Measuring angiogenesis and hemodynamics in mice

This protocol outlines methods to measure vascular parameters, including angiogenesis (e.g., vascular density, length, diameter) and hemodynamics (e.g., erythrocyte velocity), in tumors and normal vascular networks in mice. Fluorescein-isothiocyanate (FITC)-dextran is injected into the tail vein of mice to visualize microvessels within 150 μm (using single-photon microscopy) or ~600 μm (using multiphoton laser-scanning microscopy [MPLSM]) of a tumor/window interface. Randomly selected areas (three to six locations/tumor or animal) are investigated using long-working-distance objectives with appropriate magnification. During each observation period, FITC-fluorescence images are recorded for 60 sec, and the videotapes are analyzed off-line for single-photon microscopy; or a three-dimensional (3D) image stack of the vessel network is generated, and vessel properties are measured for MPLSM. If desired, red blood cell (RBC) flux can be measured on a vessel-by-vessel basis using fluorescent tracer RBCs.