In Vivo Investigation of the Cortical Microcirculation Under Ischemia/Reperfusion in Rats Using Dynamic Laser Confocal Fluorescence Microscopy

Abstract

This study describes the dynamic cerebral microcirculatory changes occurring in transient forebrain ischemia and reperfusion, associating evaluations of capillary perfusion and microvessel diameter. A laser-scanning confocal fluorescence microscope, with high-speed resolution in two dimensions, was used in combination with fluorescence-labeled erythrocytes and plasma. Pentobarbital-anesthetized rats equipped with a closed cranial window over the parietal cortex were given both diluted fluorescein isothiocyanate (FITC)-dextran and FITC-labeled erythrocytes. The microcirculation was dynamically visualized in pial microvessels and parenchymal capillaries down to 200μm underneath the brain surface. The microvessel flow was video-recorded at 50 images per second to determine the flux and velocity of labeled erythrocytes in capillaries. Forebrain ischemia was induced for 15 min under the con-focal microscope, using the four-vessel occlusion method. As soon as carotid arteries were clamped the arteriolar erythrocyte flow became extremely low and a rapid, transient arteriole diameter increase occurred for 1-2 min, immediately followed by vasoconstriction and a fall in capillary erythrocyte velocity from 0.39 ± 0.15 mm/s to approximately 0. At the unclamping, arteriolar blood flow was instantaneously rapid but with no significant change in vessel diameter and without full recovery of capillary perfusion. It is only after 5 min of reperfusion that arteriole dilatation started, further attaining a maximal value of 181% ± 27% (n = 19) at 11 min of re-perfusion. In the meantime, capillary erythrocyte velocity and flux increased (up to 1.79 ± 1.01 mm/s velocity). No capillary recruitment of erythrocytes, in its classical sense, was observed.