Spatial flow-volume dissociation of the cerebral microcirculatory response to mild hypercapnia Elizabeth B. Hutchinson, Bojana Stefanovic, Alan P. Koretsky, and Afonso C. Silva * Laboratory of Functional and Molecular Imaging, National Institute of Neurological Disorders and Stroke, National Institutes of Health, 10 Center Drive, Building 10, Room B1D114, Bethesda, MD 20892-1065, USA Received 19 September 2005; revised 7 March 2006; accepted 16 March 2006 Available online 19 May 2006 The spatial and temporal response of the cerebral microcirculation to mild hypercapnia was investigated via two-photon laser-scanning microscopy. Cortical vessels, traversing the top 200 Mm of somatosen- sory cortex, were visualized in A-chloralose-anesthetized Sprague – Dawley rats equipped with a cranial window. Intraluminal vessel diameters, transit times of fluorescent dextrans and red blood cells (RBC) velocities in individual capillaries were measured under normocapnic (PaCO 2 = 32.6 T 2.6 mm Hg) and slightly hypercapnic (PaCO 2 = 45 T 7 mm Hg) conditions. This gentle increase in PaCO 2 was sufficient to produce robust and significant increases in both arterial and venous vessel diameters, concomitant to decreases in transit times of a bolus of dye from artery to venule (14%, P < 0.05) and from artery to vein (27%, P < 0.05). On the whole, capillaries exhibited a significant increase in diameter (16 T 33%, P < 0.001, n = 393) and a substantial increase in RBC velocities (75 T 114%, P < 0.001, n = 46) with hypercapnia. However, the response of the cerebral microvasculature to modest increases in PaCO 2 was spatially heterogeneous. The maximal relative dilatation (range: 5 – 77%; mean T SD: 25 T 34%, P < 0.001, n = 271) occurred in the smallest capillaries (1.6 Mm – 4.0 Mm resting diameter), while medium and larger capillaries (4.4 Mm–6.8 Mm resting diameter) showed no significant changes in diameter (P > 0.08, n = 122). In contrast, on average, RBC velocities increased less in the smaller capillaries (39 T 5%, P < 0.002, n = 22) than in the medium and larger capillaries (107 T 142%, P < 0.003, n = 24). Thus, the changes in capillary RBC velocities were spatially distinct from the observed volumetric changes and occurred to homogenize cerebral blood flow along capillaries of all diameters. Published by Elsevier Inc. Keywords: Cerebral blood flow; Brain; Red blood cell velocity; Hyper- capnia; Two-photon microscopy Introduction The most prominent methodologies for non-invasive imaging of human brain function – functional magnetic resonance, positron emission tomography, and near-infrared spectroscopy – all rely on tight coupling between focal cerebral hemodynamics and neuronal activity (Raichle, 2003). A detailed understanding of the mecha- nisms of local cerebral circulatory regulation is thus critical for the establishment of accuracy and range of the applicability of these techniques. Like the neuronal network itself, the cerebral vascular tree exhibits both hierarchy and spatial specialization. At rest, local blood flow varies as much as 18-fold between the different regions of a rat brain (Fenstermacher et al., 1991), likely resulting from regional differences in capillary density (Patlak et al., 1984) as well as from transit time variations (Rosen et al., 1991). Moreover, CBF undergoes local (in addition to global) regulation so that, on a sub- millimeter scale, maps of hemodynamic changes closely follow those of neuronal activity (at least at the columnar level) under a variety of conditions (Cox et al., 1993; Woolsey et al., 1996; Malonek and Grinvald, 1996; Duong et al., 2001; Logothetis, 2002). A growing number of studies have suggested that both the density of the capillary beds as well as the amplitude and the temporal evolution of blood flow response to functional activation follow the cortical neuronal architecture (Cox et al., 1993; Gerrits et al., 2000; Harrison et al., 2002; Silva and Koretsky, 2002; Lu et al., 2004). While the existence of capillary level structures for very fine hemodynamic regulation has been demonstrated in various species (Ehler et al., 1995; Rodriguez-Baeza et al., 1998; Harrison et al., 2002), the spatial limit of hemodynamic adjust- ments remains unclear and is a subject of current interest (Lauritzen, 2001), as it dictates the theoretical limit on the functional specificity of flow-weighted neuroimaging techniques. Indeed, it is the microcirculatory CBF control that is of most interest to brain function investigations due to the proximity of the capillary network to the activated parenchyma, and thus it is crucial to understand how capillary diameter and red blood cell (RBC) velocities are regulated. While a heterogeneous profile of microcirculatory CBF adjustments has long been suspected (Rosenblum, 1965), data on the spatial pattern of microvascular flow regulation in the brain have been scarce, likely due to the intrinsic difficulty of achieving the required spatial resolution in vivo. Conventionally, the pial microvessels have been directly 1053-8119/$ - see front matter. Published by Elsevier Inc. doi:10.1016/j.neuroimage.2006.03.033 * Corresponding author. Fax: +1 301 480 2558. E-mail address: Silvaa@ninds.nih.gov (A.C. Silva). Available online on ScienceDirect (www.sciencedirect.com). www.elsevier.com/locate/ynimg NeuroImage 32 (2006) 520 – 530