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  • doi:10.1152/ajpheart.00417.2011 302:H1367-H1377, 2012. First published 20 January 2012;Am J Physiol Heart Circ Physiol

    SchafferSchafer, Richard T. Silver, Peter C. Doerschuk, William L. Olbricht and Chris B. Thom P. Santisakultarm, Nathan R. Cornelius, Nozomi Nishimura, Andrew I.blood flow in cortical blood vessels in micereveals cardiac- and respiration-dependent pulsatile In vivo two-photon excited fluorescence microscopy

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    ISSN: 0363-6135, ESSN: 1522-1539. Visit our website at http://www.the-aps.org/.Physiological Society, 9650 Rockville Pike, Bethesda MD 20814-3991. Copyright 2012 by the American Physiological Society. intact animal to the cellular, subcellular, and molecular levels. It is published 12 times a year (monthly) by the Americanlymphatics, including experimental and theoretical studies of cardiovascular function at all levels of organization ranging from the

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  • In vivo two-photon excited fluorescence microscopy reveals cardiac- andrespiration-dependent pulsatile blood flow in cortical blood vessels in mice

    Thom P. Santisakultarm ( ),1 Nathan R. Cornelius,1 Nozomi Nishimura,1

    Andrew I. Schafer,2 Richard T. Silver,2 Peter C. Doerschuk,1,3 William L. Olbricht,1,4

    and Chris B. Schaffer11Department of Biomedical Engineering, Cornell University, Ithaca; 2Department of Medicine, Weill Cornell MedicalCollege, New York; and Departments of 3Electrical and Computer Engineering and 4Chemical and BiomolecularEngineering, Cornell University, Ithaca, New York

    Submitted 25 April 2011; accepted in final form 13 January 2012

    Santisakultarm TP, Cornelius NR, Nishimura N, Schafer AI,Silver RT, Doerschuk PC, Olbricht WL, Schaffer CB. In vivotwo-photon excited fluorescence microscopy reveals cardiac- andrespiration-dependent pulsatile blood flow in cortical blood vessels inmice. Am J Physiol Heart Circ Physiol 302: H1367H1377, 2012. Firstpublished January 20, 2012; doi:10.1152/ajpheart.00417.2011.Sub-tle alterations in cerebral blood flow can impact the health andfunction of brain cells and are linked to cognitive decline anddementia. To understand hemodynamics in the three-dimensionalvascular network of the cerebral cortex, we applied two-photonexcited fluorescence microscopy to measure the motion of red bloodcells (RBCs) in individual microvessels throughout the vascularhierarchy in anesthetized mice. To resolve heartbeat- and respiration-dependent flow dynamics, we simultaneously recorded the electrocar-diogram and respiratory waveform. We found that centerline RBCspeed decreased with decreasing vessel diameter in arterioles, slowedfurther through the capillary bed, and then increased with increasingvessel diameter in venules. RBC flow was pulsatile in nearly allcortical vessels, including capillaries and venules. Heartbeat-inducedspeed modulation decreased through the vascular network, while thedelay between heartbeat and the time of maximum speed increased.Capillary tube hematocrit was 0.21 and did not vary with centerlineRBC speed or topological position. Spatial RBC flow profiles insurface vessels were blunted compared with a parabola and could bemeasured at vascular junctions. Finally, we observed a transientdecrease in RBC speed in surface vessels before inspiration. Inconclusion, we developed an approach to study detailed characteris-tics of RBC flow in the three-dimensional cortical vasculature, includ-ing quantification of fluctuations in centerline RBC speed due tocardiac and respiratory rhythms and flow profile measurements. Thesemethods and the quantitative data on basal cerebral hemodynamicsopen the door to studies of the normal and diseased-state cerebralmicrocirculation.

    microcirculation; hemodynamics; intravital imaging; vessel bifurca-tion; brain

    THE COMPLEX, THREE-DIMENSIONAL (3-D) vascular architecture ofthe brain presents a challenge to studies of microvascularhemodynamics. Larger arterioles form a net at the corticalsurface and give rise to penetrating arterioles that plunge intothe brain and feed capillary networks, where most nutrient andmetabolite exchange occurs (2). These capillaries coalesce intoascending venules, which return to the cortical surface anddrain into larger surface venules (22). Several lines of evidencehave suggested that even small changes in hemodynamics in

    the brain can have detrimental impacts on the health andfunction of neurons. Chronic diseases that alter the microcir-culation, such as hypertension (24) and diabetes (7), are riskfactors for dementia and have been linked to cognitive decline(33). Flow disruptions from hyperviscous blood in diseasessuch as polycythemia vera and essential thrombocythemia arelinked to neural degeneration (16). In addition, occlusion ofcerebral microvessels may cause the small, clinically silent,strokes that have been associated with cognitive impairmentand are a risk factor for dementia (34). Furthermore, decreasesin the pulsatility of blood flow result in higher systemicvascular resistance, decreased O2 metabolism, and reducedefficiency of the microcirculation (51). Novel methods thatenable the quantification of blood flow and vascular changes atthe level of individual microvessels in the complex corticalvasculature would greatly improve our understanding of thecauses and consequences of such microvascular pathologies aswell as provide insights into normal-state hemodynamic regu-lation.

    The most extensive data on blood flow dynamics in micro-vascular networks come from two-dimensional (2-D) vascularbeds, such as the cremaster muscle, omentum, and mesentery,where experimental techniques such as wide-field intravitalmicroscopy can be readily applied. In these systems, studies ofaverage blood flow speed in individual microvessels (54), thedistribution of red blood cells (RBCs) at microvascular junc-tions (36), and flow pulsatility in capillaries (54) have beenmade. These data have fueled detailed modeling of blood flowdynamics in microcirculatory networks that has elucidated thebiophysical and physiological principles that govern flow (1,11, 35, 42).

    In early studies of brain microcirculation, the average flowspeed and pulsatility of flow in cortical surface vessels wascharacterized (38), and more recent work has measured flowprofiles in pial arterioles using Doppler optical coherencetomography (46). However, because of the 3-D vascular archi-tecture of the brain, techniques with the ability to resolve flowin microvessels at different depths in the tissue are required toquantify flow dynamics throughout the vascular hierarchy.Recently, two-photon excited fluorescence (2PEF) microscopyhas emerged as an approach to quantify flow changes inindividual cortical microvessels in response to neural activity(20) and microvascular occlusion (30, 31, 40), with the poten-tial to access vessels throughout the full cortical thickness of amouse (21). However, the careful quantification of blood flowdynamics that has been done in 2-D microvascular beds has notyet been performed in the brain vascular network. For example,

    Address for reprint requests and other correspondence: C. B. Schaffer, Dept.of Biomedical Engineering, Cornell Univ., B57 Weill Hall, Ithaca, NY 14853(e-mail: cs385@cornell.edu).

    Am J Physiol Heart Circ Physiol 302: H1367H1377, 2012.First published January 20, 2012; doi:10.1152/ajpheart.00417.2011.

    0363-6135/12 Copyright 2012 the American Physiological Societyhttp://www.ajpheart.org H1367

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  • previous work has not mapped average flow speed or heart-beat-induced flow modulation in vessels throughout the corti-cal network, measured spatial flow profiles in brain microves-sels, or quantified important hemodynamic parameters, such aspulse wave velocity.

    Our work aimed to overcome the challenges of quantifyingblood flow dynamics in 3-D microvascular networks in liveanimal models. We coupled electrocardiogram (ECG) andrespiratory waveform measurements with 2PEF-based assess-ment of RBC speed in individual vessels in anesthetized mice.We quantified average centerline RBC flow speed as well astemporal flow fluctuations due to cardiac and respiratoryrhythms in cortical arterioles, capillaries, and venules. Inter-estingly, we found pulsatile RBC flow in vessels throughoutthe cortical network, including capillaries and venules, andidentified respiration-dependent changes in cortical blood flow.We quantified the tube hematocrit in brain capillaries andfound no dependence on centerline RBC speed or position inthe vascular hierarchy. We also measured time- and space-dependent RBC flow profiles in arterioles, in venules, and atvascular junctions and found that spatial flow profiles wereblunted in most vessels. These data provide a detailed quanti-fication of cerebral hemodynamics in brain microvessels, whilethe methods we developed open the door to future studies ofalterations in brain hemodynamics