אנו מודים לבני זוג. אראטה Tabuchi וולפגנג Kuebler (אוניברסיטת טורונטו) להוראה בדבר מיקרוסקופיה intravital. אנו מודים לאנדרו קייהיל (ניקון מכשירים) לסיוע בתכנון ויישום מיקרוסקופים. עבודה זו מומנה על ידי NIH / NHLBI מענקים P30 HL101295 וK08 HL105538 (למניה).

<ol>
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אין ניגודי האינטרסים הכריזו.

ביחד עם התרחבות השימוש במיקרוסקופ בvivo, יש הערכה גוברת הוא לגודל משמעותי של ESL, כמו גם התרומות הרבות שלו לתפקוד כלי דם. נתונים אלה המתעוררים, לעומת זאת, נובעים בעיקר ממחקרים של מערכת כלי הדם המערכתית. ואכן, שימוש במיקרוסקופ בvivo הריאות מאתגר מבחינה טכנית, ניתן ח...

Glycocalyx אנדותל הוא שכבה תאית של proteoglycans וglycosaminoglycans הנלווה המצפה את כלי דם אינטימה. בחי, glycocalyx הוא hydrated מאוד, ויצר שכבת האנדותל משמעותית משטח (ESL), המסדירה מגוון רחב של פונקציות אנדותל כולל חדירות נוזלי 1, נויטרופילים אנדותל- הידבקות 2, וmechanotransduction של 3 מאמץ גזירת נוזל. מבחינה הסטורית, glycocalyx היה מוערך בשל aberrance בהכנות בתרבית תאים 4, 5 והשפלתה במהלך קיבוע רקמה הסטנדרטי ועיבוד 6. השימוש הגובר 7 של מיקרוסקופיה intravital (במיקרוסקופ vivo, IVM) שעלה בקנה אחד עם עניין מדעי מוגבר בחשיבות של ESL לתפקוד כלי דם במהלך מחלה ובריאות. ESL הוא בלתי נראה למיקרוסקופ אור ולא יכול להיות מתויג בקלותvivo, בהתחשב בנטייה של קטיני ניאון glycocalyx מחייבים לגרום להתלכדות RBC 8 ותסחיפי ריאות קטלניים (תצפיות שלא פורסמו). גישות עקיפות אחדות לכן פותחו להסיק עובי ESL (ובהרחבה, יושרת glycocalyx) במיטות שאינן נעות וסקולריים כגון microcirculations cremasteric וmesenteric. טכניקות אלו כוללות מדידה של הבדלים במהירות במחזור microparticle כפונקציה של מרחק מקרום אנדותל (velocimetry תמונת microparticle 9) כמו גם המדידה של ההדרה של סמנים מגושמים, fluorescently כותרת וכלי דם (למשל dextrans) ממשטח אנדותל (טכניקת dextran הדרת 10, 11). טכניקות אלו, רק הדרת dextran מסוגלת לאמוד עובי ESL ממדידות שנעשו בנקודה אחת בזמן. על ידי מדידת רוחב כלי דם בו זמנית באמצעות מיקרוסקופיה brightfield (רוחב בclusive של ESL &quot;בלתי נראה&quot;), ומיקרוסקופיה ניאון של נותב כלי דם הוצא מESL, עובי ESL ניתן לחשב כמחצית ההפרש בין רוחב כלי דם 2. השימוש באמצעי מיידי של עובי ESL הוא מותאם היטב ללימוד glycocalyx ריאתי. מיקרוסקופיה Intravital של הריאות היא מאתגרת, בהתחשב בחפץ לב וריאות תנועה משמעותי. למרות התקדמות שחל באחרונה לאפשר לקיבוע של ריאות עכבר ב12 vivo, 13, חששות קיימים לגבי ההשפעה הפיזיולוגית של ריאות קיפאון. חוסר תנועת ריאות קשור בתחמוצת חנקן האנדותל ירד איתות 14, מסלול איתות המשפיע גם הידבקות נויטרופילים 15 ופציעה 16 ריאות. יתר על כן, קיבוע של אזור של ריאה חושפת מקיף alveoli הנייד לכוחות גזירה שפגיעתו (מה שנקרא &quot;atelectrauma&quot;), בהתאם למושגים הפיסיולוגיים הקלסיים שלתלות הדדית מכתשיים 17. בשנת 2008, אראטה Tabuchi, וולפגנג Kuebler ועמיתיו פתחו טכניקת ניתוח המאפשרת למיקרוסקופיה intravital של ריאת עכבר שנע בחופשיות-18. חפץ נשימה הנובע מטכניקה זו ניתן לשלול על ידי שימוש בהדמיה במהירות גבוהה, כולל מדידה סימולטנית של brightfield ומיקרוסקופ פלואורסצנטי. בדו&quot;ח זה, אנו פירוט כיצד הדמית הדרת dextran מיידית יכולה להיות מועסקת על מנת למדוד את עובי ESL בזרימת דם subpleural של שנע בחופשיות-, in vivo ריאות עכבר. טכניקה זו ניתן לשנות בקלות כדי לקבוע glycocalyx פונקציה ספציפית, יכולתו של שלם ESL שלא לכלול במחזור אלמנטים ממשטח אנדותל. אנחנו השתמשנו בטכניקות אלה לאחרונה כדי לקבוע את החשיבות של יושרת ESL ריאתי להתפתחות פציעת ריאות חריפה במחלות דלקתיות מערכתיות כגון אלח דם 2.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td></td><td></td><td></td><td> שם מגיב </td></tr><tr><td> FITC-dextran (150 KDA) </td><td> סיגמא </td><td> FD150S </td><td></td></tr><tr><td> TRITC-dextran (150 KDA) </td><td> סיגמא </td><td> T1287 </td><td></td></tr><tr><td> Streptavidin מצופה הניאון microspheres </td><td> מעבדות פוני </td><td> CP01F/10428 </td><td> הדרקון הירוק פלואורסצנטי (בדומה לFITC) </td></tr><tr><td> קטמין </td><td> מור רפואי </td><td></td><td></td></tr><tr><td> Xylazine </td><td> מור רפואי </td><td></td><td></td></tr><tr><td> אנטי Icam-1 נוגדן biotinylated </td><td> eBioscience </td><td> שיבוט YN1/1.7.4 </td><td> 1:50 דילול </td></tr><tr><td> אלוטיפ נוגדן biotinylated </td><td> eBioscience </td><td> IgG2b eB149/10H5 </td><td> 1:50 דילול </td></tr><tr><td></td><td></td><td></td><td> ציוד </td></tr><tr><td> הנשמה מכאנית </td><td> הרווארד Apparatus </td><td> אינספירה </td><td></td></tr><tr><td> קטטר טרכאוסטומיה </td><td> הרווארד Apparatus </td><td> 730028 </td><td></td></tr><tr><td> מנגנון electrocautery </td><td> DRE רפואי </td><td> Valleylab SSE-2L </td><td></td></tr><tr><td> דו קוטבי מלקחי כווייה </td><td> אולסן רפואי </td><td> 10-1200I </td><td> 9.9cm מקפרסון </td></tr><tr><td> הטמפרטורה control מערכה </td><td> מכשירי Precision העולם </td><td> ATC1000 </td><td></td></tr><tr><td> משאבת מזרק </td><td> הרווארד Apparatus </td><td> 11 עלית משאבה </td><td></td></tr><tr><td> מיקרוסקופ (widefield) </td><td> ניקון </td><td> LV-150 </td><td></td></tr><tr><td> מיקרוסקופ (confocal) </td><td> ניקון </td><td> A1R </td><td></td></tr><tr><td> התמונה ספליטר </td><td> Photometrics </td><td> Dv2 </td><td></td></tr><tr><td> מצלמת CCD </td><td> Photometrics </td><td> CoolSNAP HQ2 </td><td></td></tr><tr><td> תוכנת עיבוד תמונה </td><td> ניקון </td><td> אלמנטי ש&quot;ח </td><td></td></tr><tr><td> קרום polyvinylidene </td><td> ק.א.ר. Wrap </td><td></td><td></td></tr><tr><td> תלוש כיסוי חוזר </td><td> Bellco </td><td> 5CIR-1-BEL </td><td> 5 מ&quot;מ, עובי # 1 </td></tr><tr><td> דבק (לכסות להחליק לממברנה) </td><td> Pattex </td><td> Flussig (נוזלי) </td><td> להדבקת תלוש כיסוי לממברנה </td></tr><tr><td> דבק (לכסות להחליק לעכבר) </td><td> Pattex </td><td> קריש </td><td> לחיבור קרום לעכבר </td></tr><tr><td> צינורות כירורגים </td><td> Intramedic </td><td> PE50, PE10 </td><td></td></tr><tr><td> תפר </td><td> דיג </td><td></td><td> משי 04:00 </td></tr><tr><td> מכונת גילוח חשמלית </td><td> אוסטר </td><td> 78997 </td><td></td></tr><tr><td> מלקחיים כירורגיים מעוקלים </td><td> Roboz </td><td></td><td></td></tr><tr><td> מלקחיים כירורגיים ישרים </td><td> Roboz </td><td></td><td></td></tr><tr><td> מספרי כירורגים </td><td> Roboz </td><td></td><td></td></tr><tr><td> microscissors כירורגים </td><td> Roboz </td><td></td><td></td></tr><tr><td> נהג מחט כירורגים </td><td> Roboz </td><td></td><td></td></tr><tr><td> קלטת כירורגים </td><td> דיג </td><td></td><td></td></tr><tr><td> ספוגי מטבח (חתוך לפרוסות) </td><td> שונה </td><td></td><td></td></tr></tbody></table>

in vivo measurement of the mouse pulmonary endothelial surface layer

Glycocalyx אנדותל הוא שכבה של proteoglycans וglycosaminoglycans הנלווה המצפה את חלל כלי הדם. בחי, glycocalyx הוא hydrated מאוד, ויצר שכבת האנדותל משמעותית משטח (ESL) שתורמת לשמירה על תפקוד האנדותל. כglycocalyx האנדותל הוא לעתים קרובות סוטה במבחנה והוא אבד במהלך טכניקות קיבוע רקמות הרגילות, מחקר של ESL דורש שימוש במיקרוסקופ intravital. כדי להתקרב לפיסיולוגיה המורכבת ביותר של microvasculature מכתשיים, הדמית intravital ריאה מבוצעת באופן אידיאלי על ריאות באופן חופשי אל לב. הכנות אלה, עם זאת, בדרך כלל סובלות מחפץ תנועה נרחבת. אנחנו מדגימים איך מיקרוסקופיה intravital סגור חזה של ריאת עכבר שנע בחופשיות, יכולה לשמש למדידת תקינות glycocalyx באמצעות הדרה של ESL dextrans משקל המולקולרי שכותרת-fluorescently הגבוה ממשטח אנדותל. טכניקה זו אינה התאוששות אחרי ניתוח, שדורשתbrightfield בו זמנית והדמית ניאון של ריאת העכבר, מאפשרים תצפית אורך של microvasculature subpleural ללא עדות לגרימת פציעת ריאות בלבול.

הגישה הניסויית מתואר בשלבים 1-6 תאפשר לכידה של מסגרות מרובות של דסק&quot;ש סימולטני (brightfield) ותמונות ניאון. כדי לקבוע את עובי ESL, תמונות נרשמו נבדקות על ידי משקיף עיוור לאחר השלמת פרוטוקול הניסוי. שימוש במיקוד המסגרת, microvessels subpleural (&lt;20 קוטר מיקרומטר) מזוהית; לפחות 3 microvessels נ...

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In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

שכבת פני שטח glycocalyx / אנדותל האנדותל היא למד באופן אידיאלי באמצעות מיקרוסקופ intravital. מיקרוסקופיה Intravital טכני מאתגרת באיבר מרגש כגון ריאות. אנחנו מדגימים איך brightfield סימולטני ומיקרוסקופיה ניאון ניתן להשתמש כדי להעריך את עובי שכבת פני שטח האנדותל שבנע בחופשיות,<em&gt; In vivo</em&gt; ריאות עכבר.

במדידת vivo של שכבת פני שטח האנדותל ריאתי המאוסה

The endothelial glycocalyx is a layer of proteoglycans and associated glycosaminoglycans lining the vascular lumen. In vivo, the glycocalyx is highly ...

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Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

14.5K Views.  University of Colorado School of Medicine. שכבת פני שטח glycocalyx / אנדותל האנדותל היא למד באופן אידיאלי באמצעות מיקרוסקופ intravital. מיקרוסקופיה Intravital טכני מאתגרת באיבר מרגש כגון ריאות. אנחנו מדגימים איך brightfield סימולטני ומיקרוסקופיה ניאון ניתן להשתמש כדי להעריך את עובי שכבת פני שטח האנדותל שבנע בחופשיות, In ...

Video: In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

An in vivo Assay to Test Blood Vessel Permeability

This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under physiologic conditions the endothelium is impermeable to albumin, so Evans blue bound albumin remains restricted within blood vessels. In pathologic conditions that promote increased vascular permeability endothelial cells partially lose their close contacts and the endothelium becomes permeable to small proteins such as albumin. This condition allows for extravasation of Evans Blue in tissues. A healthy endothelium prevents extravasation of the dye in the neighboring vascularized tissues. Organs with increased permeability will show significantly increased blue coloration compared to organs with intact endothelium. The level of vascular permeability can be assessed by simple visualization or by quantitative measurement of the dye incorporated per milligram of tissue of control versus experimental animal/tissue. Two powerful aspects of this assay are its simplicity and quantitative characteristics. Evans Blue dye can be extracted from tissues by incubating a specific amount of tissue in formamide. Evans Blue absorbance maximum is at 620 nm and absorbance minimum is at 740 nm. By using a standard curve for Evans Blue, optical density measurements can be converted into milligram dye captured per milligram of tissue. Statistical analysis should be used to assess significant differences in vascular permeability.

An in vivo Assay to Test Blood Vessel Permeability

We are presenting an in vivo assay to test blood vessel permeability. This assay is based on intravenous injection of a dye and subsequent visualization of its diffusion into interstitial spaces.

<em&gt; In vivo</em&gt; Assay לבדיקת חדירות כלי דם

This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under ...

Implantation of Fibrin Gel on Mouse Lung to Study Lung-specific Angiogenesis

Recent significant advances in stem cell research and bioengineering techniques have made great progress in utilizing biomaterials to regenerate and repair damage in simple tissues in the orthopedic and periodontal fields. However, attempts to regenerate the structures and functions of more complex three-dimensional (3D) organs such as lungs have not been very successful because the biological processes of organ regeneration have not been well explored. It is becoming clear that angiogenesis, the formation of new blood vessels, plays key roles in organ regeneration. Newly formed vasculatures not only deliver oxygen, nutrients and various cell components that are required for organ regeneration but also provide instructive signals to the regenerating local tissues. Therefore, to successfully regenerate lungs in an adult, it is necessary to recapitulate the lung-specific microenvironments in which angiogenesis drives regeneration of local lung tissues. Although conventional in vivo angiogenesis assays, such as subcutaneous implantation of extracellular matrix (ECM)-rich hydrogels (e.g., fibrin or collagen gels or Matrigel - ECM protein mixture secreted by Engelbreth-Holm-Swarm mouse sarcoma cells), are extensively utilized to explore the general mechanisms of angiogenesis, lung-specific angiogenesis has not been well characterized because methods for orthotopic implantation of biomaterials in the lung have not been well established. The goal of this protocol is to introduce a unique method to implant fibrin gel on the lung surface of living adult mouse, allowing for the successful recapitulation of host lung-derived angiogenesis inside the gel. This approach enables researchers to explore the mechanisms by which the lung-specific microenvironment controls angiogenesis and alveolar regeneration in both normal and pathological conditions. Since implanted biomaterials release and supply physical and chemical signals to adjacent lung tissues, implantation of these biomaterials on diseased lung can potentially normalize the adjacent diseased tissues, enabling researchers to develop new therapeutic approaches for various types of lung diseases.

Recapitulation of the organ-specific microenvironment, which stimulates local angiogenesis, is indispensable for successful regeneration of damaged tissues. This report demonstrates a novel method to implant fibrin gels on the lung surface of living mouse in order to explore how the lung-specific microenvironment modulates angiogenesis and alveolar regeneration in adult mouse.

השרשה של הפיברין ג&#39;ל על עכבר ריאות לחקר אנגיוגנזה ריאות ספציפית

Recent significant advances in stem cell research and bioengineering techniques have made great progress in utilizing biomaterials to regenerate and ...

Automated Measurement of Pulmonary Emphysema and Small Airway Remodeling in Cigarette Smoke-exposed Mice

COPD is projected to be the third most common cause of mortality world-wide by 2020(1). Animal models of COPD are used to identify molecules that contribute to the disease process and to test the efficacy of novel therapies for COPD. Researchers use a number of models of COPD employing different species including rodents, guinea-pigs, rabbits, and dogs(2). However, the most widely-used model is that in which mice are exposed to cigarette smoke. Mice are an especially useful species in which to model COPD because their genome can readily be manipulated to generate animals that are either deficient in, or over-express individual proteins. Studies of gene-targeted mice that have been exposed to cigarette smoke have provided valuable information about the contributions of individual molecules to different lung pathologies in COPD(3-5). Most studies have focused on pathways involved in emphysema development which contributes to the airflow obstruction that is characteristic of COPD. However, small airway fibrosis also contributes significantly to airflow obstruction in human COPD patients(6), but much less is known about the pathogenesis of this lesion in smoke-exposed animals. To address this knowledge gap, this protocol quantifies both emphysema development and small airway fibrosis in smoke-exposed mice. This protocol exposes mice to CS using a whole-body exposure technique, then measures respiratory mechanics in the mice, inflates the lungs of mice to a standard pressure, and fixes the lungs in formalin. The researcher then stains the lung sections with either Gill&#8217;s stain to measure the mean alveolar chord length (as a readout of emphysema severity) or Masson&#8217;s trichrome stain to measure deposition of extracellular matrix (ECM) proteins around small airways (as a readout of small airway fibrosis). Studies of the effects of molecular pathways on both of these lung pathologies will lead to a better understanding of the pathogenesis of COPD.

The goal of this protocol is to provide automated methods to quantify chronic lung pathologies in a murine model of COPD. The protocol includes exposing mice to cigarette smoke (CS), measuring pulmonary function, inflating the lungs, and using morphometry methods to measure emphysema and small airway remodeling in mice.

מדידה אוטומטית של הריאות נפחתו וקטן Airway Remodeling בעכברים שנחשפו לעשן סיגריות

COPD is projected to be the third most common cause of mortality world-wide by 2020(1).  Animal models of COPD are used to identify molecules that ...

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

בידוד תרבות לתאי אנדותל ריאתי מעכברים ילודים

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

Biology

Isolation of Primary Mouse Lung Endothelial Cells

Endothelial cells play critical roles in the regulation of vascular tone, immunity, coagulation, and permeability. Endothelial dysfunction occurs in medical conditions including diabetes, atherosclerosis, sepsis, and acute lung injury. A reliable and reproducible method to isolate pure endothelial cells from mice is needed to investigate the role of endothelial cells in the pathogenesis of these and other conditions. In this protocol, lung microvascular endothelial cells were prepared from 5-7 day old neonatal mouse pups. Lungs are harvested, minced, and enzymatically digested with collagenase I, and released cells are cultured overnight. Endothelial cells are then selected using anti-PECAM1 (CD-31) IgG conjugated to magnetic beads, and cells again are cultured to confluence. A secondary cell selection then occurs with anti-ICAM2 (CD-102) IgG conjugated to magnetic beads to increase the purity of the endothelial cells, and the cells again are cultured to confluence. The entire process takes approximately 7-10 days before the cells can be used for experimentation. This simple protocol yields highly pure (purity &#62;92%) endothelial cells that can be immediately used for in vitro studies, including the studies focused on endothelial cytokine and chemokine production, leukocyte-endothelial interactions, endothelial coagulation pathways, and endothelial permeability. With many knockouts and transgenic mouse lines available, this procedure also lends itself to understanding the function of specific genes expressed by endothelial cells in healthy and pathologic responses to injury, infection, and inflammation.

In this article, primary lung endothelial cells were isolated and cultured from neonatal mice.

בידוד תאי אנדותל ריאה ראשוניים של עכבר

Endothelial cells play critical roles in the regulation of vascular tone, immunity, coagulation, and permeability. Endothelial dysfunction occurs in ...

Immunology and Infection

Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with real time imaging, it becomes feasible to determine permeability changes in individual pulmonary microvessels. Herein we describe steps to isolate rat lungs and perfuse them with autologous blood. Then, we outline steps to infuse fluorophores or agents via a microcatheter into a small lung region. Using these procedures described, we determined permeability increases in rat lung microvessels in response to infusions of bacterial lipopolysaccharide. The data revealed that lipopolysaccharide increased fluid leak across both venular and capillary microvessel segments. Thus, this method makes it possible to compare permeability responses among vascular segments and thus, define any heterogeneity in the response. While commonly used methods to define lung permeability require postprocessing of lung tissue samples, the use of real time imaging obviates this requirement as evident from the present method. Thus, the isolated lung preparation combined with real time imaging offers several advantages over traditional methods to determine lung microvascular permeability, yet is a straightforward method to develop and implement.

The isolated blood-perfused lung preparation makes it feasible to visualize microvessel networks on the lung surface. Here we describe an approach to quantify permeability of single microvessels in isolated lungs using real time fluorescence imaging.

כימות יחיד כלי דם קטנים בחדירות מבודדת-perfused דם עכברוש ריאות הכנה

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with ...

Dynamic Measurement and Imaging of Capillaries, Arterioles, and Pericytes in Mouse Heart

Coronary arterial tone along with the opening or closing of the capillaries largely determine the blood flow to cardiomyocytes at constant perfusion pressure. However, it is difficult to monitor the dynamic changes of the coronary arterioles and the capillaries in the whole heart, primarily due to its motion and non-stop beating. Here we describe a method that enables monitoring of arterial perfusion rate, pressure and the diameter changes of the arterioles and capillaries in mouse right ventricular papillary muscles. The mouse septal artery is cannulated and perfused at a constant flow or pressure with the other dynamically measured. After perfusion with a fluorescently labeled lectin (e.g., Alexa Fluor-488 or -633 labeled Wheat-Germ Agglutinin, WGA), the arterioles and capillaries (and other vessels) in right ventricle papillary muscle and septum could be readily imaged. The vessel-diameter changes could then be measured in the presence or absence of heart contractions. When genetically encoded fluorescent proteins were expressed, specific features could be monitored. For examples, pericytes were visualized in mouse hearts that expressed NG2-DsRed. This method has provided a useful platform to study the physiological functions of capillary pericytes in heart. It is also suitable for studying the effect of reagents on the blood flow in heart by measuring the vascular/capillary diameter and the arterial luminal pressure simultaneously. This preparation, combined with a state-of-the-art optic imaging system, allows one to study the blood flow and its control at cellular and molecular level in the heart under near-physiological conditions.

Presented here is a protocol to study the coronary microcirculation in living murine heart tissue by ex vivo monitoring of the arterial perfusion pressure and flow that maintains the pressure, as well as vascular tree components including the capillary beds and pericytes, as the septal artery is cannulated and pressurized.

מדידה והדמיה דינמיות של נימים, עורקים ופריציטים בלב העכבר

Coronary arterial tone along with the opening or closing of the capillaries largely determine the blood flow to cardiomyocytes at constant perfusion ...

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

Angiogenesis is the process by which new blood vessels are formed from existing vessels. New vessel growth requires coordinated endothelial cell proliferation, migration, and alignment to form tubular structures followed by recruitment of pericytes to provide mural support and facilitate vessel maturation. Current in vitro cell culture approaches cannot fully reproduce the complex biological environment where endothelial cells and pericytes interact to produce functional vessels. We present a novel application of the in vivo matrix gel plug assay to study endothelial-pericyte interactions and formation of functional blood vessels using severe combined immune deficiency mutation (SCID) mice. Briefly, matrix gel is mixed with a solution containing endothelial cells with or without pericytes followed by injection into the back of anesthetized SCID mice. After 14 days, the matrix gel plugs are removed, fixed and sectioned for histological analysis. The length, number, size and extent of pericyte coverage of mature vessels (defined by the presence of red blood cells in the lumen) can be quantified and compared between experimental groups using commercial statistical platforms. Beyond its use as an angiogenesis assay, this matrix gel plug assay can be used to conduct genetic studies and as a platform for drug discovery. In conclusion, this protocol will allow researchers to complement available in vitro assays for the study of endothelial-pericyte interactions and their relevance to either systemic or pulmonary angiogenesis.

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

We present a protocol to study human endothelial-pericyte interactions in mouse using a variation of the matrix gel plug angiogenesis assay.

In vivo לחקר האדם האנדותל-pericyte אינטראקציה באמצעות assay Plug מטריקס ג&#39;ל עכבר

Angiogenesis is the process by which new blood vessels are formed from existing vessels. New vessel growth requires coordinated endothelial cell ...

Utilizing the Precision-Cut Lung Slice to Study the Contractile Regulation of Airway and Intrapulmonary Arterial Smooth Muscle

Smooth muscle cells (SMC) mediate the contraction of the airway and the intrapulmonary artery to modify airflow resistance and pulmonary circulation, respectively, hence playing a critical role in the homeostasis of the pulmonary system. Deregulation of SMC contractility contributes to several pulmonary diseases, including asthma and pulmonary hypertension. However, due to limited tissue access and a lack of culture systems to maintain in vivo SMC phenotypes, molecular mechanisms underlying the deregulated SMC contractility in these diseases remain fully identified. The precision-cut lung slice (PCLS) offers an ex vivo model that circumvents these technical difficulties. As a live, thin lung tissue section, the PCLS retains SMC in natural surroundings and allows in situ tracking of SMC contraction and intracellular Ca2+ signaling that regulates SMC contractility. Here, a detailed mouse PCLS preparation protocol is provided, which preserves intact airways and intrapulmonary arteries. This protocol involves two essential steps before subjecting the lung lobe to slicing: inflating the airway with low-melting-point agarose through the trachea and infilling pulmonary vessels with gelatin through the right ventricle. The PCLS prepared using this protocol can be used for bioassays to evaluate Ca2+-mediated contractile regulation of SMC in both the airway and the intrapulmonary arterial compartments. When applied to mouse models of respiratory diseases, this protocol enables the functional investigation of SMC, thereby providing insight into the underlying mechanism of SMC contractility deregulation in diseases.

The present protocol describes preparing and utilizing mouse precision-cut lung slices to assess the airway and intrapulmonary arterial smooth muscle contractility in a nearly in vivo milieu.

שימוש בפרוסת הריאה החתוכה באופן מדויק כדי לחקור את ויסות ההתכווצות של שריר חלק עורקי דרכי הנשימה והפנים-פולמונרי

Smooth muscle cells (SMC) mediate the contraction of the airway and the intrapulmonary artery to modify airflow resistance and pulmonary circulation, ...

In Vivo Vascular Permeability Detection in Mouse Submandibular Gland

Saliva plays an important role in oral and overall health. The intact endothelial barrier function of blood vessels enables saliva secretion, whereas the endothelial barrier dysfunction is related to many salivary gland secretory disorders. The present protocol describes an in vivo paracellular permeability detection method to evaluate the function of endothelial tight junctions (TJs) in mouse submandibular glands (SMG). First, fluorescence-labeled dextrans with different molecular weights (4 kDa, 40 kDa, or 70 kDa) were injected into the angular veins of mice. Afterward, the unilateral SMG was dissected and fixed in the customized holder under a two-photon laser-scanning microscope, and then images were captured for blood vessels, acini, and ducts. Utilizing this method, the real-time dynamic leakage of the different-sized tracers from blood vessels into the basal sides of the acini and even across the acinar epithelia into the ducts was monitored to evaluate the alteration of the endothelial barrier function under physiological or pathophysiological conditions.

In Vivo Vascular Permeability Detection in Mouse Submandibular Gland

In the present protocol, the endothelial barrier function of the submandibular gland (SMG) was evaluated by injecting different molecular weighted fluorescent tracers into the angular veins of test animal models in vivo under a two-photon laser-scanning microscope.

אין ויוו זיהוי חדירות כלי דם בבלוטה תת-מנדיבולרית של עכבר

Saliva plays an important role in oral and overall health. The intact endothelial barrier function of blood vessels enables saliva secretion, whereas the ...

במדידת vivo של שכבת פני שטח האנדותל ריאתי המאוסה

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השרשה של הפיברין ג'ל על עכבר ריאות לחקר אנגיוגנזה ריאות ספציפית