TY - JOUR
T1 - A novel nonradioactive method to evaluate vascular barrier breakdown and leakage
AU - Trichonas, George
AU - Manola, Akrivi
AU - Morizane, Yuki
AU - Thanos, Aristomenis
AU - Koufomichali, Xanthi
AU - Papakostas, Thanos D.
AU - Montezuma, Sandra
AU - Young, Lucy
AU - Miller, Joan W.
AU - Gragoudas, Evangelos
AU - Vavvas, Demetrios
PY - 2010/3
Y1 - 2010/3
N2 - Purpose. To identify a novel, sensitive, nonradioactive leakage assay that can be used in the assessment of retinal vascular permeability in rats and mice. Methods. Breakdown of the vascular barrier was induced by vascular endothelial growth factor (VEGF), lipopolysaccharide (LPS), or diabetes. Biotinylated bovine serum albumin (bBSA) was administered as a tracer. After perfusion with lactated Ringer's solution, extravasated bBSA was detected with immunoprecipitation and Western blot analysis or sandwich ELISA. The results were then normalized against the final bBSA plasma concentration, the circulation time, and the protein concentration of the tissue. Results. Six hours after VEGF injection, BRB breakdown was quantified in the injected eye and was 2.5-fold higher than in the contralateral phosphate-buffered saline (PBS)-injected eye (n = 6 rats, P < 0.01). Intravitreal LPS injection induced severe inflammation in the directly injected eye and moderate inflammation in the contralateral untreated eye. Leakage was six- and threefold higher, respectively, compared with that in the untreated control animals (n = 5 rats, P < 0.01). Nine-month diabetic rats had a threefold increase in vascular leakage compared with age-matched control animals (n = 6 retinas, P < 0.05). Twenty-four hours after intraperitoneal administration of LPS in mice, the animals showed increased vascular leakage in all tissue organs examined (retina, 1.7-fold; brain, 1.5-fold; and kidney, 1.3-fold). Conclusions. bBSA can serve as an effective alternative to the current methods used for quantitating vascular leakage and especially the blood-retinal barrier breakdown. It is reasonably easy to perform, low in cost, and adaptable to experiments in mice.
AB - Purpose. To identify a novel, sensitive, nonradioactive leakage assay that can be used in the assessment of retinal vascular permeability in rats and mice. Methods. Breakdown of the vascular barrier was induced by vascular endothelial growth factor (VEGF), lipopolysaccharide (LPS), or diabetes. Biotinylated bovine serum albumin (bBSA) was administered as a tracer. After perfusion with lactated Ringer's solution, extravasated bBSA was detected with immunoprecipitation and Western blot analysis or sandwich ELISA. The results were then normalized against the final bBSA plasma concentration, the circulation time, and the protein concentration of the tissue. Results. Six hours after VEGF injection, BRB breakdown was quantified in the injected eye and was 2.5-fold higher than in the contralateral phosphate-buffered saline (PBS)-injected eye (n = 6 rats, P < 0.01). Intravitreal LPS injection induced severe inflammation in the directly injected eye and moderate inflammation in the contralateral untreated eye. Leakage was six- and threefold higher, respectively, compared with that in the untreated control animals (n = 5 rats, P < 0.01). Nine-month diabetic rats had a threefold increase in vascular leakage compared with age-matched control animals (n = 6 retinas, P < 0.05). Twenty-four hours after intraperitoneal administration of LPS in mice, the animals showed increased vascular leakage in all tissue organs examined (retina, 1.7-fold; brain, 1.5-fold; and kidney, 1.3-fold). Conclusions. bBSA can serve as an effective alternative to the current methods used for quantitating vascular leakage and especially the blood-retinal barrier breakdown. It is reasonably easy to perform, low in cost, and adaptable to experiments in mice.
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U2 - 10.1167/iovs.09-4193
DO - 10.1167/iovs.09-4193
M3 - Article
C2 - 19875655
AN - SCOPUS:77949888782
SN - 0146-0404
VL - 51
SP - 1677
EP - 1682
JO - Investigative Ophthalmology and Visual Science
JF - Investigative Ophthalmology and Visual Science
IS - 3
ER -