Name: rat mesentery angiogenesis assay
Uploaded: 2011-06-18T14:00:00
Duration: 18 min 30 s
Description: Watch this Scientific Journal Video about Rat Mesentery Angiogenesis Assay at JoVE.com

Financial support for most of the studies was provided by the Swedish Medical Research Council (grant 5942).

1. Animals
<ol>
	<li>Small rodents, such as rats, mice, and Guinea pigs, have been used as the animal models. Unfortunately, adult mice (many mouse strains have been assessed) tend to lack or have only a very low number of potential-parent microvessels (from which angiogenesis can originate) within their mesenteric windows, which makes studies using mice unreliable. Therefore, the assay has been developed primarily for rats.</li>
	<li>The rats, which are acclimatized to a standard environment for at least 7 days following delivery from the breeder, are usually housed 
	in pairs in a standard cage under a 12-hr light/dark cycle with free access to water and pellets. Each animal is marked. We use mostly 
	young adult rats (typically 7-10 weeks of age of various strains but usually male Sprague-Dawley rats; in rats younger than 5-6 weeks of age the number of native microvessels tend to be low) obtained from diverse breeders. In the period prior to 
	the experiment and throughout the experiment, the animals are weighed every second day, and always on the day of sacrifice. When 
	animals are treated concurrently i.p. (for induction of angiogenesis, see below) and s.c. or i.v. or p.o., they are weighed each
	day. This enables the establishment of experimental and control groups of equal body weight at the start of the experiment, and 
	allows monitoring of the influence of the treatment on body-weight-gain during the course of the experiment. Since adult male rats 
	grow robustly physiologically (gaining approximately 40-60 g per week depending on type of strain), weight-gain retardation is a sensitive substitute marker 
	of toxicity, systemic well-being, anorexia, and failure to thrive.</li>
</ol>
We work in a high-standard animal care facility and considerable efforts are made to minimize the levels of 
stress experienced by the animals. The current ethical guidelines are those established by Workman P. et al. (Guidelines 
for the welfare and use of animals in cancer research. Br. J. Cancer 102, 1555-77, 2010). The Animal Ethics Committee of the University of Gothenburg approved these studies.

2. Pro-angiogenic Intraperitoneal Treatment
<ol>
 <li>The candidate pro-angiogenic factor to be tested is dissolved and diluted in the endotoxin-free saline used for infusion of patients (even at extremely low levels, endotoxin is pro-angiogenic). All solutions are made up freshly on the day of injection, sterile-filtered (Millipore 0.22-&mu;m filtration), checked for neutral pH (7.1-7.3), and used at room temperature. 
 The test agent is injected at low or very low concentrations. For example, rat rVEGF164 (564-RV/CF; R&amp;D Systems Europe, Ltd., Oxon, UK), which is the predominant VEGF-A isoform in rats, is diluted to 96 pmole/ml in endotoxin-free saline, frozen and thawed and a volume of 5 ml is injected i.p. into the rat. We use a 5-ml syringe with 0.8-mm needle (green needle, 21G) for i.p. injection. This treatment, given twice daily for 4.5 days, i.e., from Monday morning (Day 0) to Friday morning (Day 4), induces a robust sprouting angiogenesis response in the mesenteric test tissue, peaking at around Day 21. 
 Administering the solution i.p. ensures that the entire volume is delivered into the peritoneal cavity (and not into the intestines, bladder or any other organ). The rapid injection of 5 ml allows tactile confirmation by the operator that the fluid jet has passed through the abdominal wall into the abdominal cavity. 
 Following, prior to or concurrently with the i.p. pro-angiogenic treatment, any test agent of choice can be administered systemically.
 </li>
</ol>
3. Systemic Administation of Angiogenesis-modulating Agents 
<ol>
	<li>Any route other than the i.p. route can be used, since i.p. treatment may affect the ongoing angiogenic reaction through the induction of inflammation or possibly by activating mast cells in the test tissue, which can lead to a strong pro-angiogenic response. Besides oral administration or single injection s.c. or i.v., we often use s.c. administration using osmotic minipumps (one or more, with varying volumes and pumping times) to deliver the solution at a constant rate for up to two or several weeks.</li>
	<li>Continuous subcutaneous infusion of test agents 
	Filling and implantation of osmotic minipumps 
	Usually on Day 5, i.e., one day after the end of the i.p. VEGF treatment, osmotic minipumps (for example, Model 2ML2, with constant pumping rate of 5.0 &mu;l/h for 14-15 days; Alzet Osmotic Pumps, Mountain View, CA, USA) are filled under sterile conditions with the test agent or the appropriate vehicle. After storage in sterile 0.9% (w/v) NaCl overnight at 37&deg;C, the pump(s) are surgically implanted s.c. on the backs (at the side of the spinal column in the region of the scapulae) of rats that have been anesthetized using isoflurane gas (Isoba vet; Schering-Plough Animal Health, Merck &amp; Co., Inc., Whitehouse Station, NJ, USA), initially in an airtight transparent chamber and subsequently via a mask (to minimize the stress caused to the following animal, the chamber is flushed with air before it is reused). The skin incision made for pump implantation is sutured immediately post-implantation using silk thread. 
	Since the animals gain weight physiologically during the experimental period and the test agents are administered at a constant rate, the actual dosage selected for a test drug per kg of body weight is higher than the average dosage at the beginning of the infusion period and is lower than the average dosage at the end of the infusion period. Continuous infusion, as described here, can be viewed as an extended form of metronomic treatment.
	</li>
</ol>
 4. Ending the Experiment
 <ol>
 <li>One animal at a time is placed in a transparent airtight chamber, which is flushed with CO2 gas, which rapidly kills the animal. The chamber is flushed with air before being used again.</li>
</ol>
5. Harvesting Tissue Samples
<ol>
 <li>As shown in the DVD, the abdominal wall is opened and the small-gut mesentery is identified, including the most distal part that reaches the ileo-cecal valve (the junction of the small intestine and the large intestine). Four (or more) of the most distally located "windows" are numbered and spread onto objective standard untreated Superfrost Plus-slides (DAKO Denmark A/S, Glostrup, Denmark). It is important to avoid, if possible, contaminating the samples with blood, gut contents or other bodily fluids, as these materials may be stained non-specifically, although this does not invalidate the subsequent immunohistochemical visualization of microvessels.</li>
 <li>Each mesenteric window specimen with attached small gut segment is spread on a slide and dried at room temperature for 20 minutes. Then, the intestinal stump is cut off, while the intact membranous mesentery specimens are stored at -20&deg;C for extended periods. This is shown in the DVD.</li> 
</ol>
It should be mentioned that the tissue and patent vasculature can be visualized prior to harvest with transmitted light intravital microscopy.
6. Protocol for Immunohistochemical Staining of Microvessels
<ol>
	<li>Day 0 
	The specimens are allowed to thaw from -20&deg;C to room temperature, dried in a cabinet at 60&deg;C for 1 hr, and kept at room temperature overnight.
	</li>
	<li>Day 1 
	Place the slides in staining slide holders (in every second track) in a staining jar and wash twice with TBS (pH 7.8) for 10 minutes at room temperature. The solution is discarded and replaced with the next solution, and so on for all the following steps.</li>
	<li>Trypsin treatment 
	Fill the staining jar that will harbor the staining slide holder with trypsin solution (25&deg; C for 20 minutes). Discard the solution and incubate twice with 2.5% sucrose solution (room temperature for 10 minutes each time). Discard the solution and rinse a couple of times in aqua dest (room temperature for a few minutes each).
	</li>
	<li>Blocking 
	Fill the staining jar that will harbor the staining slide holder with 0.5% H2O2 in methanol (room temperature for 30 minutes). Discard the solution and rinse with aqua dest (room temperature for a few minutes). Discard the solution and rinse twice with TBS (pH 7.4) (room temperature for 8 minutes each time).
	</li>
	<li>
	Incubation 
	All incubations are in a humidified chamber at room temperature. Approximately 150 &mu;l of each of the solutions listed below are used per slide. The slides are placed in a humidified chamber and the solutions are pipetted onto the specimens (the non-membranous parts do not need to be covered by the solutions). 
	For each of the following steps, carefully shake off all of the solution before the subsequent solution is added.
		<ol>
			<li>Incubation with Normal (blocking) serum (horse) (ABC kit)
			3 drops yellow solution/10 ml dilution buffer for 20 minutes.
			</li>
			<li>Incubation with Primary antibody ARU0181, which labels rat vascular endothelial cells. The antibody is diluted 1:600 with dilution buffer (Biosource) (100 &mu;l diluted 40-fold [frozen] plus 900 &mu;l dilution buffer) and incubation takes place overnight at 4&deg;C.</li>
		</ol>
	</li>
	<li>Day 2 
	The slides are removed from the humidified chamber, the primary antibody is discarded, and the slides are placed in a staining slide holder, which is submerged in the staining jar and rinsed twice with TBS (pH 7.8) for 8 minutes.
		<ol>
			<li>Incubation with biotinylated antibody (ABC kit) 
			The slides are placed horizontally in a humidified chamber and incubated with 1 drop of blue solution/10 ml dilution buffer for 30 minutes. Discard the solution and place the slides in a staining slide holder submerged in the staining jar and rinse twice with TBS (pH 7.4) for 8 minutes.
			</li><li> Incubation with the ABC reagent 
The slides are again placed horizontally in a humidified chamber and the following solutions are pipetted onto the membranous parts of the specimens: 2 drops of orange solution plus 2 drops of brown solution/10 ml TBS (pH 7.4) for 30 minutes. Mix carefully 30 minutes before use! Discard the solution and place the slides in the slide holder in the staining jar and rinse twice with TBS (pH 7.4) for 8 minutes.
</li>
</ol>
</li>
<li>
Staining with DAB (0.05%) 
This step must be performed in a ventilated chemical hood! 
Place the slides and add the 3,3-diaminobenzidine (DAB; Sigma-Aldrich) solution using a pipette: 0.1% DAB solution in TBS (pH 7.4) [frozen] is diluted 1:1 with a mixture of 10 &mu;l H2O2 and 7.5 ml TBS (pH 7.4). This solution is sterile-filtered just before use (to eliminate any particles that may obscure the microscopic picture). Stain the specimens for 8 minutes. Discard the solution and place the slides in the staining slide holder submerged in the staining jar. Rinse under running tap water and then in aqua dest for a few minutes.
</li>
<li>Dehydration 
The slides are kept in the staining slide holder. Rinse by discarding and refilling the staining jar containing aqua dest/ethanol: aqua dest, 70% alcohol, 95% alcohol, absolute alcohol, with 2 minutes in each solution.</li>
</ol>
7. Imaging and Assessing the Microvascular Network in Each Mesenteric Window Specimen After Immunohistochemical Staining of Vascular Endothelial Cells
As shown in the movie, microvessels are microscopically visualized in situ in the intact tissue.
<ol>
	<li>First, we use a drawing microscope with magnification range from 14 to 34 (Leica Wild M 3Z) to pencil on white paper the entire window area, as well as the entire vascularized area (VA) per mesenteric window. VA, as a percentage of the entire area, is easily measured using computerized or non-computerized planimetry (or simply by cutting out and weighting the image of the whole window, and the images of all its vascularized parts).</li>
	<li>Using another drawing microscope, individual microvessel profiles, which are readily identified, are drawn in black ink on white paper at 80 magnification within randomly chosen fields. This is the starting point for the subsequent computerized morphometric assessment of microvascular length (MVL), which is basically a measurement of microvascular density.</li>
	<li>Optionally, variables related to microvascular pattern formation, as well as those for the assessment of the number of microvascular sprouts and length of individual microvascular sprouts (No. SP and Le. SP, respectively) can be determined. -The optimal contrast between the depicted black vessel structures and the white background facilitates detailed analyses of the images in most commercial morphometric computerized programs.</li>
	<li>In most cases, the major variables are VA and MVL, which when multiplied together generate the total microvascular length (TMVL).</li>
</ol> 
8. Statistical Analysis
We usually use the standard non-parametric Mann-Whitney U-test to analyze unpaired (two-tailed) observations. A mean of four mesenteric windows per animal is used as the independent data-point for each variable of the mesenteric window. The criterion for statistical significance is P &le; 0.05.

<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>

No conflicts of interest declared.

The assay was introduced in 19863 and has successively been further developed and refined in our 
laboratory7,10-12. As discussed in review papers (invited)1, 2, the assay compares well with other mammalian in 
vivo angiogenesis assay in regard particularly to important features such as the adult test tissue is natively vascularized and lacks 
physiologic angiogenesis; minimal trauma, if any, is inflicted upon the tissue; truly quantitative variables are measured, which 
allows sound statistical analysis regarding dose-response and molecular-activity studies; sprouting angiogenesis occurs, which is 
predominating in normal tissues and tumors; and toxicity data are easily accumulated in rats that grow robustly physiologically 
in adulthood. Disadvantageous features may include the fact that the assay is relatively time-consuming, especially when assessing 
the number and length of individual microvessel segments and microvessel sprouts; that it does not allow real-time/serial observations; and that it is 
hardly suitable for mice since many mesenteric windows in mice lack potential parent microvessels from which new capillaries can originate.
Real-time observations are, however, enabled by intravital microscopy where the exteriorized but intact mesenteric windows are splayed out over a microscope stage while the animal is anesthetized, as discussed in1.
Significant advances in angiogenesis research have been achieved in the past decades using models such as the corneal 
micropocket, chick chorioallantoic membrane, and s.c. Matrigel plug assays, which are useful but ultimately limited tools. Future clinical 
applications of anti-angiogenic and pro-angiogenic therapies necessitate the establishment and validation of more sensitive and biologically 
relevant, truly quantitative pre-clinical models, such as the one described in the current report.

Buffers and reagents
0.5 M Tris-HCl (pH 7.8) stock solution 
Tris (hydroxymethyl) aminomethan (60.57 g) is dissolved in ~900 ml aqua dest and ~45 ml of 25% HCl (concentrated) is added to achieve pH 7.8 followed by dilution to 1 liter with aqua dest.

Tris-buffer saline (TBS; pH 7.8) 
To 200 ml of the stock solution is added 18 g NaCl dissolved in ~1800 ml aqua dest with adjustment of pH to 7.8 using the stock solution, followed by dilution to 2 liter with aqua dest.

TBS (pH 7.4) 
To 200 ml of the stock solution is added 18 g NaCl dissolved in dissolved in ~1800 ml aqua dest with adjustment of pH to 7.4 using the stock solution, followed by dilution to 2 liter with aqua dest.

Trypsin solution (0.05% trypsin, 0.1% CaCl2) 
Contains 0.1 g trypsin and 0.2 g CaCl2 in 200 ml TBS (pH 7.8).

Sucrose solution 2.5% 
Contains 5 g sucrose in 200 ml TBS (pH 7.8).

Stock solution of 1% Sodium azide in TBS (pH 7.4) 
Contains 1 g Na-azide in 100 ml with TBS (pH 7.4).

Diluting buffer: 0.1% BSA, 0.01% Na-azide 
(Used to dilute antibodies in the Vectastain ABC kit). Contains 0.1 g bovine serum albumin (BSA) and 1 ml of 1% Na-azide, diluted to 100 ml with TBS (pH 7.4).

ABC kit Vectastain PK 4002, ABC peroxidase mouse IgG 
Normal horse serum; blocking serum; yellow solution: 3 drops/10 ml dilution buffer. 
Biotinylated antibody; anti-mouse IgG; blue solution: 1 drop/10 ml dilution buffer. ABC-reagent: orange solution, 2 drops plus brown solution 2 drops/10 ml TBS (pH 7.4). 

Mix these solutions 20 minutes before use!

Mouse (monoclonal) anti-rat endothelium(Biosource International, Camarillo, CA, U.S.A.)
<table border="1"><tbody><tr><td>Catalog #</td><td> ARU0181</td></tr>
<tr><td>Clone #</td><td> MRC OX-43</td></tr></tbody></table>
This antibody is directed against all vascular endothelial cells in the rat, with the exception of those in the brain.
Vectastain peroxidase Avidin Biotin Complex (ABC) kit 
The kit contains primary antibody, biotinylated secondary antibody, avidin-biotinylated enzyme complex (ABC), and enzyme substrate. See <a href="http://www.vectorlabs.com/">www.vectorlabs.com</a> or <a href="http://www.immunkemi.se/">www.immunkemi.se</a> for protocols.

Bovine serum albumin(BSA) (Sigma-Aldrich, catalog # A-4503).
 DAB= 3,3'-Diaminobenzidine Isopac (Sigma-Aldrich, catalog # D 9015).
 Trypsinfrom Bovine Pancreas (Sigma-Aldrich, catalog # T 4665).
 Vectastain peroxidase ABC-kit, Mouse IgG (Immunkemi F &amp; D AB, Jarfalla, Sweden)

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

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The adult rat mesentery window angiogenesis assay is biologically appropriate and is exceptionally well suited to the 
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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).

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.

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.

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.

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.

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.

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.