עבודה זו נתמכה על ידי NIH מענק R01-3155-53502 ו-אוניברסיטת וושינגטון פייזר הסכם Biomedical Research.

1. בעלי חיים הכנה: פרוטוקול זה משתמש הנקבה הבוגרת LysM-eGFP עכברים בוגרים, בהם נויטרופילים מקרופאגים הם שכותרתו fluorescently ידי הביטוי של eGFP 1. <ol class="lalpha" style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> סימון כלי הדם: כדי להמחיש את כלי הדם, 15μl הלא ממוקד נקודות קוונטיות (655nm) הם מדולל לתוך μl 100 של PBS ו מוזרק לווריד תוך דקות את העכבר 10-30 לפני הדמיה. </li><li style=";text-align:right;direction:rtl"> הרדמה: <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> מניחים את העכבר בתא אינדוקציה של מערכת הרדמה וטרינרי משאיפת (נובל לרפואה Service, Inc). </li><li style=";text-align:right;direction:rtl"> הגדר את מאדה כדי% 3-4 Isoflurane והספק של 100% חמצן גז קצב הזרימה ל ~ 1 L / min. כדי למנוע מינון יתר מקרי הרדמה, לעקוב מקרוב אחר בעלי חיים עד רפלקס אחורי קמצוץ הבוהן הולך לאיבוד. </li><li style=";text-align:right;direction:rtl"> הרדמה נשמר 1.5 ~ 2% Isoflurane ו בסדר מכוון במהלך הדמיה כדי למזער את החפצים נשימה לפי הצורך. קצב הנשימה של העכבר הוא במעקב צמוד לאורך כל הפגישה הדמיה כדי להבטיח מטוס נאותה הרדמה. </li><li style=";text-align:right;direction:rtl"> משחה Puralube וטרינרי מוחל על העיניים כדי למנוע התייבשות. </li><li style=";text-align:right;direction:rtl"> העכבר ממוקם על 36 מעלות צלזיוס מוסדר כרית חימום (Braintree Scientific) במהלך זריקות הדמיה כדי למנוע היפותרמיה. </li><li style=";text-align:right;direction:rtl"> כדי למנוע התייבשות, העכבר מקבל 100 μl של PBS sc כל 1-2 שעות. </li></ol></li><li style=";text-align:right;direction:rtl"> מתן גירוי דלקתי לגרום גיוס לויקוציטים: <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> מניחים את העכבר על הבמה ניתוח ספוגית שודד דרכים עם אתנול 70%. </li><li style=";text-align:right;direction:rtl"> כופף את המחט של מזרק אינסולין ~ 90 מעלות ליד שפוע כדי להקל על הזרקת ולשפר את הדיוק של עומק הלידה. </li><li style=";text-align:right;direction:rtl"> טען את המזרק עם 0.5 מיקרוגרם של E.coli bioparticles מדולל 1μl PBS. ניתן לעשות זאת במכסה של צינור microfuge כדי להקל על העומס רביב. </li><li style=";text-align:right;direction:rtl"> החזק את הבוהן עם מלקחיים מעוקל להכניס את המחט דרך העור בעדינות עם שפוע פונה כלפי מעלה עד שהוא ממש מתחת לעור. </li><li style=";text-align:right;direction:rtl"> הזרק באיטיות ולהחזיק במקום למשך 5 שניות כדי למנוע סומק בחזרה. </li></ol></li></ol> 2. הדמיה שלב ההתקנה: פלטפורמת הדמיה מורכבת צלחת אלומיניום עם חור עגול לחתוך למרכז. זכוכית לכסות גדול מודבק בתחתית הצלחת גומי O-Ring מודבק על גבי כדי ליצור תא נוזל חזק הפסקה כדי להכיל את כפה האחורית. צלחת אלומיניום הוא חימם עד 36 ° C באמצעות שני גופי חימום המחוברים העליון של הצלחת. <ol class="lalpha" style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הנח את העכבר על שלב טרום חימם את ההדמיה ולשמור על הגוף הליבה של העכבר טמפרטורה באמצעות 36 ° C כרית חימום. </li><li style=";text-align:right;direction:rtl"> אבטח את כפה coverglass של חדר הדמיה עם הסרט דקה של דבק מגע. </li><li style=";text-align:right;direction:rtl"> שטפו את כפה פעמיים כדי להסיר כל חתיכות צפות של דבק ולאחר מכן למלא את התא עם PBS טרי (מחומם מראש ל -36 ° C). </li></ol> עכברים ניתן הדמיה של עד 4 שעות ללא המהווים הכדאיות של החיה. כף הרגל ניתן לשחרר בעדינות עם כמות קטנה של אצטון עכברים לתחייה ללימודי הדמיה האורך. עם זאת, במקרה זה, אנו ממליצים על תקופת הדמיה &lt;2 שעות ההמתנה 24-48 שעות בין אימון כדי למזער את הסיכון של הרדמה ממנת יתר או התייבשות. 3. הדמיה רכישה מיקרוסקופ שני הפוטונים (מיקרוסקופ 2P) משמש פרוטוקול זה שהותקן כפול לייזר וידאו שער המערכת המורכבת מיקרוסקופ זקוף. המערכת מצויד בשתי טי: לייזרים ספיר, ארבעה חזיתית Bi-אלקלי PMTs לרכישות 3 ו -4 סימולטני ערוץ 20x טבילה במים אובייקטיבי (אולימפוס, 20x/0.95 NA). <ol class="lalpha" style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> Tune XR Chameleon טי: ספיר לייזר (קוהירנט בע&quot;מ) עד 890-900nm. </li><li style=";text-align:right;direction:rtl"> שימוש במראות dichroic 480nm ו 560nm (SemRoc) להפריד שלושה ערוצים: כחול (&lt;480 ננומטר, הדור השני אות הרמוני), ירוק (480-560nm, LysM-eGFP נויטרופילים חיובי) ואדום (&gt; 560 ננומטר, נקודה קוונטית כלי הדם שכותרתו ). לחלופין, מסנן 560nm ניתן להחליף מסנן 570nm להבחין E. coli bioparticles (576nm bioparticles יופיעו כתום) מ 655nm נקודות קוונטיות (יופיעו אדום עמוק). </li><li style=";text-align:right;direction:rtl"> בזהירות להוריד את המטרה לתוך PBS ולהתמקד כלי של הבוהן. </li><li style=";text-align:right;direction:rtl"> עם ההפעלה הרכישה מהירות תמונה בקצב-video (30f/sec) ושימוש אות הרמוני second הנובעות קולגן כציון דרך רקמות, סקר במהירות את הרקמה (במהירות וידאו שער הרכישה) כדי לאתר כלי דם של עניין (אחד עם lysM-eGFP נויטרופילים גלוי). </li><li style=";text-align:right;direction:rtl"> התאם את הרווח על PMTs כדי לייעל הפרדת צבעים ולמזער את כמות לייזר הנדרש כדי להשיג אות מספיק על הרקע (להיותלהיזהר לא להרוות את התמונה!). </li><li style=";text-align:right;direction:rtl"> ברגע האזור שטח של האינטרסים נמצא, להתחיל זמן לשגות הדמיה. זמן לשגות הדמיה ניתן לבצע עם שתי החלטות הזמני שונים. עבור מתגלגל הדמיה התא הידבקות בזמן אמת בתוך כלי הדם, אנו רוכשים 30f/sec על מטוס-z יחיד. לתיעוד extravasation התא הגירה parenchyma אנו מבצעים הדמיה 3D זמן לשגות. פרוטוקול רכישה טיפוסית תכלול ממוצעים של 15 מסגרות לצעד-Z כל רכישת 21 2.5μm רציפים Z-צעדים בהיקף של 200-225 50μm ב 30 מרווחי שניות. </li><li style=";text-align:right;direction:rtl"> לאחר קבצי הנתונים מאוחסנים בשרת המעבדה, תוכנה או Imaris Volocity ניתן להשתמש כדי לבצע רב מימדי עיבוד התא מעקב 2, 3. </li></ol> 4. נציג תוצאות 1.Real בזמן (30f/sec) מדמיינת של נויטרופילים מתגלגל לאורך כלי ב 200x. זמן לשגות הדמיה מראה נויטרופילים, בירוק, מתגלגלים לאורך האנדותל של כלי הדם על 10mins לאחר זיהום חיידקי ליסטריה עם, באדום. סיבי הקולגן ברקמת החיבור מופיעים בכחול. <a href="http://www.jove.com/files/ftp_upload/2062/2062_Miller_Movie1.mov">אנא לחץ כאן כדי לראות את הסרטון.</a> 2.Time לשגות 3D הדמיה של דינמיקה נויטרופילים גיוס 200x. גיוס נויטרופילים אופיינית מהמחזור והגירה דרך רקמה דלקתית מוצג. <a href="http://www.jove.com/files/ftp_upload/2062/2062_Miller_Movie2.mov">אנא לחץ כאן כדי לראות את הסרטון.</a>

<ol>
	<li>Faust, N., Varas, F., Kelly, L. M., Heck, S., Graf, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insertion+of+enhanced+green+fluorescent+protein+into+the+lysozyme+gene+creates+mice+with+green+fluorescent+granulocytes+and+macrophages.">Insertion of enhanced green fluorescent protein into the lysozyme gene creates mice with green fluorescent granulocytes and macrophages.</a> Blood. 96, 719-726 (2000).</li><li>Zinselmeyer, B. H., Lynch, J. N., Zhang, X., Aoshi, T., Miller, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Video-rate+two-photon+imaging+of+mouse+footpad+-+a+promising+model+for+studying+leukocyte+recruitment+dynamics+during+inflammation.">Video-rate two-photon imaging of mouse footpad - a promising model for studying leukocyte recruitment dynamics during inflammation.</a> Inflamm Res. 57, 93-96 (2008).</li><li>Zinselmeyer, B. H., Dempster, J., Wokosin, D. L., Cannon, J. J., Pless, R., Parker, I., Miller, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+16.+Two-photon+microscopy+and+multidimensional+analysis+of+cell+dynamics.">Chapter 16. Two-photon microscopy and multidimensional analysis of cell dynamics.</a> Methods Enzymol. 461, 349-378 (2009).</li><li>Gray, D. F., Jennings, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allergy+in+experimental+mouse+tuberculosis.">Allergy in experimental mouse tuberculosis.</a> Am Rev Tuberc. 72, 171-195 (1955).</li><li>Lin, A., Loughman, J. A., Zinselmeyer, B. H., Miller, M. J., Caparon, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Streptolysin+S+inhibits+neutrophil+recruitment+during+the+early+stages+of+Streptococcus+pyogenes+infection.+Infect+Immun.">Streptolysin S inhibits neutrophil recruitment during the early stages of Streptococcus pyogenes infection. Infect Immun.</a> 77, 5190-5201 (2009).</li><li>Smith, C. J., Zhang, Y., Koboldt, C. M., Muhammad, J., Zweifel, B. S., Shaffer, A., Talley, J. J., Masferrer, J. L., Seibert, K., Isakson, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+analysis+of+cyclooxygenase-1+in+inflammation.">Pharmacological analysis of cyclooxygenase-1 in inflammation.</a> Proc Natl Acad Sci U S A. 95, 13313-13318 (1998).</li><li>Wipke, B. T., Allen, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Essential+role+of+neutrophils+in+the+initiation+and+progression+of+a+murine+model+of+rheumatoid+arthritis.">Essential role of neutrophils in the initiation and progression of a murine model of rheumatoid arthritis.</a> J Immunol. 167, 1601-1608 (2001).</li><li>Baez, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+open+cremaster+muscle+preparation+for+the+study+of+blood+vessels+by+in+vivo+microscopy.">An open cremaster muscle preparation for the study of blood vessels by in vivo microscopy.</a> Microvasc Res. 5, 384-394 (1973).</li><li>Mempel, T. R., Scimone, M. L., Mora, J. R., von Andrian, U. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=in+vivo+imaging+of+leukocyte+trafficking+in+blood+vessels+and+tissues.">in vivo imaging of leukocyte trafficking in blood vessels and tissues.</a> Curr Opin Immunol. 16, 406-417 (2004).</li><li>Miller, M. J., Wei, S. H., Cahalan, M. D., Parker, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autonomous+T+cell+trafficking+examined+in+vivo+with+intravital+two-photon+microscopy.">Autonomous T cell trafficking examined in vivo with intravital two-photon microscopy.</a> Proc Natl Acad Sci U S A. 100, 2604-2609 (2003).</li></ol>

כל הניסויים בוצעו על פי הפרוטוקולים שאושרו על ידי ועדת מחקרים בבעלי חיים של אוניברסיטת וושינגטון בסנט לואיס.

בפרוטוקול הווידאו הזאת אנחנו מדגימים את ההליכים עבור הדמיה בלתי פולשנית 2P גיוס לויקוציטים בתגובה לדלקת. שודד הוא אתר קלאסי פיזיולוגיים לחקר דלקת כגון מחלה, אלרגיה דלקת אוטואימונית 4-7. 2P הדמיה מספק תמונה מפורטת של צעדים ברו?...

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		<th>Comment</th>
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<tr>
<td>Isoflurane, USP</td>
<td>&nbsp;</td>
<td>Butler Animal Health Supply</td>
<td>029405</td>
<td>&nbsp;</td>
<tr>
<td>655nm non-targeted quantum dots</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>Q21021MP</td>
<td>&nbsp;</td>
<tr>
<td>Tetramathylrhodamine conjugated E. coli BioParticles </td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>E2862</td>
<td>&nbsp;</td>
<tr>
<td>Listeria monocytogenes (Lm)</td>
<td>&nbsp;</td>
<td>Reference 2</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Quick gel</td>
<td>&nbsp;</td>
<td>Duro</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Puralube Vet Ointment </td>
<td>&nbsp;</td>
<td>Pharmaderm Animal Health</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Insulin syringes</td>
<td>&nbsp;</td>
<td>BD</td>
<td>08290-3284-38</td>
<td>&nbsp;</td>
<tr>
<td>PBS</td>
<td>&nbsp;</td>
<td>Thermo Scirntific</td>
<td>SH30256.02</td>
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non-invasive imaging of leukocyte homing and migration in vivo

שני הפוטונים (2P) מיקרוסקופיה היא הדמיה ברזולוציה גבוהה טכניקה הותאם באופן רחב על ידי ביולוגים. הערך של מיקרוסקופיה 2P היא שזה מספק מידע עשיר spatiotemporal לגבי התנהגויות בתוך רקמות תאים שלמים של עכברים חיים. גיוס ליקוציט משחק תפקיד משמעותי בהגנה מפני זיהומים מארח וכאשר מסומנת, יכול לתרום מחלות דלקתיות אוטואימוניות. לימוד גיוס לויקוציטים in vivo מבחינה טכנית מאתגרת מאז התאים עוברים במהירות בתוך כלי הנמצא עמוק בתוך רקמות פיזור אור. עד כה, מחקרי הדמיה intravital ביותר דורשים הכנה כירורגי כדי לחשוף את כלי הדם והרקמות. כדי למנוע נזק לרקמות ודלקת הנגרמת על ידי הניתוח עצמו, כאן, אנחנו מתארים פולשני תא בודד הדמיה הגישה כי ניתן להשתמש כדי ללמוד סחר ליקוציט ב שודד את העכבר הפלנגות. אנו דנים בהיבטים הטכניים של ההכנה שלנו 2P הדמיה ללכת את הקורא דרך בניסוי אופייני הראשונית להגדיר לאוסף ביצוע ונתונים.

Watch this Scientific Journal Video about Non-invasive Imaging of Leukocyte Homing and Migration in vivo at JoVE.com

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

כאן אנו מתארים פולשני שני הפוטונים (2P) הגישה מיקרוסקופיה ללמוד ליקוציט ביות ב שודד את העכבר. אנו דנים בהיבטים הטכניים של ההכנה שלנו רקמות הדמיה ללכת את הקורא דרך בניסוי אופייני הראשונית להגדיר לאוסף ביצוע ונתונים.

לא פולשני הדמיה של ביות ליקוציט והגירה 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 ...

non-invasive-imaging-of-leukocyte-homing-and-migration-in-vivo

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Immunology and Infection

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

12.7K Views.  Washington University in St. Louis. כאן אנו מתארים פולשני שני הפוטונים (2P) הגישה מיקרוסקופיה ללמוד ליקוציט ביות ב שודד את העכבר. אנו דנים בהיבטים הטכניים של ההכנה שלנו רקמות הדמיה ללכת את הקורא דרך בניסוי אופייני הראשונית להגדיר לאוסף ביצוע ונתונים.

Video: Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Competitive Homing Assays to Study Gut-tropic T Cell Migration

In order to exert their function lymphocytes need to leave the blood and migrate into different tissues in the body. Lymphocyte adhesion to endothelial cells and tissue extravasation is a multistep process controlled by different adhesion molecules (homing receptors) expressed on lymphocytes and their respective ligands (addressins) displayed on endothelial cells 1 2. Even though the function of these adhesion receptors can be partially studied ex vivo, the ultimate test for their physiological relevance is to assess their role during in vivo lymphocyte adhesion and migration. Two complementary strategies have been used for this purpose: intravital microscopy (IVM) and homing experiments. Although IVM has been essential to define the precise contribution of specific adhesion receptors during the adhesion cascade in real time and in different tissues, IVM is time consuming and labor intensive, it often requires the development of sophisticated surgical techniques, it needs prior isolation of homogeneous cell populations and it permits the analysis of only one tissue/organ at any given time. By contrast, competitive homing experiments allow the direct and simultaneous comparison in the migration of two (or even more) cell subsets in the same mouse and they also permit the analysis of many tissues and of a high number of cells in the same experiment.
Here we describe the classical competitive homing protocol used to determine the advantage/disadvantage of a given cell type to home to specific tissues as compared to a control cell population. We chose to illustrate the migratory properties of gut-tropic versus non gut-tropic T cells, because the intestinal mucosa is the largest body surface in contact with the external environment and it is also the extra-lymphoid tissue with the best-defined migratory requirements. Moreover, recent work has determined that the vitamin A metabolite all-trans retinoic acid (RA) is the main molecular mechanism responsible for inducing gut-specific adhesion receptors (integrin a4b7and chemokine receptor CCR9) on lymphocytes. Thus, we can readily generate large numbers of gut-tropic and non gut-tropic lymphocytes ex vivoby activating T cells in the presence or absence of RA, respectively, which can be finally used in the competitive homing experiments described here.

Competitive homing experiments allow to directly assessing the migratory properties of two different cell populations in a single mouse. Here we illustrate this procedure by comparing the migration of ex vivo-generated gut-tropic versus non-gut tropic T cells.

מבחני ביות תחרותי לחקר נדידת גוט-טרופי T Cell

In order to exert their function lymphocytes need to leave the blood and migrate into different tissues in the body. Lymphocyte adhesion to endothelial ...

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.

הדמיה הדבקה ליקוציט כדי האנדותל דם בלחץ גבוה Intraluminal

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

Non-invasive Imaging of Disseminated Candidiasis in Zebrafish Larvae

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 to 40% attributable mortality6. Systemic candidiasis is normally controlled by innate immunity, and individuals with genetic defects in innate immune cell components such as phagocyte NADPH oxidase are more susceptible to candidemia7-9. Very little is known about the dynamics of C. albicans interaction with innate immune cells in vivo. Extensive in vitro studies have established that outside of the host C. albicans germinates inside of macrophages, and is quickly destroyed by neutrophils10-14. In vitro studies, though useful, cannot recapitulate the complex in vivo environment, which includes time-dependent dynamics of cytokine levels, extracellular matrix attachments, and intercellular contacts10, 15-18. To probe the contribution of these factors in host-pathogen interaction, it is critical to find a model organism to visualize these aspects of infection non-invasively in a live intact host. 
The zebrafish larva offers a unique and versatile vertebrate host for the study of infection. For the first 30 days of development zebrafish larvae have only innate immune defenses2, 19-21, simplifying the study of diseases such as disseminated candidiasis that are highly dependent on innate immunity. The small size and transparency of zebrafish larvae enable imaging of infection dynamics at the cellular level for both host and pathogen. Transgenic larvae with fluorescing innate immune cells can be used to identify specific cells types involved in infection22-24. Modified anti-sense oligonucleotides (Morpholinos) can be used to knock down various immune components such as phagocyte NADPH oxidase and study the changes in response to fungal infection5. In addition to the ethical and practical advantages of using a small lower vertebrate, the zebrafish larvae offers the unique possibility to image the pitched battle between pathogen and host both intravitally and in color. 
The zebrafish has been used to model infection for a number of human pathogenic bacteria, and has been instrumental in major advances in our understanding of mycobacterial infection3, 25. However, only recently have much larger pathogens such as fungi been used to infect larva5, 23, 26, and to date there has not been a detailed visual description of the infection methodology. Here we present our techniques for hindbrain ventricle microinjection of prim25 zebrafish, including our modifications to previous protocols. Our findings using the larval zebrafish model for fungal infection diverge from in vitro studies and reinforce the need to examine the host-pathogen interaction in the complex environment of the host rather than the simplified system of the Petri dish5.

The rapid development, small size and transparency of zebrafish are tremendous advantages for the study of innate immune control of infection1-4. Here we demonstrate techniques for infecting zebrafish larvae using the fungal pathogen Candida albicans by microinjection, methodology recently used to implicate phagocyte NADPH oxidase activity in control of fungal dimorphism5.

הדמיה לא פולשנית של פטרת מופצת הזחלים דג הזברה

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 ...

Bioluminescent Bacterial Imaging In Vivo

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of bacteria in gene and cell therapy for cancer. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumors following systemic administration. Bacteria engineered to express the lux gene cassette permit BLI detection of the bacteria and concurrently tumor sites. The location and levels of bacteria within tumors over time can be readily examined, visualized in two or three dimensions. The method is applicable to a wide range of bacterial species and tumor xenograft types. This article describes the protocol for analysis of bioluminescent bacteria within subcutaneous tumor bearing mice. Visualization of commensal bacteria in the Gastrointestinal tract (GIT) by BLI is also described. This powerful, and cheap, real-time imaging strategy represents an ideal method for the study of bacteria in vivo in the context of cancer research, in particular gene therapy, and infectious disease. This video outlines the procedure for studying lux-tagged E. coli in live mice, demonstrating the spatial and temporal readout achievable utilizing BLI with the IVIS system.

Bioluminescent Bacterial Imaging In Vivo

This article describes the administration of lux-tagged bacteria to mice and subsequent in vivo analysis using IVIS bioluminescence imaging.

הדמיה בקטריאלי bioluminescent<em&gt; In Vivo</em

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of ...

Non-invasive Optical Imaging of the Lymphatic Vasculature of a Mouse

The lymphatic vascular system is an important component of the circulatory system that maintains fluid homeostasis, provides immune surveillance, and mediates fat absorption in the gut. Yet despite its critical function, there is comparatively little understanding of how the lymphatic system adapts to serve these functions in health and disease1. Recently, we have demonstrated the ability to dynamically image lymphatic architecture and lymph "pumping" action in normal human subjects as well as in persons suffering lymphatic dysfunction using trace administration of a near-infrared fluorescent (NIRF) dye and a custom, Gen III-intensified imaging system2-4. NIRF imaging showed dramatic changes in lymphatic architecture and function with human disease. It remains unclear how these changes occur and new animal models are being developed to elucidate their genetic and molecular basis. In this protocol, we present NIRF lymphatic, small animal imaging5,6 using indocyanine green (ICG), a dye that has been used for 50 years in humans7, and a NIRF dye-labeled cyclic albumin binding domain (cABD-IRDye800) peptide that preferentially binds mouse and human albumin8. Approximately 5.5 times brighter than ICG, cABD-IRDye800 has a similar lymphatic clearance profile and can be injected in smaller doses than ICG to achieve sufficient NIRF signals for imaging8. Because both cABD-IRDye800 and ICG bind to albumin in the interstitial space8, they both may depict active protein transport into and within the lymphatics. Intradermal (ID) injections (5-50 &mu;l) of ICG (645 &mu;M) or cABD-IRDye800 (200 &mu;M) in saline are administered to the dorsal aspect of each hind paw and/or the left and right side of the base of the tail of an isoflurane-anesthetized mouse. The resulting dye concentration in the animal is 83-1,250 &mu;g/kg for ICG or 113-1,700 &mu;g/kg for cABD-IRDye800. Immediately following injections, functional lymphatic imaging is conducted for up to 1 hr using a customized, small animal NIRF imaging system. Whole animal spatial resolution can depict fluorescent lymphatic vessels of 100 microns or less, and images of structures up to 3 cm in depth can be acquired9. Images are acquired using V++ software and analyzed using ImageJ or MATLAB software. During analysis, consecutive regions of interest (ROIs) encompassing the entire vessel diameter are drawn along a given lymph vessel. The dimensions for each ROI are kept constant for a given vessel and NIRF intensity is measured for each ROI to quantitatively assess "packets" of lymph moving through vessels.

Recently developed imaging techniques using near-infrared fluorescence (NIRF) may help elucidate the role the lymphatic system plays in cancer metastasis, immune response, wound repair, and other lymphatic-associated diseases.

דימות אופטי לא פולשנית של כלי דם הלימפה של עכבר

The lymphatic  vascular system is an important component of the circulatory system that  maintains fluid homeostasis, provides immune surveillance, and ...

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal features of the natural infection. This protocol describes procedures for the establishment and evaluation of systemic infection due to neuropathogenic Escherichia coli K1 in the neonatal rat. Colonization of the gastrointestinal tract leads to dissemination of the pathogen along the gut-lymph-blood-brain course of infection and the model displays strong age dependency. A strain of E. coli O18:K1 with enhanced virulence for the neonatal rat produces exceptionally high rates of colonization, translocation to the blood compartment and invasion of the meninges following transit through the choroid plexus. As in the human host, penetration of the central nervous system is accompanied by local inflammation and an invariably lethal outcome. The model is of proven utility for studies of the mechanism of pathogenesis, for evaluation of therapeutic interventions and for assessment of bacterial virulence.

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Here, a procedure is described for the establishment of systemic infection in the neonatal rat with cultures of Escherichia coli K1. This non-invasive procedure permits colonization of the gastrointestinal tract, translocation of the pathogen to the systemic circulation, and invasion of the central nervous system at the choroid plexus.

דגם לא פולשנית של Neuropathogenic<em&gt; Coli Escherichia</em&gt; זיהום בעכברוש הילודים

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal ...

Complete Thymectomy in Adult Rats with Non-invasive Endotracheal Intubation

Thymectomy in neonatal rodents is an established and reliable procedure for immunological studies. However, in adult rats, complications of hemorrhage and pneumothorax from pleural disruption can result in a significant mortality rate. This protocol is a simple method of rat thymectomy that utilizes a mini-sternotomy and endotracheal intubation. Intubation is accomplished with a non-invasive and easily reproducible method and allows for positive pressure ventilation to prevent pneumothorax and a controlled airway that allows sufficient time for careful thymus dissection to minimize pleural disruption. A 1.5 cm sternal incision decreases contact with mediastinal vessels and pleura, while still providing full visualization of the thymus. Following exposure of the mediastinum, the thymus is removed by blunt dissection under magnification. The pleural space is then sealed by suture closure of the pre-tracheal muscles followed by the application of surgical glue. The thorax is then closed by suture closure of the sternum, followed by suture closure of the skin. All thymectomies were complete as evidenced by immunohistochemical (IHC) staining of mediastinal tissue, and absence of na&#239;ve T-cells by flow cytometry, and the procedure had a 96% survival rate. This method is suitable when complete thymectomy with minimal complications is desired for further immunological studies in athymic adult rats.

Rodent thymectomy is a valuable technique in immunological research. Here, a protocol for complete thymectomy in adult rats using a mini-sternotomy along with non-invasive intubation and positive pressure ventilation to minimize perioperative morbidity and mortality is described.

Thymectomy מלא בחולדות מבוגרות עם לא פולשני endotracheal אינטובציה

Thymectomy in neonatal rodents is an established and reliable procedure for immunological studies. However, in adult rats, complications of hemorrhage and ...

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection.
The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle.

הדמיה לא פולשנית של התגובה החיסונית המולדת בדגם דג הזברה הזחל של<em&gt; Streptococcus iniae</em&gt; זיהום

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of ...

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.

אינטראקציות מיקרוסקופית Intravital של יקוציט-האנדותל וטסיות דם-יקוציט בMesenterial ורידים בעכברים

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

Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral tissues through an as-yet undefined mechanism. The application of multiphoton microscopy to the study of the immune system provides a tool to measure the dynamics of immune responses within intact tissues. Here we present a protocol for non-invasive intravital multiphoton imaging of CD4 T cells in the inflamed mouse ear dermis. Use of a custom imaging platform and a venous catheter allows for the visualization of CD4 T cell dynamics in the dermal interstitium, with the ability to interrogate these cells in real-time via the addition of blocking antibodies to key molecular components involved in motility. This system provides advantages over both in vitro models and surgically invasive imaging procedures. Understanding the pathways used by CD4 T cells for motility may ultimately provide insight into the basic function of CD4 T cells as well as the pathogenesis of both autoimmune diseases and pathology from chronic infections.

The mechanisms that govern the interstitial motility of CD4 effector T cells at sites of inflammation are relatively unknown. We present a non-invasive approach to visualize and manipulate in vitro-primed CD4 T cells in the inflamed ear dermis, allowing for study of the dynamic behavior of these cells in situ.

הדמית תא CD4 T גירת ביניים בדרמיס המוסת

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral ...