Финансовую поддержку для большинства исследований были предоставлены шведским Советом по медицинским исследованиям (грант 5942).

<ol>
	<li>Norrby, K. <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+models+of+angiogenesis.">In vivo models of angiogenesis.</a> J. Cell. Mol. Med. 10, 588-612 (2006).</li><li>Norrby, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+testing+with+angiogenesis+models.">Drug testing with angiogenesis models.</a> Expert Opin. Drug. Discov. 3, 533-549 (2008).</li><li>Norrby, K., Jakobsson, A., Sorbo, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mast-cell-mediated+angiogenesis:+a+novel+experimental+model+using+the+rat+mesentery.">Mast-cell-mediated angiogenesis: a novel experimental model using the rat mesentery.</a> Virchows Arch. B Cell. Pathol. Incl. Mol. Pathol. 52, 195-206 (1986).</li><li>Norrby, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mast+cells+and+angiogenesis.">Mast cells and angiogenesis.</a> APMIS. 110, 355-371 (2002).</li><li>Albertsson, P., Lennernas, B., Norrby, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+meteronomic+chemotherapy:+modulation+of+angiogenesis+mediated+by+VEGF-A.">On meteronomic chemotherapy: modulation of angiogenesis mediated by VEGF-A.</a> Acta Oncol. 45, 144-155 (2006).</li><li>Norrby, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+apo-lactoferrin+enhances+angiogenesis+mediated+by+vascular+endothelial+growth+factor+A+in+vivo.">Human apo-lactoferrin enhances angiogenesis mediated by vascular endothelial growth factor A in vivo.</a> J. Vasc. Res. 41, 293-304 (2004).</li><li>Norrby, K., Mattsby-Baltzer, I., Innocenti, M., &amp;amp, T. u. n. e. b. e. r. g., S, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orally+administered+bovine+lactoferrin+systemically+inhibits+VEGF(165)-mediated+angiogenesis+in+the+rat.">Orally administered bovine lactoferrin systemically inhibits VEGF(165)-mediated angiogenesis in the rat.</a> Int. J. Cancer. 91, 236-240 (2001).</li><li>Norrby, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-molecular-weight+heparins+and+angiogenesis.">Low-molecular-weight heparins and angiogenesis.</a> APMIS. 114, 79-102 (2006).</li><li>Norrby, K., Nordenhem, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dalteparin,+a+low-molecular-weight+heparin,+promotes+angiogenesis+mediated+by+heparin-binding+VEGF-A+in+vivo.">Dalteparin, a low-molecular-weight heparin, promotes angiogenesis mediated by heparin-binding VEGF-A in vivo.</a> APMIS. 118, 949-957 (2010).</li><li>Norrby, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+fibroblast+growth+factor+and+de+novo+mammalian+angiogenesis.">Basic fibroblast growth factor and de novo mammalian angiogenesis.</a> Microvasc. Res.. 48, 96-113 (1994).</li><li>Norrby, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+endothelial+growth+factor+and+de+novo+mammalian+angiogenesis.">Vascular endothelial growth factor and de novo mammalian angiogenesis.</a> Microvasc. Res. 51, 153-163 (1996).</li><li>Norrby, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microvascular+density+in+terms+of+number+and+length+of+microvessel+segments+per+unit+tissue+volume+in+mammalian+angiogenesis.">Microvascular density in terms of number and length of microvessel segments per unit tissue volume in mammalian angiogenesis.</a> Microvasc. Res. 55, 43-53 (1998).</li></ol>

Нет конфликта интересов объявлены.

Анализ был введен в 1986 году и имеет 3 последовательно получили дальнейшее развитие и уточнение в нашей лаборатории 7,10-12. Как отмечалось в обзорных статьях (приглашен) 1, 2, анализа сравнивает хорошо с других млекопитающих в естественных условиях анализа ангиогенеза в отношении особенно важны такие функции, как взрослые ткани тест изначально васкуляризированной и недостатки физиологического ангиогенеза; минимальные травмы, если таковые имеются, нанесенных ткани; действительно количественные переменные измерены, которая позволяет звуковой статистический анализ по доза-реакция и молекулярно-активность исследований; прорастания кровеносных сосудов происходит, который преобладает в нормальных тканях и опухолей, а также данных о токсичности легко накапливаются в крысах, которые растут энергично физиологически во взрослом возрасте. Невыгодные особенности могут включать то, что анализ является сравнительно много времени, особенно при оценке количества и длины отдельных сегментов капилляров и микрососудов ростки, что оно не позволяет real-time/serial наблюдений, и что она вряд ли подойдет для мышей так как многие брыжеечной окнами у мышей отсутствие потенциальных микрососудов родителей, из которых новые капилляры могут возникать. В режиме реального времени наблюдения, однако, включены по прижизненной микроскопии, где экстериоризируется но нетронутыми брыжеечной окна растопыренными над столике микроскопа в то время как животное под наркозом, как описано в 1. Значительные достижения в области ангиогенеза исследования были достигнуты в последние десятилетия с использованием моделей, таких как роговицы micropocket, цыпленок chorioallantoic мембраны, и подкожно анализов Матригель вилки, которые являются полезными, но в конечном счете, ограниченные средства. Будущее клинического применения антиангиогенного и проангиогенных терапии необходимость создания и проверки более чувствительным и биологически значимым, по-настоящему количественные доклинических моделях, например, описанный в настоящем докладе.

Буферы и реагенты 0,5 М Трис-HCl (рН 7,8) исходного раствора Трис (гидроксиметил) aminomethan (60,57 г) растворяют в ~ 900 мл аква Dest и ~ 45 мл 25% HCl (концентрированный) добавляется для достижения рН 7,8 с последующим разбавлением до 1 литра аква Dest. Трис-буфер физиологического раствора (TBS, рН 7,8) К 200 мл исходного раствора добавляют 18 г NaCl, растворенных в ~ 1800 мл аква Dest с регулировкой рН до 7,8 использованием маточного раствора, с последующим разбавлением до 2 л с аква Dest. TBS (рН 7,4) К 200 мл исходного раствора добавляют 18 г NaCl, растворенных в растворяется в ~ 1800 мл аква Dest с регулировкой рН до 7,4 использованием маточного раствора, с последующим разбавлением до 2 л с аква Dest. Трипсин раствора (0,05% трипсина, 0,1% CaCl 2) Содержит 0,1 г трипсина и 0,2 г CaCl 2 в 200 мл TBS (рН 7,8). Раствором сахарозы 2,5% Содержит 5 г сахарозы в 200 мл TBS (рН 7,8). Исходный раствор 1% натрия азид в TBS (рН 7,4) Содержит в 1 г Na-азид в 100 мл с TBS (рН 7,4). Разводящий буфер: 0,1% BSA, 0,01% Na-азид (Используется для разбавления антител в Vectastain ABC комплект). Содержит 0,1 г бычьего сывороточного альбумина (БСА) и 1 мл 1% Na-азид, разбавленный до 100 мл TBS (рН 7,4). ABC комплект Vectastain PK 4002, ABC пероксидазы мышь IgG Нормальной лошадиной сыворотки; блокирование сыворотке крови; желтый раствор: 3 drops/10 мл буфером для разведения. Биотинилированные антител, анти-IgG мыши, синий раствор: 1 мл drop/10 буфером для разведения. ABC-реагент: оранжевый раствор, 2 капли плюс коричневый раствор 2 мл drops/10 TBS (рН 7,4). Смешайте эти решения за 20 минут до использования! Мышь (моноклональные) анти-крыса эндотелия (Биосурс International, Camarillo, Калифорния, США) <table border="1"><tbody><tr><td> Каталог # </td><td> ARU0181 </td></tr><tr><td> Клон # </td><td> MRC ОХ-43 </td></tr></tbody></table> Это антитело направлено против всех сосудистых эндотелиальных клеток у крыс, за исключением тех, в головном мозге. Vectastain пероксидазой авидин Биотин комплекса (ABC) комплект Набор состоит из первичного антитела, биотинилированного вторичными антителами, авидин-биотинилированного ферментный комплекс (ABC), и фермента субстратом. Смотрите <a href="http://www.vectorlabs.com/">www.vectorlabs.com</a> или <a href="http://www.immunkemi.se/">www.immunkemi.se</a> для протоколов. Бычий сывороточный альбумин (BSA) (Sigma-Aldrich, каталог #-4503). DAB = 3,3 &#39;-диаминобензидина Isopac (Sigma-Aldrich, каталог # D 9015). Трипсин из бычьей поджелудочной железы (Sigma-Aldrich, каталог № Т 4665). Vectastain пероксидазы ABC-кит, мышь IgG (Immunkemi F &amp; D AB, Jarfalla, Швеция)

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).

Watch this Scientific Journal Video about Rat Mesentery Angiogenesis Assay at JoVE.com

Rat Mesentery Angiogenesis Assay

Нормальный взрослый васкуляризированной ткани млекопитающих, что не хватает физиологических ангиогенеза и который не подвергался хирургическому вмешательству используется для изучения: (я) возникновение и развитие ангиогенеза после внутрибрюшинного введения тестовых агентов, и (II) изменение ангиогенеза следующие системные администрации выбранного теста агентов.

Крыса Брыжейка Ангиогенез Пробирной

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

Research

JoVE Journal

Biology

13.8K Views.  University of Gothenburg. Нормальный взрослый васкуляризированной ткани млекопитающих, что не хватает физиологических ангиогенеза и который не подвергался хирургическому вмешательству используется для изучения: (я) возникновение и развитие ангиогенеза после внутрибрюшинного введ...

Video: Rat Mesentery Angiogenesis Assay

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.

Кристофер Хьюз: в пробирке модель для изучения ангиогенеза (Интервью)

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

Optimized Fibrin Gel Bead Assay for the Study of Angiogenesis

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These steps include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, branching, lumen formation, anastomosis, and formation of a new basement membrane.  Many in vitro assays have been developed to study this process, but most only mimic certain stages of angiogenesis, and morphologically the vessels within the assays often do not resemble vessels in vivo.  Based on earlier work by Nehls and Drenckhahn, we have optimized an in vitro angiogenesis assay that utilizes human umbilical vein EC and fibroblasts.  This model recapitulates all of the key early stages of angiogenesis and, importantly, the vessels display patent intercellular lumens surrounded by polarized EC. EC are coated onto cytodex microcarriers and embedded into a fibrin gel.  Fibroblasts are layered on top of the gel where they provide necessary soluble factors that promote EC sprouting from the surface of the beads.  After several days, numerous vessels are present that can easily be observed under phase-contrast and time-lapse microscopy. This video demonstrates the key steps in setting up these cultures.

This video demonstrates the protocol of an in vitro angiogenesis assay that recapitulates several stages of angiogenesis. Time-lapse images of sprouting, lumen formation, branching and anastomosis - key features of angiogenesis - are shown.

Оптимизированная фибринового геля из бисера тест для изучения ангиогенеза

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These ...

Organotypic Slice Culture of E18 Rat Brains

Organotypic slice cultures from embryonic rodent brains are widely used to study brain development. While there are often advantages to an in-vivo system, organotypic slice cultures allow one to perform a number of manipulations that are not presently feasible in-vivo. To date, organtotypic embryonic brain slice cultures have been used to follow individual cells using time-lapse microscopy, manipulate the expression of genes in the ganglionic emanances (a region that is hard to target by in-utero electroporation), as well as for pharmacological studies. In this video protocol we demonstrate how to make organotypic slice cultures from rat embryonic day 18 embryos. The protocol involves dissecting the embryos, embedding them on ice in low melt agarose, slicing the embedded brains on the vibratome, and finally plating the slices onto filters in culture dishes. This protocol is also applicable in its present form to making organotypic slice cultures from different embryonic ages for both rats and mice. 

Органотипической культуре Кусочек E18 Мозги крысы

Organotypic slice cultures from embryonic rodent brains are widely used to study brain development. While there are often advantages to an in-vivo system, ...

Optical Mapping of Langendorff-perfused Rat Hearts

Optical mapping of the cardiac surface with voltage-sensitive fluorescent dyes has become an important tool to investigate electrical excitation in experimental models that range in scale from cell cultures to whole-organs[1, 2]. Using state-of-the-art optical imaging systems, generation and propagation of action potentials during normal cardiac rhythm or throughout initiation and maintenance of arrhythmias can be visualized almost instantly[1]. The latest commercially-available systems can provide information at exceedingly high spatiotemporal resolutions and were based on custom-built equipment initially developed to overcome the obstacles imposed by more conventional electrophysiological methods[1]. Advancements in high-resolution and high-speed complementary metal-oxide-semiconductor (CMOS) cameras and intensely-bright, light-emitting diodes (LEDs) as well as voltage-sensitive dyes, optics, and filters have begun to make electrical signal acquisition practical for cardiovascular cell biologists who are more accustomed to working with microscopes. Although the newest generation of CMOS cameras can acquire 10,000 frames per second on a 16,384 pixel array, depending on the type of sample preparation, long-established fluorescence acquisition technologies such as photodiode arrays, laser scanning systems, and cooled charged-coupled device (CCD) cameras still have some distinct advantages with respect to dynamic range, signal-to-noise ratio, and quantum efficiency[1, 3]. In the present study, Lewis rat hearts were perfused ex vivo with a crystalloid perfusate (Krebs-Henseleit solution) at 37&deg;C on a modified Langendorff apparatus. After a 20 minute stabilization period, the hearts were intermittently perfused with 11 mMol/L 2,3-butanedione monoxime to eliminate contraction-associated motion during image acquisition. For optical mapping, we loaded hearts with the fast-response potentiometric probe di-8-ANEPPS[4] (5 &mu;Mol/L) and briefly illuminated the preparation with 475&plusmn;15 nm excitation light. During a typical 2 second period of illumination, &gt;605 nm light emitted from the cardiac preparation was imaged with a high-speed CMOS camera connected to a horizontal macroscope. For this demonstration, hearts were paced at 300 beats per minute with a coaxial electrode connected to an isolated electrical stimulation unit. Simultaneous bipolar electrographic recordings were acquired and analyzed along with the voltage signals using readily-available software. In this manner, action potentials on the surface of Langendorff-perfused rat hearts can be visualized and registered with electrographic signals.

This article describes a high temporal and spatial resolution technique to optically image action potential movement on the surface of Langendorff-perfused rat hearts using a potentiometric dye (di-8-ANEPPS).

Оптический Картирование Langendorff-перфузии сердца крысы

Optical mapping of the cardiac surface with voltage-sensitive fluorescent dyes has become an important tool to investigate electrical excitation in ...

Primary Culture of Adult Rat Heart Myocytes

Cultured primary adult rodent heart cells are an important model system for cardiovascular research. Nevertheless, establishment of robust, viable cultured adult myocytes can be a technically challenging, rate-limiting step for many researchers. Here we described a protocol to obtain a high yield of adult rat heart myocytes that remain viable in culture for several days. The heart is isolated and perfused with collagenase and protease under low Ca2+ conditions to recover single myocytes. Ca2+-tolerant cells are obtained by stepwise increases in extracellular Ca2+ concentration in three subsequent wash steps. Cells are filtered, resuspended in culture medium, and plated on laminin coated slips. Cultured myocytes obtained using this protocol are viable for up to four days and are suitable for most experiments including electrophysiology, biochemistry, imaging and molecular biology.

In this paper, we described a typical way to isolate and culture adult rat heart myocytes. Collagenase and protease are used to digest and isolate single myocytes. Myocytes cultured follow this protocol meet most experiment requirements.

Первичные культуры взрослых Миоциты сердца крысы

Cultured primary adult rodent heart cells are an important model system for cardiovascular research.  Nevertheless, establishment of robust, viable ...

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

Microvacular network growth and remodeling are critical aspects of wound healing, inflammation, diabetic retinopathy, tumor growth and other disease conditions1, 2. Network growth is commonly attributed to angiogenesis, defined as the growth of new vessels from pre-existing vessels. The angiogenic process is also directly linked to arteriogenesis, defined as the capillary acquisition of a perivascular cell coating and vessel enlargement. Needless to say, angiogenesis is complex and involves multiple players at the cellular and molecular level3. Understanding how a microvascular network grows requires identifying the spatial and temporal dynamics along the hierarchy of a network over the time course of angiogenesis. This information is critical for the development of therapies aimed at manipulating vessel growth.
The exteriorization model described in this article represents a simple, reproducible model for stimulating angiogenesis in the rat mesentery. It was adapted from wound-healing models in the rat mesentery4-7, and is an alternative to stimulate angiogenesis in the mesentery via i.p. injections of pro-angiogenic agents8, 9. The exteriorization model is attractive because it requires minimal surgical intervention and produces dramatic, reproducible increases in capillary sprouts, vascular area and vascular density over a relatively short time course in a tissue that allows for the two-dimensional visualization of entire microvascular networks down to single cell level. The stimulated growth reflects natural angiogenic responses in a physiological environment without interference of foreign angiogenic molecules. Using immunohistochemical labeling methods, this model has been proven extremely useful in identifying novel cellular events involved in angiogenesis. Investigators can readily correlate the angiogenic metrics during the time course of remodeling with time specific dynamics, such as cellular phenotypic changes or cellular interactions4, 5, 7, 10, 11.

This article describes a simple model for stimulating angiogenesis in the rat mesentery. The model produces dramatic increases in capillary sprouting, vascular area and vascular density over a relatively short time course in a tissue that allows en face visualization of entire microvascular networks down to the single cell level.

Крысы Брыжейка экстериоризации: модель для исследования Клеточная динамика, вовлеченных в ангиогенез

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

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using this protocol, angiogenesis may be measured in vitro in a fast, quantifiable manner. Primary or immortalized endothelial cells are mixed with conditioned media and plated on basement membrane matrix. The endothelial cells form capillary like structures in response to angiogenic signals found in conditioned media. The tube formation occurs quickly with endothelial cells beginning to align themselves within 1&#160;hr and lumen-containing tubules beginning to appear within 2 hr. Tubes can be visualized using a phase contrast inverted microscope, or the cells can be treated with calcein AM prior to the assay and tubes visualized through fluorescence or confocal microscopy. The number of branch sites/nodes, loops/meshes, or number or length of tubes formed can be easily quantified as a measure of in vitro angiogenesis. In summary, this assay can be used to identify genes and pathways that are involved in the promotion or inhibition of angiogenesis in a rapid, reproducible, and quantitative manner.

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

The tube formation assay is a fast, quantifiable method for measuring in vitro angiogenesis. Endothelial cells are combined with conditioned media and plated on basement membrane extract. Tube formation occurs within hours and newly formed tubules easily quantified.

Эндотелия Формирование Анализ сотового трубка для<em&gt; In Vitro</em&gt; Изучение ангиогенеза

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using ...

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 Техника по расследованию Правила микрососудов проницаемость у крыс брыжейки

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

In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver

Unrepaired DNA damage can lead to genetic instability, which in turn may enhance cancer development. Therefore, identifying potential DNA damaging agents is important for protecting public health. The in vivo alkaline comet assay, which detects DNA damage as strand breaks, is especially relevant for assessing the genotoxic hazards of xenobiotics, as its responses reflect the in vivo absorption, tissue distribution, metabolism and excretion (ADME) of chemicals, as well as DNA repair process. Compared to other in vivo DNA damage assays, the assay is rapid, sensitive, visual and inexpensive, and, by converting oxidative DNA damage into strand breaks using specific repair enzymes, the assay can measure oxidative DNA damage in an efficient and relatively artifact-free manner. Measurement of DNA damage with the comet assay can be performed using both acute and subchronic toxicology study designs, and by integrating the comet assay with other toxicological assessments, the assay addresses animal welfare requirements by making maximum use of animal resources. Another major advantage of the assays is that they only require a small amount of cells, and the cells do not have to be derived from proliferating cell populations. The assays also can be performed with a variety of human samples obtained from clinically or occupationally exposed individuals.

In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver

The alkaline comet assay measures DNA strand breaks in eukaryotic cells. By adding an Endonuclease III or human 8-oxoguanine-DNA-N-glycosylase digestion step, the assay can efficiently detect oxidative DNA damage. We describe methods for using these assays to detect DNA damage in rat liver.

В Vivo щелочной Comet Assay и энзим-модифицированной щелочной Comet Анализ для Измерение разрывов ДНК и окислительный ДНК повреждений в печени крысы

Unrepaired DNA damage can lead to genetic instability, which in turn may enhance cancer development. Therefore, identifying potential DNA damaging agents ...

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.