Res.</a> 69, 1034-1041 (1991).</li><li>Bernhagen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MIF+is+a+noncognate+ligand+of+CXC+chemokine+receptors+in+inflammatory+and+atherogenic+cell+recruitment.">MIF is a noncognate ligand of CXC chemokine receptors in inflammatory and atherogenic cell recruitment.</a> Nat Med. 13, 587-596 (2007).</li><li>Woollard, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathophysiological+levels+of+soluble+P-selectin+mediate+adhesion+of+leukocytes+to+the+endothelium+through+Mac-1+activation.">Pathophysiological levels of soluble P-selectin mediate adhesion of leukocytes to the endothelium through Mac-1 activation.</a> Circ Res. 103, 1128-1138 (2008).</li><li>Eberth, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Importance+of+pulsatility+in+hypertensive+carotid+artery+growth+and+remodeling.">Importance of pulsatility in hypertensive carotid artery growth and remodeling.</a> J Hypertens. 27, 2010-2021 (2009).</li><li>Cooke, J. P., Rossitch, E., Andon, N. A., Loscalzo, J., Dzau, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+activates+an+endothelial+potassium+channel+to+release+an+endogenous+nitrovasodilator.">Flow activates an endothelial potassium channel to release an endogenous nitrovasodilator.</a> J Clin Invest. 88, 1663-1671 (1991).</li></ol>

Il protocollo dello studio è stato approvato dal Medical Alfred Ricerca e l&#39;Educazione degli animali Precinct Comitato Etico e l&#39;Alfred Hospital Comitato Etico.

Si tratta di un metodo modificato per studiare l&#39;adesione dei leucociti all&#39;endotelio in intatti i vasi sanguigni isolato in condizioni di pressione in tempo reale. Perfusione della camera solo recipiente consente una rapida convalida dei pro-infiammatorie ceppi di topi e grandi vasi di ratto. L&#39;attivazione consente la manipolazione di pressione dinamica delle interazioni delle cellule che devono essere osservati dal basso verso l&#39;intraluminale pressioni molto elevate, quindi meglio imitare-mento fisiologico e condizioni fisiopatologiche. Diametro dei vasi può essere misurata con un sufficiente cellulare imaging programma e quindi il flusso di taglio e la velocità può essere determinato e quindi manipolata. Con le sue capacità miografo, interventi farmacologici nel bagno aggiungere un&#39;altra dimensione alle condizioni sperimentali possibile con questo modello che permette studi meccanicistici e dei percorsi di segnalazione. Mentre la conservazione endoteliali non può essere confermata durante la manipolazione di pressione, le risposte a ACh e PE può essere condotta perfusione post 9. Va notato che questa impostazione dimostra gli effetti della pressione intraluminale sulla cellula-cellula non interazioni gli effetti del flusso ematico pulsatile né sistolica o diastolica. Inoltre, mentre le variazioni di pressione acuta sulla adesione dei leucociti sono stati osservati, questa configurazione può essere utilizzata anche per guardare gli effetti della pressione cronica (cioè aumentando i tempi di incubazione e utilizzando un modello animale cronico di pressione). Sprague Dawley arterie carotidi comuni sono dimostrate in questo set up, ma altri ceppi e le specie possono essere utilizzati con gli opportuni adeguamenti alle dimensioni cannula. Infatti, è importante notare che l&#39;età e il peso degli animali sulle dimensioni del vaso e quindi che il set up deve essere personalizzato per ciascuna nave. Dissezione stretta e attenta del tessuto connettivo in grado di migliorare la visualizzazione dei leucociti immensamente.

<table border="1"><tbody><tr><th> Nome del reagente / attrezzature </th><th> Azienda </th><th> Numero di catalogo </th><th> Commenti </th></tr><tr><td> Microscopio </td><td> Carl Zeiss, Inc </td><td> STEREO Discovery V.20 </td><td></td></tr><tr><td> PHD 2000 pompa siringa </td><td> Harvard Apparatus </td><td> 70-2016 </td><td></td></tr><tr><td> Fotocamera Digitale e Controller </td><td> Hamamatsu ORCA-ER </td><td> C4742-95 </td><td></td></tr><tr><td> Sistema di illuminazione a fluorescenza </td><td> Lumen Dynamics </td><td> X-Cite 120 </td><td></td></tr><tr><td> Nave da Camera </td><td> Vivere Strumentazione Sistemi </td><td> CH/1/SH </td><td></td></tr><tr><td> Pressione Servo Controller e pompa peristaltica </td><td> Vivere Strumentazione Sistemi </td><td> PS - 200 </td><td></td></tr><tr><td> Monitor di pressione di perfusione </td><td> Vivere Strumentazione Sistemi </td><td> PM - 4 </td><td></td></tr><tr><td> 2 x Trasduttore di pressione </td><td> Vivere Strumentazione Sistemi </td><td> PT - F </td><td></td></tr><tr><td> Controllore di temperatura </td><td> Vivere Strumentazione Sistemi </td><td> TC-01 </td><td></td></tr><tr><td> Pompa peristaltica </td><td> Instech Laboratories, Inc. </td><td> P720 </td><td></td></tr><tr><td> VybrantDil cellula di etichettatura soluzione </td><td> Invitrogen </td><td> V-22885 </td><td> Usa 1:1000 </td></tr></tbody></table>

imaging leukocyte adhesion to the vascular endothelium at high intraluminal pressure

In tutto il mondo, l&#39;ipertensione è segnalato per essere in circa un quarto della popolazione ed è il fattore principale di rischio biomedici per la mortalità a livello mondiale. Nella ipertensione vascolare è associata a disfunzione endoteliale e l&#39;infiammazione che porta a una maggiore aterosclerosi e patologie diverse come la malattia renale cronica 2, 3, ictus e scompenso cardiaco 4. Un primo passo nel processo infiammatorio vascolari che determinano l&#39;aterogenesi è la cascata di adesione che prevede la laminazione, tethering, aderenza e conseguente trasmigrazione dei leucociti attraverso l&#39;endotelio. Reclutamento e l&#39;accumulo dei leucociti all&#39;endotelio è mediata da un sovraregolazione delle molecole di adesione come la molecola di adesione delle cellule vascolari-1 (VCAM-1), l&#39;adesione cellulare molecola intracellulare-1 (ICAM-1) ed E-selectina così come gli aumenti in rilascio di citochine e chemochine e una sovraregolazione di specie reattive dell&#39;ossigeno 5. Metodi in vitro come test adesione statica aiutare a determinare i meccanismi coinvolti nella cellula-cellula l&#39;adesione, nonché l&#39;analisi delle molecole di adesione delle cellule. Metodi utilizzati in precedenti studi in vitro hanno dimostrato che un aumento acuto della pressione sul endotelio può portare ad adesione dei monociti, un sovraregolazione di molecole di adesione e marker infiammatori 6 Tuttavia, simile a molti test in vitro, questi risultati non sono stati eseguiti in tempo reale in condizioni di flusso fisiologico, né con sangue intero. Pertanto, in vivo sono sempre più utilizzati in modelli animali per dimostrare l&#39;infiammazione vascolare e lo sviluppo della placca. Microscopia intravitale è ora ampiamente utilizzato per valutare l&#39;adesione dei leucociti, lamiere, migrazione e trasmigrazione 7-9. Quando si combinano gli effetti della pressione sui leucociti per l&#39;adesione endoteliale studi in vivo sono meno estese. Uno di questi studi prende in esame gli effetti in tempo reale del flusso di taglio e sulla crescita arteriosa e marker infiammatori rimodellamento, ma sono stati valutati solo attraverso immunoistochimica 10. Qui vi presentiamo un modello per la registrazione di adesione dei leucociti in tempo reale intatta vasi sanguigni sotto pressione, utilizzando la perfusione di sangue intero. La metodologia è una modifica di un modello di nave ex camera di perfusione in vivo 9 che permette analisi in tempo reale delle interazioni leucociti-endotelio adesivo in vasi intatte. La nostra modifica consente la manipolazione della pressione intraluminale fino a 200 mmHg consentendo lo studio non solo in condizioni di flusso fisiologico ma anche condizioni di pressione. Mentre i sistemi myography pressione sono state precedentemente dimostrato di osservare nave parete e lume diametro 11 così come la contrazione nave questa è la prima volta dimostrando leucociti-endotelio interazioni in tempo reale. Qui mostriamo la tecnica utilizzando carotidi raccolti da ratti e cannulata ad una misura camera di flusso accoppiato ad un microscopio a fluorescenza. La camera di imbarcazione è equipaggiata con il coprioggetti fondo permettendo una grande lente di grande diametro obiettivo a breve distanza di lavoro per l&#39;immagine della nave. Inoltre, agonista selezionati e / o antagonisti può essere utilizzata per approfondire i meccanismi che controllano l&#39;adesione cellulare. I vantaggi di questo metodo rispetto microscopia intravitale includono alcun coinvolgimento di un intervento chirurgico invasivo e quindi un throughput più elevato può essere ottenuto. Questo metodo consente anche l&#39;utilizzo di inibitori della localizzato l&#39;imbarcazione desiderata mentre intravitale consente solo trattamento sistemico inibitore.

Watch this Scientific Journal Video about Imaging Leukocyte Adhesion to the Vascular Endothelium at High Intraluminal Pressure at JoVE.com

Imaging Leukocyte Adhesion to the Vascular Endothelium at High Intraluminal Pressure

Questo è un metodo per visualizzare l&#39;adesione dei leucociti all&#39;endotelio in raccolte serbatoi in pressione. La tecnica consente di studiare l&#39;adesione vascolare in condizioni di flusso di taglio con differenti pressioni intraluminali fino a 200 mmHg così imitare-zione le condizioni fisiopatologiche di alta pressione sanguigna.

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. ...

imaging-leukocyte-adhesion-to-vascular-endothelium-at-high

Research

JoVE Journal

Immunology and Infection

15.5K Views.  Monash University. Questo è un metodo per visualizzare l'adesione dei leucociti all'endotelio in raccolte serbatoi in pressione. La tecnica consente di studiare l'adesione vascolare in condizioni di flusso di taglio con differenti pressioni intraluminali fino a 200 mmHg così imitare-zione le condizioni fisiopatologiche di alta pressione sanguigna.

Video: Imaging Leukocyte Adhesion to the Vascular Endothelium at High Intraluminal Pressure

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non invasiva di imaging di homing dei leucociti e la migrazione In vivo</em

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

Ricerca

Immunologia e infezioni

Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of guard cells, which are able to close the stomatal pore in response to a number of external factors including light intensity, carbon dioxide concentration, and relative humidity (RH). The stomatal pore is also the main route for pathogen entry into leaves, a crucial step for disease development. Recent studies have unveiled that closure of the pore is effective in minimizing bacterial disease development in Arabidopsis plants; an integral part of plant innate immunity. Previously, we have used epidermal peels to assess stomatal response to live bacteria (Melotto et al. 2006); however maintaining favorable environmental conditions for both plant epidermal peels and bacterial cells has been challenging. Leaf epidermis can be kept alive and healthy with MES buffer (10 mM KCl, 25 mM MES-KOH, pH 6.15) for electrophysiological experiments of guard cells. However, this buffer is not appropriate for obtaining bacterial suspension. On the other hand, bacterial cells can be kept alive in water which is not proper to maintain epidermal peels for long period of times. When an epidermal peel floats on water, the cells in the peel that are exposed to air dry within 4 hours limiting the timing to conduct the experiment. An ideal method for assessing the effect of a particular stimulus on guard cells should present minimal interference to stomatal physiology and to the natural environment of the plant as much as possible. We, therefore, developed a new method to assess stomatal response to live bacteria in which leaf wounding and manipulation is greatly minimized aiming to provide an easily reproducible and reliable stomatal assay. The protocol is based on staining of intact leaf with propidium iodide (PI), incubation of staining leaf with bacterial suspension, and observation of leaves under laser scanning confocal microscope. Finally, this method allows for the observation of the same live leaf sample over extended periods of time using conditions that closely mimic the natural conditions under which plants are attacked by pathogens.

We have developed a simple and reproducible protocol to access stomatal response to live bacteria. This method minimizes wounding and manipulation of the leaf as compared to the use of epidermal peels reported previously.

Valutazione della risposta stomatica a Live cellule batteriche utilizzando Imaging Whole Leaf

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

L&#39;introduzione di shear stress nello studio di adesione batterica

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

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

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

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

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

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

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 ...

Human Neutrophil Flow Chamber Adhesion Assay

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion involves many different adhesion molecules including members of the &beta;2 integrin family and their counter-receptors of the ICAM family. Recently, naturally occurring genetic variants in both &beta;2 integrins and ICAMs are reported to be associated with autoimmune disease. Thus, the quantitative adhesive capacity of neutrophils from individuals with varying allelic forms of these adhesion molecules is important to study in relation to mechanisms underlying development of autoimmunity. Adhesion studies in flow chamber systems can create an environment with fluid shear stress similar to that observed in the blood vessel environment in vivo. Here, we present a method using a flow chamber assay system to study the quantitative adhesive properties of human peripheral blood neutrophils to human umbilical vein endothelial cell (HUVEC) and to purified ligand substrates. With this method, the neutrophil adhesive capacities from donors with different allelic variants in adhesion receptors can be assessed and compared. This method can also be modified to assess adhesion of other primary cell types or cell lines.

A method of quantitating neutrophil adhesion is reported. This method creates a dynamic flow environment similar to that encountered in a blood vessel. It allows the investigation of neutrophil adhesion to either purified adhesion molecules (ligand) or endothelial cell substrate (HUVEC) in a context similar to the in vivo environment with sheer stress.

Umano camera di flusso neutrofili saggio

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion ...

Scalable High Throughput Selection From  Phage-displayed Synthetic Antibody Libraries

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. However, it is apparent that traditional monoclonal technologies are not alone up to this task. This has led to the development of alternate methods to satisfy the demand for high quality and renewable affinity reagents to all accessible elements of the proteome. Toward this end, high throughput methods for conducting selections from phage-displayed synthetic antibody libraries have been devised for applications involving diverse antigens and optimized for rapid throughput and success. Herein, a protocol is described in detail that illustrates with video demonstration the parallel selection of Fab-phage clones from high diversity libraries against hundreds of targets using either a manual 96 channel liquid handler or automated robotics system. Using this protocol, a single user can generate hundreds of antigens, select antibodies to them in parallel and validate antibody binding within 6-8 weeks. Highlighted are: i) a viable antigen format, ii) pre-selection antigen characterization, iii) critical steps that influence the selection of specific and high affinity clones, and iv) ways of monitoring selection effectiveness and early stage antibody clone characterization. With this approach, we have obtained synthetic antibody fragments (Fabs) to many target classes including single-pass membrane receptors, secreted protein hormones, and multi-domain intracellular proteins. These fragments are readily converted to full-length antibodies and have been validated to exhibit high affinity and specificity. Further, they have been demonstrated to be functional in a variety of standard immunoassays including Western blotting, ELISA, cellular immunofluorescence, immunoprecipitation and related assays. This methodology will accelerate antibody discovery and ultimately bring us closer to realizing the goal of generating renewable, high quality antibodies to the proteome.

A method is described with visual accompaniment for conducting scalable, high throughput selections from phage-displayed combinatorial synthetic antibody libraries against hundreds of antigens simultaneously. Using this parallel approach, we have isolated antibody fragments that exhibit high affinity and specificity for diverse antigens that are functional in standard immunoassays.

Scalabile High Throughput Selection Da sintetici Biblioteche Antibody fagi-visualizzato

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. ...

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the shear stress of flowing blood.
To identify the bacterial and host factors that contribute to vascular adhesion of microorganisms, appropriate models that study these interactions under physiological shear conditions are needed. Here, we describe an in vitro flow chamber model that allows to investigate bacterial adhesion to different components of the extracellular matrix or to endothelial cells, and an intravital microscopy model that was developed to directly visualize the initial adhesion of bacteria to the splanchnic circulation in vivo. These methods can be used to identify the bacterial and host factors required for the adhesion of bacteria under flow. We illustrate the relevance of shear stress and the role of von Willebrand factor for the adhesion of Staphylococcus aureus using both the in vitro and in vivo model.

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

To study the interaction of bacteria with the blood vessels under shear stress, a flow chamber and an in vivo mesenteric intravital microscopy model are described that allow to dissect the bacterial and host factors contributing to vascular adhesion.

In vitro e in vivo modello per studiare l&#39;adesione batterica alla parete del serbatoio in condizioni di flusso

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the ...

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

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

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

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

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

Assay of Adhesion Under Shear Stress for the Study of T Lymphocyte-Adhesion Molecule Interactions

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and interaction with antigen presenting cells (APC) in the formation of immunological synapses. These two contexts of T cell adhesion differ in that T cell-APC interactions can be considered static, while T cell-blood vessel interactions are challenged by the shear stress generated by circulation itself. T cell-APC interactions are classified as static in that the two cellular partners are static relative to each other. Usually, this interaction occurs within the lymph nodes. As a T cell interacts with the blood vessel wall, the cells arrest and must resist the generated shear stress.1,2 These differences highlight the need to better understand static adhesion and adhesion under flow conditions as two distinct regulatory processes. The regulation of T cell adhesion can be most succinctly described as controlling the affinity state of integrin molecules expressed on the cell surface, and thereby regulating the interaction of integrins with the adhesion molecule ligands expressed on the surface of the interacting cell. Our current understanding of the regulation of integrin affinity states comes from often simplistic in vitro model systems. The assay of adhesion using flow conditions described here allows for the visualization and accurate quantification of T cell-epithelial cell interactions in real time following a stimulus. An adhesion under flow assay can be applied to studies of adhesion signaling within T cells following treatment with inhibitory or stimulatory substances. Additionally, this assay can be expanded beyond T cell signaling to any adhesive leukocyte population and any integrin-adhesion molecule pair.

This flow adhesion assay provides a simple, high impact model of T cell-epithelial cell interactions. A syringe pump is used to generate shear stress, and confocal microscopy captures images for quantification. The goal of these studies is to effectively quantify T cell adhesion using flow conditions.

Saggio di adesione Sotto Shear stress per lo studio di linfociti T-Adhesion Molecule Interazioni

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and ...