Automated measurement of blood flow velocity and direction and hemoglobin oxygen saturation in the rat lung using intravital microscopy

Abstract

Intravital microscopy of the pulmonary microcirculation in research animals is of great scientific interest for its utility in identifying regional changes in pulmonary microcirculatory blood flow. Although feasibility studies have been reported, the pulmonary window can be further refined into a practical tool for pharmaceutical research and drug development. We have established a method to visualize and quantify dynamic changes in three key features of lung function: microvascular red blood cell velocity, flow direction, and hemoglobin saturation. These physiological parameters were measured in an acute closed-chest pulmonary window, which allows real-time images to be captured by fluorescence and multispectral absorption microscopy; images were subsequently quantified using computerized analysis. We validated the model by quantifying changes in microcirculatory blood flow and hemoglobin saturation in two ways: 1) after changes in inspired oxygen content and 2) after pharmacological reduction of pulmonary blood flow via treatment with the β1 adrenergic receptor blocker metoprolol. This robust and relatively simple system facilitates pulmonary intravital microscopy in laboratory rats for pharmacological and physiological research.

Fig. 1.

Surgical procedures for pulmonary window fenestration. A: Plexiglas window holder with socket and window glued to it, and rat restrainer to eliminate z-directional movement. B: anesthetized intubated rat with implanted pulmonary window. C: rat with window in a restrainer, placed on a heating pad under a fluorescence upright microscope.

Fig. 2.

Hemodynamic and microcirculatory measurements using pulmonary intravital microscopy. A: brightfield reflectance image at 590 nm (top), color-encoded hemoglobin oxygen saturation maps under 100% O₂ (middle), and 16% O₂ (botom). B: time course of concomitant measurements of HbO₂ in the lung using pulmonary intravital microscopy and in the arteries of the hindlimb using pulse oximetry after 15 min at 100% O₂, 15 min of hypoxia, and 15 min at 100% O₂. Hypoxia was 12% and 16% O₂. Each data point represents an average from three sequential measurements per animal, and an average between three (12% O₂) and four (16% O₂) animals. C: color encoded maps of red blood cell velocity (top) and directionality (bottom) in the pulmonary microcirculation. Image pairs were acquired in 5-min intervals. The arrow marks what is likely a single vessel where blood flow has stalled in between imaging events. The dashed line marks an area of venous blood flow, as visible by converging movement of blood cells. The colored compass wheel is the legend for the directionality map, with colors indicating the direction in which the blood flows. D: changes in heart rate and red blood cell velocity in the pulmonary microcirculation after repeated injection of metoprolol (10 mg/kg). For each individual rat, three measurements were made, before/after injections, separated by 5-min intervals. Sequential measurements from each pre-/posttreatment period were averaged per animal, and each data point on the graph represents an average from all three rats for the respective period. Significant changes in heart rates and flow are indicated with a star (paired t-test, P < 0.05, n = 3).