Figure 3. CD73 deficiency causes increased paracellular permeability of ruthenium red in vivo.
Ten- to fourteen-week-old proestrus-staged C57BL/6 and CD73-deficient (Cd73–/–) mice were exposed to hypoxia (7% O2) or room air for 4 hours. Uterine horns were perfusion fixed in vivo with 0.2% ruthenium red and 2% glutaraldehyde. Ruthenium red paracellular permeability was quantified by assessing approximately 200 consecutive paracellular spaces per uterine section by transmission electron microscopy. The percentage of permeability is the ratio of paracellular spaces containing ruthenium red to the total number of paracellular spaces assessed. (A) Electron photomicrograph shows the presence (black arrows) and absence (white arrow) of electron-dense ruthenium red (black deposit) in the paracellular space of a uterine section. (B) Percentage of ruthenium red paracellular permeability for uterine sections (n = 6 mice). (C) Representative electron photomicrographs of uterine sections from C57BL/6 and Cd73–/– mice exposed to hypoxia (7% O2) for 4 hours. Black arrow (bottom panel) indicates the paracellular space with ruthenium red; white arrow (top panel) indicates the paracellular space showing no ruthenium red. M, microvilli. (D) Cd73–/– mice were i.p. injected with 0.1 mg/kg NECA or DMSO prior to hypoxia exposure (7% O2) for 4 hours. Data represent the mean ± SEM. (B) **P < 0.005 compared with hypoxic C57BL/6 mice; #P < 0.005 compared with normoxic Cd73–/– mice; 1-way ANOVA with Tukey’s post test. (D) *P = 0.019; unpaired, 2-tailed t test. Scale bars and original magnification: (A) 500 nm, ×25,000; (C and D) 500 nm, ×50,000.