An intravital microscopy study of radiation-induced changes in permeability and leukocyte–endothelial cell interactions in the microvessels of the rat pia mater and cremaster muscle

Using intravital microscopy and a closed window method, we measured irradiation-induced changes in the vascular permeability and cell interactions in microcirculation networks of the rat pia mater; the same effects were monitored in the cremaster muscle as a control. The closed cranial window has many advantages, including long-term direct visualization of microcirculation. The method allows for repeated testing of the same vessel or network, thereby reducing variability. The method also allows for measurement of permeability changes and the accompanying leukocyte–endothelial cell interactions in the same network or vessel, which permits correlative studies of these phenomena. However, this method is not without challenges. The optical conditions are difficult, because the brain is three-dimensional and its parenchyma is more complex than the thinner, flatter peripheral tissues. To overcome this limitation, we performed a dynamic background subtraction. The background is dynamically related to vessel intensity, and changes in intensity were determined by eliminating the effects of neighboring and underlying vessels. We applied this method to studying the effects of ionizing radiation on the blood–brain barrier (BBB) permeability and cell interactions and the modulation of these effects by anti-ICAM-1 antibodies. Our results demonstrate that this method is sensitive to changes in these properties of the BBB.