Vascular Permeability

Vascular permeability in the mouse retina was measured using an Evans blue quantitation technique as previously described (26). Briefly, mice received an intravenous injection of 45 mg/kg body wt Evans blue dye, which upon injection into the bloodstream rapidly and irreversibly binds to plasma albumin. Two hours later, animals were anesthetized, a 0.3-mL blood sample was obtained, and mice were perfused via the left ventricle with PBS, followed by 1% paraformaldehyde. The retinas were collected in PBS, dried for 2 h, and weighed. The Evans blue dye was extracted from the dried retinas with formamide at 70°C for 18 h and centrifuged through a 30,000 molecular weight filter. The absorbance of the retinal extract and a 1:1,000 dilution of plasma were measured by spectrophotometry at 620 and 740 nm. The concentration of dye in the extract and plasma was calculated from a standard curve of Evans blue dye. The blood-retinal barrier permeability was calculated as follows: (Evans blue from retina [ng]/retina wet weight [g])/Evans blue plasma [ng/μL] × circulation time [h]. Data were expressed as microliters of plasma/(g retinal wet weight × h circulation time).