Name: an in vivo assay to test blood vessel permeability
Uploaded: 2013-03-16T13:00:00
Description: Watch this Scientific Journal Video about An in vivo Assay to Test Blood Vessel Permeability at JoVE.com

This work was supported by a grant from the National Institutes of Health, R01CA142928.

1. Intravenous Injection of Evans Blue in the Mouse Lateral Tail Vein
	<ol>
	 <li>Prepare a 0.5% sterile solution of Evans blue in PBS. If necessary, filter-sterilize the solution to remove any particulate matter that has not dissolved.	 </li>
 <li>Aspirate 200 &mu;l Evans Blue solution into a syringe. Avoid all air bubbles that might have escaped into the syringe. </li>
 <li>Place mice that are 8-12 weeks old into a restraint device so that the animal is not freely mobile but its tail can be handled. </l...

<ol>
	<li>Beck, K. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inducible+NO+synthase:+role+in+cellular+signaling.">Inducible NO synthase: role in cellular signaling.</a> J. Exp. Biol. 202, 645-653 (1999).</li><li>Bertglia, S., Giusti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+nitric+oxide+in+capillary+perfusion+and+oxygen+delivery+regulation+during+systemic+hypoxia.">Role of nitric oxide in capillary perfusion and oxygen delivery regulation during systemic hypoxia.</a> Am. J. Physiol. Heart Circ. Physiol. 288, H525-H531 (2005).</li><li>Miles, A. A., Miles, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+reactions+to+histamine,+histamine-liberator+and+leutaxine+in+the+skin+of+guinea+pigs.">Vascular reactions to histamine, histamine-liberator and leutaxine in the skin of guinea pigs.</a> J. Physiol. (London). 118, 228-257 (1952).</li><li>Weis, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+permeability+in+cardiovascular+disease+and+cancer.">Vascular permeability in cardiovascular disease and cancer.</a> Curr. Opin. Hematol. 15, 243-249 (2008).</li><li>Kumar, P., Shen, Q., Pivetii, C. D., Lee, E. S., We, M. H., Yuan, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+endothelial+hyperpermeability:+implications+in+inflammation.">Molecular mechanisms of endothelial hyperpermeability: implications in inflammation.</a> Expert Rev. Mol. Med. 30, 11-19 (2009).</li><li>Viazzi, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+permeability,+blood+pressure,+and+organ+damage+in+primary+hypertension.">Vascular permeability, blood pressure, and organ damage in primary hypertension.</a> Hypertens. Res. 31, 873-879 (2008).</li><li>Scheppke, ., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+vascular+permeability+suppression+by+topical+application+of+a+novel+VEGFR2/Src+kinase+inhibitor+in+mice+and+rabbits.">Retinal vascular permeability suppression by topical application of a novel VEGFR2/Src kinase inhibitor in mice and rabbits.</a> J. Clin. Invest. 118, 2337-2346 (2008).</li><li>Blanchet, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+CD34+Leads+To+Exacerbated+Autoimmune+Arthritis+through+Increased+Vascular+Permeability.">Loss of CD34 Leads To Exacerbated Autoimmune Arthritis through Increased Vascular Permeability.</a> J. Immunol. 184, 1292-1299 (2010).</li><li>Dvorak, A. M. <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-derived+mediators+of+enhanced+microvascular+permeability,+vascular+permeability+factor/vascular+endothelial+growth+factor,+histamine,+and+serotonin,+cause+leakage+of+macromolecules+through+a+new+endothelial+cell+permeability+organelle,+the+vesiculo-vacuolar+organelle.">Mast cell-derived mediators of enhanced microvascular permeability, vascular permeability factor/vascular endothelial growth factor, histamine, and serotonin, cause leakage of macromolecules through a new endothelial cell permeability organelle, the vesiculo-vacuolar organelle.</a> Chem. Immunol. Allergy. 85, 185-204 (2005).</li><li>Le Guelte, A., Gavard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+endothelial+cell-cell+junctions+in+endothelial+permeability.">Role of endothelial cell-cell junctions in endothelial permeability.</a> Methods Mol. Biol. 763, 265-279 (2011).</li><li>Martins-Green, M., Petreaca, M., Yao, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+assay+system+for+in+vitro+detection+of+permeability+in+human+"endothelium".">An assay system for in vitro detection of permeability in human "endothelium".</a> Methods Enzymol. 443, 137-153 (2008).</li></ol>

Vascular permeability is a critical marker for blood vessel status. Increased vascular permeability has been shown to be present in several systemic diseases, including diabetes, hypertension, and autoimmune diseases 6,7,8. Increased vascular permeability has been shown to be mediated by shear stress, growth factors like vascular endothelial growth factor and fibroblast growth factor, inflammatory mediators like serotonin, histamine and bradykinin 9. Extravasation of water and small molecules is tho...

Formation and maintenance of selective permeable barriers are essential for proper organ development and performance 1,2. Endothelial cells line the blood vessel lumen and form a semi-permeable barrier that is essential in the selective transport between blood and the interstitial space of all organs. An adequate permeability barrier is maintained through tight cell-to-cell junctions that are strictly controlled by growth factors, cytokines and other stress related molecules 3. Disruption of the endothelial cell barrier can result in increased permeability and vascular leakage. These effects are seen in various disease states and the understanding of the underlining molecular signaling requires multidisciplinary methods 4,5. In this article, we describe an in vivo method to measure vessel permeability using a mouse model.
The assay we are describing, also known as Miles assay, is a well-established method to test vascular permeability in vivo. The assay is based on the fact that, under basal physiological conditions, albumin does not cross the endothelial barrier. Evans Blue, an azo dye with high affinity for albumin, is injected in the blood stream of an experimental animal, and under physiologic conditions, it is expected to be restricted within blood vessels. When a vascular permeability stimulus is added, either topically or systemically, blood vessels start to leak protein and thus, also the Evans blue that is bound to albumin. This results in a rapid bluish coloration of tissues that have permeable vessels.
Successful injection of the dye in the mouse lateral tail vein is critical for the good outcome of the experiment. Tail vein injection technique requires extensive practice and should be mastered prior to starting the experiment.
 Vessel permeability is highly dependent on the age and weight of the animal, so when comparing different mouse strains it is imperative that the mice or other test subjects have close to identical birth dates and weight. Other factors that influence the permeability of the endothelial barrier are environmental conditions such as temperature, humidity, and very importantly, the handling stress of the mouse. Due to the multitude of factors that can influence the outcome of the experiment it is always advisable that the experiment is repeated at least three times and statistical analysis performed. 
This assay can be used or to compare vessel permeability of mice that have been genetically modified, as well as mice with different genetic backgrounds. Permeability can be assessed in the presence or absence of a stimulus, depending on the function of the gene that is modulated. This assay can also be used or to test the effect of different compounds on vessel permeability.

<table border="1">
	<tbody>
 	<tr>
 	<td></td>
 <td></td>
 <td></td>
 <td>REAGENT</td>
 </tr>
 <tr>
 	<td>EVANS BLUE</td>
 <td>SIGMA	</td>
 <td>E2129	</td>
 <td></td>
 </tr>
 <tr>
 	<td>FORMAMIDE</td>
 <td>INVITROGEN</td>
 <td>15515-026	</td>
 <td></td>
 </tr>
 <tr>
 	<td>PBS</td>
 <td></td>
 <td></td>
 <td>0.2M Phosphate 
				1.5M NaCl 
				pH 7.4</td>
 </tr>
 <tr>
 	<td></td>
 <td></td>
 <td></td>
 <td>EQUIPMENT</td>
 </tr>
 <tr>
 	<td>SPECTROPHOTOMETER</td>
 <td>EPPENDORF</td>
 <td>952000006	</td>
 <td></td>
 </tr>
 <tr>
 	<td>MOUSE RESTRAINT DEVICE</td>
 <td>HARVARD APPARATUS</td>
 <td>340012	</td>
 <td></td>
 </tr>
 <tr>
 	<td>SYRINGE</td>
 <td>BD</td>
 <td>309659	</td>
 <td></td>
 </tr>
 <tr>
 	<td>NEEDLES</td>
 <td>BD</td>
 <td>305106</td>
 <td>The gauge of the needle depends on the size of the animal.</td>
 </tr>
 <tr>
 	<td>BALANCE</td>
 <td>DENVER INSTRUMENT</td>
 <td>TP-64	</td>
 <td></td>
 </tr>
 </tbody>
 </table>

an in vivo assay to test blood vessel permeability

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

We used an in vivo permeability assay to test vessel leakage in mice 8-12 weeks old. This test is useful in comparing the relative vascular permeability between animals of different genetic background or in a single strain of mice subjected to treatments that affect the vasculature. Our results show that a strain of genetically modified mice we created in our lab has a more permeable endothelium compared to wild-type mice. These changes are evident at macroscopic level in many organs (Figure 1)...

Watch this Scientific Journal Video about An in vivo Assay to Test Blood Vessel Permeability at JoVE.com

An in vivo Assay to Test Blood Vessel Permeability

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

An in vivo Assay to Test Blood Vessel Permeability

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

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