Total organ vascular leakage was assessed by intravascular administration of Evan's blue (Sigma-Aldrich) as described previously (23 (link)). In brief, Evan's blue (0.2 ml of 0.5% in PBS) was injected intravenously into Cd73−/− or littermate control Cd73+/+ animals that were exposed to normobaric hypoxia (8% O2, 92% N2) or room temperature air for 4 h (n = 4–6 animals per condition). After experimental exposure, the animals were killed, and the colon, skeletal muscle (gluteus maximus), kidney, brain, heart, liver, and lungs were harvested. Evan's blue concentrations in organs were quantified after formamide extraction (55°C for 2 h) by measuring absorbances at 610 nm with subtraction of reference absorbances at 450 nm. This protocol was in accordance with National Institutes of Health (NIH) guidelines for use of live animals and was approved by the Institutional Animal Care and Use Committee at Brigham and Women's Hospital.
In subsets of experiments, mice were reconstituted with 5′-NT purified from Crotalus atrox venom (Sigma-Aldrich). Pilot dosing experiments revealed that 5′-NT could be used at concentrations as high as 500 U/kg i.p. without deleterious effects. After administration of 5′-NT, animals were subjected to normoxia or hypoxia, and examined for vascular leakage using Evan's blue as described before.
In other experiments, mice were administered the adenosine A2A receptor antagonist 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385; Tocris Cookson Inc.; dosage of 1 mg/kg i.p. plus 1 mg/kg s.c.), adenosine A2B receptor antagonist N-(4-cyanophenyl)-2-[4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)-phenoxy]acetamide (MRS1754; a gift from K. Jacobson, Molecular Recognition Section, NIH, Bethesda, MD; dosage of 1 mg/kg i.p. plus 1 mg/kg s.c.) or the nonmetabolizable adenosine analogue NECA (Sigma-Aldrich; dosage of 0.1 mg/kg i.p. plus 0.1 mg/kg s.c. based on previous work [28 (link)]). After administration of drug, animals were subjected to normoxia or hypoxia, as indicated, and examined for vascular leakage using Evan's blue as described before. For assessment of pulmonary edema, lungs were collected, weighed, and dried by speed-vac. Weight differences before and after drying were used to calculate lung water content.
In subsets of experiments, mice were reconstituted with 5′-NT purified from Crotalus atrox venom (Sigma-Aldrich). Pilot dosing experiments revealed that 5′-NT could be used at concentrations as high as 500 U/kg i.p. without deleterious effects. After administration of 5′-NT, animals were subjected to normoxia or hypoxia, and examined for vascular leakage using Evan's blue as described before.
In other experiments, mice were administered the adenosine A2A receptor antagonist 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385; Tocris Cookson Inc.; dosage of 1 mg/kg i.p. plus 1 mg/kg s.c.), adenosine A2B receptor antagonist N-(4-cyanophenyl)-2-[4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)-phenoxy]acetamide (MRS1754; a gift from K. Jacobson, Molecular Recognition Section, NIH, Bethesda, MD; dosage of 1 mg/kg i.p. plus 1 mg/kg s.c.) or the nonmetabolizable adenosine analogue NECA (Sigma-Aldrich; dosage of 0.1 mg/kg i.p. plus 0.1 mg/kg s.c. based on previous work [28 (link)]). After administration of drug, animals were subjected to normoxia or hypoxia, as indicated, and examined for vascular leakage using Evan's blue as described before. For assessment of pulmonary edema, lungs were collected, weighed, and dried by speed-vac. Weight differences before and after drying were used to calculate lung water content.
