Santisakultarm TP, Cornelius NR, Nishimura N, Schafer AI, Silver RT, Doerschuk PC, Olbricht WL, Schaffer CB. In vivo two-photon excited fluorescence microscopy reveals cardiac-and respiration-dependent pulsatile blood flow in cortical blood vessels in mice. Am J Physiol Heart Circ Physiol 302: H1367-H1377, 2012. First published January 20, 2012; doi:10.1152/ajpheart.00417.2011.-Subtle alterations in cerebral blood flow can impact the health and function of brain cells and are linked to cognitive decline and dementia. To understand hemodynamics in the three-dimensional vascular network of the cerebral cortex, we applied two-photon excited fluorescence microscopy to measure the motion of red blood cells (RBCs) in individual microvessels throughout the vascular hierarchy in anesthetized mice. To resolve heartbeat-and respirationdependent flow dynamics, we simultaneously recorded the electrocardiogram and respiratory waveform. We found that centerline RBC speed decreased with decreasing vessel diameter in arterioles, slowed further through the capillary bed, and then increased with increasing vessel diameter in venules. RBC flow was pulsatile in nearly all cortical vessels, including capillaries and venules. Heartbeat-induced speed modulation decreased through the vascular network, while the delay between heartbeat and the time of maximum speed increased. Capillary tube hematocrit was 0.21 and did not vary with centerline RBC speed or topological position. Spatial RBC flow profiles in surface vessels were blunted compared with a parabola and could be measured at vascular junctions. Finally, we observed a transient decrease in RBC speed in surface vessels before inspiration. In conclusion, we developed an approach to study detailed characteristics of RBC flow in the three-dimensional cortical vasculature, including quantification of fluctuations in centerline RBC speed due to cardiac and respiratory rhythms and flow profile measurements. These methods and the quantitative data on basal cerebral hemodynamics open the door to studies of the normal and diseased-state cerebral microcirculation. microcirculation; hemodynamics; intravital imaging; vessel bifurcation; brain THE COMPLEX, THREE-DIMENSIONAL (3-D) vascular architecture of the brain presents a challenge to studies of microvascular hemodynamics. Larger arterioles form a net at the cortical surface and give rise to penetrating arterioles that plunge into the brain and feed capillary networks, where most nutrient and metabolite exchange occurs (2). These capillaries coalesce into ascending venules, which return to the cortical surface and drain into larger surface venules (22). Several lines of evidence have suggested that even small changes in hemodynamics in the brain can have detrimental impacts on the health and function of neurons. Chronic diseases that alter the microcirculation, such as hypertension (24) and diabetes (7), are risk factors for dementia and have been linked to cognitive decline (33). Flow disruptions from hyperviscous blood in diseases such as...