עבודה זו נתמכה על ידי מועצת העוצרים של מדינת לואיזיאנה LEQSF (2009-12), RD-A-19 (PI: WL Murfee) לבין יתר לחץ דם טוליין מרכז כליות מצוינות ממומן על ידי מענק P20RR017659 NIH-08 (PI : ל &#39;גבריאל Navar).

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אין לנו מה למסור.

המודל החצנה דווח בשנת 2006 והוא מותאם החל מה מכניים חולדה פגיעה מודלים לפדר של אנגיוגנזה 4-7 ומייצרת תוצאות דומות שהוקמו גם דגמים הזרקת ה-IP לנצל עכברוש לפדר 9. השעה החצנה 20 דקות נקבע באופן ניסיוני כדי לייצר תגובה angiogenic חזקים. אמנם זה פרק הזמן יכול להיות מגוונים, זה לאפשר יישום מקומי של מעכבי angiogenic 4 ללימודי מכניסטיים יישום ישיר של תאים חיצוניים ללימודי השושלת סלולריים. הכדאיות של שילוב תאים לתוך הרקמה שיפוץ mesenteric נתמכת על ידי מחקרים ראשוניים במעבדה שלנו באמצעות מראש שכותרתו מח עצם ותאי גזע mesenchymal (איור 7), ועל ידי ההצלחה של חוקרת את גורלו של האדם IP השומן המופק בתאי סטרומה הזרקת 14 . במעבדה שלנו, השתמשנו במודל זה כדי לזהות phen pericyteשינויים otypic במהלך זמן התגובה angiogenic 10 ולהעריך את הפוטנציאל angiogenic במהלך במצבים פתולוגיים, דוגמת יתר לחץ דם 12. בתגובה angiogenic ו פנוטיפ התא שינויים הקשורים מודל זה יכול להיות גם ציין אחרות mesenteric מודלים חולדה angiogenic כולל חשיפה היפוקסיה כרונית 10, 11. הגבלה של המודל החצנה היא מפעילה את המנגנונים המדויקים של אנגיוגנזה אינם ידועים. החצנה של לפדר נקשר degranulation תא התורן רמות היסטמין גבוהות 6, אך חקירה נוספת נדרשת כדי לקבל תובנה רבה יותר. גירוי angiogenic הוא ללא ספק רב העצרת, ייצור תגובה שיפוץ חזקים מעבר בהיררכיה של רשת כלי הדם. אמנם לא ידועים המנגנונים להישאר הביקורת העיקרי של מודל זה, שחזור שלה ופשטות להפוך אותו למושך לזיהוי הסלולר דינמיקה involved בתהליך מורכב מיסודו הנבטה נימי. שחזור של מודל נתמך על ידי מדדים angiogenic דומים במהלך תקופה של צמיחה רשת כלי הדם על פני מספר רב של זנים של עכברים (Wistar זכר ונקבה Sprague-Dawley) במחקרים שפורסמו בעבר מהמעבדה שלנו, 10, 12. מאז, רוב הרקמות mesenteric עכברים בוגרים הם vascularized, המודל גם מאפשר מספר רב של רקמות כדי להיבדק לכל בעל חיים. למרבה הצער, מודל זה אינו ישים בעליל מודלים העכבר גנטיות כמו mesenteric Windows העכבר יש כלי הדם יליד פחות, מניסיוננו, בדרך כלל אין רשתות הסתעפות הנצפה. יישומים עתידיים כוללים את החקירה של פונקציונליות כלי במהלך אנגיוגנזה באמצעות מיקרוסקופ תוך חיוני בנקודות זמן ספציפיות חקירת הדינמיקה הסלולר הקשורות המעורבים lymphangiogenesis ו neurogenesis. למרות היקף כלי הדם יליד לכל חלון mesenteric נראה המשוגעיחסי ghly עם הגיל, ראינו הסתעפות רשתות כלי הדם אצל הזכר חולדות Wistar החל מגיל 4-5 שבועות. תצפיות מראות כי המודל החצנה יכול להיות מיושם גם להשוות את ההבדלים angiogenic על פני המון זמן.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם </td><td> חברה </td><td> מספר קטלוגי </td><td> הערות נוספות </td></tr><tr><td> וילון </td><td> הקרדינל הבריאות </td><td> 4012 </td><td> 12 &quot;x12&quot; Bio-Shield עיקור רגיל כורכת </td></tr><tr><td> נויס מספריים Micro </td><td> Roboz כירורגית כלי </td><td> RS-5677 </td><td> נויס מיקרו הביתור מספריים אביב; ישרים, שארפ-בלאנט נקודות; חדשניות 13 מ&quot;מ, רוחב עצה 0.25mm, 4 1/2 &quot;אורך כולל </td></tr><tr><td> גרפה המלקחיים </td><td> Roboz כירורגית כלי </td><td> RS-5135 </td><td> מיקרו הביתור מלקחיים, משונן, עיקול קל, רוחב עצה 0.8mm; 4 &quot;אורך </td></tr><tr><td> גרפה המלקחיים </td><td> Roboz כירורגית כלי </td><td> RS-5130 </td><td> מלקחיים המבתרים מיקרו, משונן, סטרייט, רוחב 0.8mm, עצה 4 &quot;אורך </td></tr&gt; <tr><td> 4-0 תפר </td><td> Ethicon </td><td> 699G </td><td> (1.5 מדד) קשירת ETHILON ניילון שחור monofilament </td></tr><tr><td> 5-0 תפר </td><td> Ethicon </td><td> 8556 </td><td> (1.0 מדד) PROLENE Polyprolene קשירת monofilament כחול </td></tr><tr><td> 7-0 תפר </td><td> Ethicon </td><td> 1647G </td><td> (0.5 מדד) קשירת ETHILON ניילון שחור monofilament </td></tr><tr><td> Castroviejo מיקרו מחזיק מחט </td><td> מדע כלים עדינים </td><td> 12060-02 </td><td> רוחב טיפ: 0.6 מ&quot;מ לחיצה אורך: 5 מ&quot;מ אורך: 9cm ישר עצה </td></tr><tr><td> Castroviejo מחזיק מחט </td><td> מדע כלים עדינים </td><td> 12565-14 </td><td> צורה טיפ: סטרייט רוחב טיפ: 1.5mm לחיצה אורך: 10 מ&quot;מ מספריים: אין נעילה: כן אורך: 14cm משוננת: כן </td></tr><tr><td> אזמל ידית </td><td> רובוz כירורגית כלי </td><td> RS-9843 </td><td> אזמל ידית, # 3, מוצק; 4 &quot;אורך </td></tr><tr><td> סכין כירורגית סטרילית </td><td> סינסינטי כירורגי </td><td> 0110 </td><td> נירוסטה, גודל 10 </td></tr><tr><td> צלחת פטרי </td><td> פישר סיינטיפיק </td><td> 08-757-13 </td><td> רכס beveled, Slippable </td></tr></tbody></table> טבלת חומרים כירורגית כלים ספציפיים. <table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם </td><td> חברה </td><td> מספר קטלוגי </td><td> הערות נוספות </td></tr><tr><td> Beuthanasia </td><td> Schering-Plough בעלי חיים בריאות ברית קורפ (שהוזמנו אספקה ​​MWI וטרינרית) </td><td> MWI #: 011168 </td><td> המרכיב הפעיל: לכל 100 מ&quot;ל, 390 מ&quot;ג נתרן pentobarbital, 50 מ&quot;ג נתרן פניטואין </td></tr><tr><td> קטמין </td><td> Fort Dodge לבריאות בעלי חיים (שהוזמנו אספקה ​​MWI וטרינרית) </td><td> MWI #: 000680 </td><td> Kateset 100 מ&quot;ג / מ&quot;ל </td></tr><tr><td> Xylazine </td><td> לויד. בע&quot;מ (שהוזמנו אספקה ​​MWI וטרינרית) </td><td> MWI #: 009307 </td><td> Anased 100 מ&quot;ג / מ&quot;ל </td></tr><tr><td> מלוח </td><td> Hospira בע&quot;מ </td><td> 94-217-JT </td><td></td></tr><tr><td> PBS </td><td> SIGMA </td><td> 011M8207 </td><td></td></tr><tr><td> Saponin </td><td> סיגמא </td><td> BCBB4080 </td><td></td></tr><tr><td> PECAM (CD31) </td><td> BD Pharmingen </td><td> 553371 </td><td></td></tr><tr><td> Streptavidin-CY3 </td><td> ג&#39;קסון ImmunoResearch </td><td> 016-160-084 </td><td></td></tr><tr><td> BSA </td><td> ג&#39;קסון ImmunoResearch </td><td> 096555 </td><td></td></tr><tr><td> VECTASTAIN עלית ABC </td><td> וקטור מעבדות </td><td> PK-6100 </td><td></td></tr><tr><td> וקטור נובה האדום </td><td> וקטור מעבדות </td><td> SK-4800 </td><td></td></tr><tr><td> VectaMount </td><td> וקטור מעבדות </td><td> H-500 </td><td></td></tr></tbody></table> טבלה של חומרים כימיים ספציפיים

rat mesentery exteriorization: a model for investigating the cellular dynamics involved in angiogenesis

הצמיחה לרשת שיפוץ Microvacular והם היבטים קריטיים של ריפוי פצעים, דלקות, רטינופתיה סוכרתית, הגידול ותנאי מחלות אחרות 1, 2. הצמיחה לרשת מיוחסת בדרך כלל אנגיוגנזה, כהגדרתו צמיחה של כלי דם חדשים מתוך קיימים כלי. תהליך angiogenic הוא גם מקושר ישירות אל arteriogenesis, כהגדרתו רכישת נימי של ציפוי תא perivascular והגדלת כלי. למותר לציין, אנגיוגנזה הוא מורכב ומעורבים בו מספר שחקנים ברמה תאית ומולקולרית 3. ההבנה כיצד רשת כלי הדם גדל דורש לזהות את הדינמיקה במרחב ובזמן, יחד היררכיה של הרשת במהלך תקופה של אנגיוגנזה. מידע זה הוא קריטי להתפתחות של טיפולים שמטרתם מניפולציה צמיחה כלי. המודל החצנה המתוארת במאמר זה מהווה מודל פשוט, לשחזור של stimulating אנגיוגנזה ב לפדר את העכברוש. זה הותאם מפצע ריפוי מודלים בחולדה לפדר 4-7, והוא אלטרנטיבה לעורר אנגיוגנזה ב לפדר באמצעות זריקות IP של Pro-angiogenic סוכני 8, 9. המודל החצנה הוא אטרקטיבי כי זה דורש התערבות כירורגית מינימלית ומפיק דרמטיים, עולה ב לשחזור נבטים נימי, אזור כלי הדם וצפיפות כלי הדם במשך זמן קצר יחסית ברקמות המאפשר הדמיה דו ממדית של רשתות שלמות כלי הדם עד יחיד התא ברמה. הגידול משקף מגורה תגובות angiogenic טבעיים בסביבה ללא הפרעה פיזיולוגית של מולקולות angiogenic זרים. באמצעות שיטות תיוג immunohistochemical, מודל זה הוכח מאוד שימושי בזיהוי אירועים הסלולר הרומן המעורבים אנגיוגנזה. החוקרים יכולים בקלות לתאם את המדדים angiogenic במהלך תקופה של שיפוצים עם הזמן specific Dynamics, כגון שינויים פנוטיפי סלולריים או אינטראקציות הסלולר 4, 5, 7, 10, 11.

Watch this Scientific Journal Video about Rat Mesentery Exteriorization: A Model for Investigating the Cellular Dynamics Involved in Angiogenesis at JoVE.com

Rat Mesentery Exteriorization: A Model for Investigating the Cellular Dynamics Involved in Angiogenesis

מאמר זה מתאר מודל פשוט של גירוי אנגיוגנזה ב לפדר את העכברוש. המודל מייצר עליות דרמטיות באזור נימי, מצמיח כלי דם וצפיפות כלי הדם במשך זמן קצר יחסית ברקמות המאפשר en להדמיה פני רשתות שלמות כלי הדם עד לרמה תא בודד.

לפדר החצנה חולדה: דגם לחקירת דינמיקה סלולר המעורבים אנגיוגנזה

Microvacular network growth and remodeling are critical aspects of wound healing, inflammation, diabetic retinopathy, tumor growth and other disease ...

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19.0K Views.  Tulane University. מאמר זה מתאר מודל פשוט של גירוי אנגיוגנזה ב לפדר את העכברוש. המודל מייצר עליות דרמטיות באזור נימי, מצמיח כלי דם וצפיפות כלי הדם במשך זמן קצר יחסית ברקמות המאפשר en להדמיה פני רשתות שלמות כלי הדם עד לרמה תא בודד.

Video: Rat Mesentery Exteriorization: A Model for Investigating the Cellular Dynamics Involved in Angiogenesis

Christopher Hughes:  An in vitro model for the Study of Angiogenesis (Interview)

Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro.  He explains the importance of fibroblasts that secrete a critical, yet unidentified, soluble factor that allow endothelial cells to form vessels in culture that branch, form proper lumens, and undergo anastamosis.

Christopher Hughes:  An in vitro model for the Study of Angiogenesis Interview

Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro. He explains the importance of fibroblasts that secrete a critical, yet unidentified, soluble factor that allow endothelial cells to form vessels in culture that branch, form proper lumens, and undergo anastamosis.

כריסטופר יוז: במודל חוץ גופית לחקר אנגיוגנזה (ראיון)

Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro.  He explains the importance of fibroblasts that ...

Rat Mesentery Angiogenesis Assay

The adult rat mesentery window angiogenesis assay is biologically appropriate and is exceptionally well suited to the 
study of sprouting angiogenesis in vivo [see review papers], which is the dominating form of angiogenesis in human tumors and non-tumor 
tissues, as discussed in invited review papers1,2. Angiogenesis induced in the membranous mesenteric parts by intraperitoneal (i.p.) injection of a pro-angiogenic factor can be modulated
by subcutaneous (s.c.), intravenous (i.v.) or oral (p.o.) treatment with modifying agents of choice. Each membranous part of the mesentery is 
translucent and framed by fatty tissue, giving it a window-like appearance.
The assay has the following advantageous features: (i) the test tissue is natively vascularized, albeit 
sparsely, and since it is extremely thin, the microvessel network is virtually two-dimensional, which allows the entire network 
to be assessed microscopically in situ; (ii) in adult rats the test tissue lacks significant physiologic angiogenesis, which characterizes
 most normal adult mammalian tissues; the degree of native vascularization is, however, correlated with age, as discussed in1; 
(iii) the negligible level of trauma-induced angiogenesis ensures high sensitivity; (iv) the 
assay replicates the clinical situation, as the angiogenesis-modulating test drugs are administered systemically and the responses 
observed reflect the net effect of all the metabolic, cellular, and molecular alterations induced by the treatment; (v) the assay 
allows assessments of objective, quantitative, unbiased variables of microvascular spatial extension, density, and network pattern 
formation, as well as of capillary sprouting, thereby enabling robust statistical analyses of the dose-effect and molecular 
structure-activity relationships; and (vi) the assay reveals with high sensitivity the toxic or harmful effects of treatments in 
terms of decreased rate of physiologic body-weight gain, as adult rats grow robustly.
Mast-cell-mediated angiogenesis was first demonstrated using this assay3,4. The model demonstrates a high level of 
discrimination regarding dosage-effect relationships and the measured effects of systemically administered chemically or functionally closely related drugs and proteins, 
including: (i) low-dosage, metronomically administered standard chemotherapeutics that yield diverse, drug-specific effects (i.e., 
angiogenesis-suppressive, neutral or angiogenesis-stimulating activities5); (ii) natural iron-unsaturated human lactoferrin, which stimulates 
VEGF-A-mediated angiogenesis6, and natural iron-unsaturated bovine lactoferrin, which inhibits VEGF-A-mediated angiogenesis7; and (iii) 
low-molecular-weight heparin fractions produced by various means8,9. Moreover, the assay is highly suited to studies of the combined 
effects on angiogenesis of agents that are administered systemically in a concurrent or sequential fashion.
The idea of making this video originated from the late Dr. Judah Folkman when he visited our laboratory and witnessed the methodology being demonstrated.
Review papers (invited) discussing and appraising the assay
Norrby, K. In vivo models of angiogenesis. J. Cell. Mol. Med. 10, 588-612 (2006).
Norrby, K. Drug testing with angiogenesis models. Expert Opin. Drug. Discov. 3, 533-549 (2008).

Normal adult vascularized mammalian tissue that lacks physiologic angiogenesis and that has not been exposed to surgical intervention is used to study: (i) the initiation and development of angiogenesis following intraperitoneal administration of test agents; and (ii) modification of angiogenesis following systemic administration of selected test agents.

עכברוש mesentery Assay אנגיוגנזה

The adult rat mesentery window angiogenesis assay is biologically appropriate and is exceptionally well suited to the 
study of sprouting angiogenesis in ...

An Explant Assay for Assessing Cellular Behavior of the Cranial Mesenchyme

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the neural tube in a process known as neurulation. During neurulation, morphogenesis of the mesenchyme that underlies the neural plate is believed to drive neural fold elevation. The cranial mesenchyme is comprised of the paraxial mesoderm and neural crest cells. The cells of the cranial mesenchyme form a pourous meshwork composed of stellate shaped cells and intermingling extracellular matrix (ECM) strands that support the neural folds. During neurulation, the cranial mesenchyme undergoes stereotypical rearrangements resulting in its expansion and these movements are believed to provide a driving force for neural fold elevation. However, the pathways and cellular behaviors that drive cranial mesenchyme morphogenesis remain poorly studied. Interactions between the ECM and the cells of the cranial mesenchyme underly these cell behaviors. Here we describe a simple ex vivo explant assay devised to characterize the behaviors of these cells. This assay is amendable to pharmacological manipulations to dissect the signaling pathways involved and live imaging analyses to further characterize the behavior of these cells. We present a representative experiment demonstrating the utility of this assay in characterizing the migratory properties of the cranial mesenchyme on a variety of ECM components.

The cranial mesenchyme undergoes dramatic morphogenic movements that likely provides a driving force for elevation of the neural folds1,2. Here we describe a simple ex vivo explant assay to characterize the cellular behaviors of the cranial mesenchyme during neurulation. This assay has numerous applications including being amenable to pharmacological manipulations and live imaging analyses.

Assay Explant להערכת התנהגות ניידת של mesenchyme הגולגולת

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the ...

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems including plants, invertebrates and vertebrates. There are various methods to achieve RNAi in vivo4,5. For example, the target gene may be transformed into an RNAi vector, and then either permanently or transiently transformed into cell lines or primary cells to achieve gene knockdown effects; alternatively synthesized double-strand oligonucleotides from specific target genes (RNAi oligos) may be transiently transformed into cell lines or primary cells to silence target genes; or synthesized double-strand RNA molecules may be microinjected into an organism. Since the nematode C. elegans uses bacteria as a food source, feeding the animals with bacteria expressing double-strand RNA against target genes provides a viable strategy6. Here we present an RNAi feeding method to score body size phenotype. Body size in C. elegans is regulated primarily by the TGF- &beta; - like ligand DBL-1, so this assay is appropriate for identification of TGF-&beta; signaling components7. We used different strains including two RNAi hypersensitive strains to repeat the RNAi feeding experiments. Our results showed that rrf-3 strain gave us the best expected RNAi phenotype. The method is easy to perform, reproducible, and easily quantified. Furthermore, our protocol minimizes the use of specialized equipment, so it is suitable for smaller laboratories or those at predominantly undergraduate institutions.

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

We demonstrate how to use the RNAi feeding technique to knock down target genes and score body size phenotype in C. elegans. This method could be used for a large scale screen to identify potential genetic components of interest, such as those involved in body size regulation by DBL-1/TGF-&beta; signaling.

באמצעות אסטרטגית האכלת התערבות RNA בתיווך למסך לגנים מעורבים בויסות משקל גוף בנמטודות<em&gt; ג elegans</em

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

One-channel Cell-attached Patch-clamp Recording

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate depends on properties intrinsic to each channel protein as well as the mechanism by which it is generated and controlled and represents an important area of current research. Information about the operational dynamics of ion channel proteins can be obtained by observing long stretches of current produced by a single molecule. Described here is a protocol for obtaining one-channel cell-attached patch-clamp current recordings for a ligand gated ion channel, the NMDA receptor, expressed heterologously in HEK293 cells or natively in cortical neurons. Also provided are instructions on how to adapt the method to other ion channels of interest by presenting the example of the mechano-sensitive channel PIEZO1. This method can provide data regarding the channel&#8217;s conductance properties and the temporal sequence of open-closed conformations that make up the channel&#8217;s activation mechanism, thus helping to understand their functions in health and disease.

Described here is a procedure for obtaining long stretches of current recording from one ion channel with the cell-attached patch-clamp technique. This method allows for observing, in real time, the pattern of open-close channel conformations that underlie the biological signal. These data inform about channel properties in undisturbed biological membranes.

הקלטת תיקון מהדק מצורף תא אחד ערוצים

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate ...

Microperfusion Technique to Investigate Regulation of Microvessel Permeability in Rat Mesentery

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular mechanisms regulating vascular permeability in cultured endothelial cell monolayers and the functional exchange properties of whole microvascular beds. A method to cannulate and perfuse venular microvessels of rat mesentery and measure the hydraulic conductivity of the microvessel wall is described. The main equipment needed includes an intravital microscope with a large modified stage that supports micromanipulators to position three different microtools: (1) a beveled glass micropipette to cannulate and perfuse the microvessel; (2) a glass micro-occluder to transiently block perfusion and enable measurement of transvascular water flow movement at a measured hydrostatic pressure, and (3) a blunt glass rod to stabilize the mesenteric tissue at the site of cannulation. The modified Landis micro-occlusion technique uses red cells suspended in the artificial perfusate as markers of transvascular fluid movement, and also enables repeated measurements of these flows as experimental conditions are changed and hydrostatic and colloid osmotic pressure difference across the microvessels are carefully controlled. Measurements of hydraulic conductivity first using a control perfusate, then after re-cannulation of the same microvessel with the test perfusates enable paired comparisons of the microvessel response under these well-controlled conditions. Attempts to extend the method to microvessels in the mesentery of mice with genetic modifications expected to modify vascular permeability were severely limited because of the absence of long straight and unbranched microvessels in the mouse mesentery, but the recent availability of the rats with similar genetic modifications using the CRISPR/Cas9 technology is expected to open new areas of investigation where the methods described herein can be applied.

The modified Landis technique enables paired measurement of the hydraulic conductivity of individual microvessels in the mesentery of normal and genetically modified rats under control and test conditions using microperfusion techniques. It provides a convenient method to evaluate mechanisms that regulate microvessel permeability and transvascular exchange under physiological conditions.

Microperfusion טכניקה לחקירת הסדרת כלי דם קטן חדירות בחולדה לפדר

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular ...

A Simple Bioassay for the Evaluation of Vascular Endothelial Growth Factors

The analysis of receptor tyrosine kinases and their interacting ligands involved in vascular biology is often challenging due to the constitutive expression of families of related receptors, a broad range of related ligands and the difficulty of dealing with primary cultures of specialized endothelial cells. Here we describe a bioassay for the detection of ligands to the vascular endothelial growth factor receptor-2 (VEGFR-2), a key transducer of signals that promote angiogenesis and lymphangiogenesis. A cDNA encoding a fusion of the extracellular (ligand-binding) region of VEGFR-2 with the transmembrane and cytoplasmic regions of the erythropoietin receptor (EpoR) is expressed in the factor-dependent cell line Ba/F3. This cell line grows in the presence of interleukin-3 (IL-3) and withdrawal of this factor results in death of the cells within 24 hr. Expression of the VEGFR-2/EpoR receptor fusion provides an alternative mechanism to promote survival and potentially proliferation of stably transfected Ba/F3 cells in the presence of a ligand capable of binding and cross-linking the extracellular portion of the fusion protein (i.e., one that can cross-link the VEGFR-2 extracellular region). The assay can be performed in two ways: a semi-quantitative approach in which small volumes of ligand and cells permit a rapid result in 24 hr, and a quantitative approach involving surrogate markers of a viable cell number. The assay is relatively easy to perform, is highly responsive to known VEGFR-2 ligands and can accommodate extracellular inhibitors of VEGFR-2 signaling such as monoclonal antibodies to the receptor or ligands, and soluble ligand traps.

We describe a simple cell-based bioassay for detecting, quantifying and monitoring the activity of members of the vascular endothelial growth factor family of ligands. The assay uses chimeric receptors expressed in a factor-dependent cell line to provide a semi-quantitative or quantitative assessment of receptor binding and cross-linking by the ligand.

מבדק פשוט עבור הערכת גורמי הגדילה של אנדותל כלי דם

The analysis of receptor tyrosine kinases and their interacting ligands involved in vascular biology is often challenging due to the constitutive ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

זמן לשגות הדמיה ברזולוציה גבוהה וניתוח אוטומטי של Dynamics microtubule In Living האדם טבורי וריד לתאי אנדותל

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as well as pathological changes in endothelial phenotype and gene expression. In particular, maladaptive effects of periodic, unidirectional flow induced shear stress can trigger a variety of effects on several vascular cell types, particularly endothelial cells. While by now endothelial cells from diverse anatomic origins have been cultured, in-depth analyses of their responses to fluid shear have been hampered by the relative complexity of shear models (e.g., parallel plate flow chamber, cone and plate flow model). While these all represent excellent approaches, such models are technically complicated and suffer from drawbacks including relatively lengthy and complex setup time, low surface areas, requirements for pumps and pressurization often requiring sealants and gaskets, creating challenges to both maintenance of sterility and an inability to run multiple experiments. However, if higher throughput models of flow and shear were available, greater progress on vascular endothelial shear responses, particularly periodic shear research at the molecular level, might be more rapidly advanced. Here, we describe the construction and use of shear rings: a novel, simple-to-assemble, and inexpensive tissue culture model with a relatively large surface area that easily allows for a high number of experimental replicates in unidirectional, periodic shear stress studies on endothelial cells.

The Assembly and Application of &#039;Shear Rings&#039;: A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Different levels and patterns of fluid shear are known to modulate endothelial gene expression, phenotype and susceptibility to disease. We discuss the assembly and use of 'shear rings': a model that produces unidirectional, periodic shear stress patterns. Shear rings are simple to assemble, economical and can produce high cell yields.

האסיפה והיישום של &#39;טבעות Shear&#39;: מודל האנדותל חדשני Orbital, חד-כיוונית תקופתית נוזלי זרימת מתח שאר

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as ...