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...

The cortical microvasculature plays a key role in cortical tissue health by transporting important molecules via blood. Disruptions to blood flow in the microvasculature due to events such as stroke can thus induce damage to the cortex. Recent developments in two-photon microscopy have enabled in vivo imaging of anesthetized rat cortex in three dimensions. The microscopy data provide information about the geometry of the cortical microvasculature, length and diameter of the vessels in the imaged microvasculature network, and blood flow through a subset of those vessels. We demonstrate a model that achieves three goals. First, given a network of interconnected vessels and flow measurements on a subset of those vessels, we can estimate the flows in the remaining vessels. Second, we can determine which and how many vessels should have blood flow measurements taken to provide sufficient information to predict the unmeasured flows. Finally, the model enables us to predict effects of blockages in one or more vessels, indicating which vessels are most important to overall flow in the network.

Computations are described which estimate flows in all branches of the cortical surface arteriole network from two-photon excited fluorescence (2PEF) microscopy images which provide the network topology and, in selected branches red blood cell (RBC) speeds and lumen diameters. Validation is done by comparing the flow predicted by the model with experimentally measured flows and by comparing the predicted flow redistribution in the network due to single-vessel strokes with experimental observations. The model predicts that tissue is protected from RBC flow decreases caused by multiple occlusions of surface arterioles but not penetrating arterioles. The model can also be used to study flow rerouting due to vessel dilations and constrictions.

The same assembly of tools from the toolkit but with different parameter values is able to describe effects that are considered distinctive, such as the presence or absence of an initial decrease in oxygenated hemoglobin concentration, indicating that the differences might be due to unique parameter values in a subject rather than different fundamental mechanisms.