Effects of hyperoxia on the behavior of leukocytes in rat pulmonary microcirculation assessed by confocal laser scanning microscopy

To study the dynamic interaction between blood cells and endothelial cells in the pulmonary microcirculation, we developed a method for observing leukocytes and erythrocytes in the microcirculation of perfused rat lungs by confocal laser scanning microscopy. We examined the behavior of leukocytes in the microcirculation of rat lungs exposed to hyperoxia, because oxygen toxicity is known to be associated with leukocyte infiltration and endothelial cell damage. Rats were divided into two groups: control (21% O₂) and hyperoxia (90% O₂ for 48 hours). Lungs were perfused with Krebs- Henseleit solution equilibrated with 21% O₂ and 5% CO₂ through an artificial lung. Leukocytes stained with carboxyfluorescein diacetate succinimidyl ester and erythrocytes stained with fluorescein isothiocyanate were added to the perfusate, and images of the cells were observed and recorded with a confocal laser scanning microscope and a high-speed video camera. The mean velocities of erythrocytes (Vr) in arterioles, capillaries, and venules of the control group were 1.52, 0.50, and 1.61 mm/sec, respectively. Also in the control group, the velocities of the leukocytes were divided by the mean velocities of the erythrocytes in the same arterioles, capillaries, and venules (Vw/Vr) and the results were 0.96, 0.97, and 0.98, respectively. The Vw/Vr values for arterioles in the hyperoxia group were not significantly different from those in the control group, but the Vw/Vr values for capillaries and venules were 27% and 37% lower than their respective control values. Leukocyte sequestration was seen mainly in capillaries in the hyperoxic group. These results suggest that an increase in adhesion in capillaries and venules, such as that caused by adhesion molecules, might play a key role in the behavior of leukocytes in the pulmonary microcirculation during hyperoxia.