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NeuroImage 32 (2006) 520 – 530
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 (PaCO2 = 32.6 T 2.6 mm Hg) and slightly hypercapnic
(PaCO2 = 45 T 7 mm Hg) conditions. This gentle increase in PaCO2 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 PaCO2 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
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: [email protected] (A.C. Silva).
Available online on ScienceDirect (www.sciencedirect.com).
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
E.B. Hutchinson et al. / NeuroImage 32 (2006) 520–530 521
observed via intravital microscopy in animals equipped with closed
cranial windows (Navari et al., 1978) and the vessel diameters
measured by a calibrated video microscaler (Morii et al., 1986;
Wagerle and Degiulio, 1994; Parfenova et al., 1995; Ishimura et al.,
1996; Hudetz, 1997; Takenaka et al., 2000, 2003; Iliff et al., 2003;
Xu et al., 2004). Although important information on the effects of a
range of vasoactive agents has been collected, these studies have
been constrained to the pial surface and, typically, to vessels with
diameters larger than 10 Am.
The advent of scanning confocal microscopy enabled imaging
of vessels below the cortical surface, nonetheless with limited
penetration capabilities. Microvascular CBF increases elicited by
hypercapnia (Villringer et al., 1994; Seylaz et al., 1999), as well as
decreases under ischemic conditions (Morris et al., 1999; Pinard et
al., 2000), have been investigated with scanning confocal
microscopy. Two-photon laser-scanning microscopy, on the other
hand, brought in an additional number of important advantages,
particularly for in vivo biological applications, that have afforded
widespread use (Denk et al., 1990) and made significant impact on
in vivo microscopy. Recently, the applicability of two-photon
microscopy to the investigation of cerebral microcirculation has
been demonstrated in rat somatosensory cortex following vibrissae
or hindlimb stimulation (Kleinfeld et al., 1998), rat dorsal olfactory
bulb during odor stimulation (Chaigneau et al., 2003), mouse
somatosensory cortex during bicuculline-induced focal epilepti-
form activity (Hirase et al., 2004) as well as freely moving awake
rats (Helmchen et al., 2001).
The purpose of the present work was to investigate the spatial
and temporal evolution of microvascular CBF regulation using
two-photon laser-scanning microscopy. To elicit changes in vessel
diameter and in RBC velocities, we employed mild hypercapnia
while imaging the microcirculation of the rat somatosensory
cortex. Mild hypercapnia was chosen as an effective and simple
way to elicit robust increases in global resting blood flow, thereby
obviating the need for localization of the affected region and hence
greatly facilitating the use of two-photon microscopy. Using
fluorescently labeled dextrans, we quantified the changes in transit
times from arteries to venules and veins. The concomitant
vasodilatation was quantified by estimating the diameter of
arteries, veins and capillaries from stack maximum intensity
projections. Special attention was given to capillaries. The present
experiments yield new insight into the spatial distribution of CO2-
induced changes in hemodynamics, in terms of both RBC velocity
and intraluminal diameter, and provide detailed information on
microvascular CBF regulation.
Materials and methods
Animal preparation
Twenty male Sprague–Dawley rats, weighing 146–300 g, were
anesthetized with isoflurane (3% for initial induction and 1.5% for
maintenance) in O2-enriched medical air, orally intubated and
mechanically ventilated throughout the remainder of the experi-
ments (with 1.5% isoflurane concentration). Respiratory parame-
ters, including end-tidal CO2 levels, were carefully monitored by a
capnograph (BCI 300 Capnocheck, BCI, Inc, Waukesha, Wiscon-
sin). Rectal temperature was monitored and maintained at 37.0-Cby means of a feedback-controlled warm water bath. The right
femoral artery and vein were cannulated for blood gas analyses and
intravenous administration drugs. After surgery, all wounds were
treated with 2% lidocaine and closed.
Closed cranial window
The rats were secured to a custom-built stereotaxic equipped
with plastic ear pieces and a bite-bar. The dorsal area of the scalp
was shaved, and a rostral-to-caudal incision was made to expose
the skull. Connective tissue above the skull was removed, and local
bleeding was controlled either by cauterization or by coating the
skull with bone wax. Some portions of the temporal muscle on the
side of the cranial window were excised, and the location of a
cranial window over the primary somatosensory cortex was
determined based on stereotaxic coordinates (center at 4 mm
lateral to bregma, 0 mm caudal to bregma, diameter 5 mm). Using
a dremel with a burr bit, the perimeter around the window was
carefully thinned until pial vessels were visible. Ice-cold saline
solution was constantly applied during drilling to avoid over-
heating of the skull. The area of skull within the perimeter was then
removed with no. 7 forceps. In most cases (animal body weight
BW <250 g, n = 14), the dura matter below the cranial window
remained intact and was left in place. In a few cases, however (BW
�250 g, n = 6), it was necessary to remove the dura matter by
careful dissection, leaving the pial surface of the cortex exposed.
Once the craniotomy was completed, the ear bars were removed
and a metal frame was fixed around the craniotomy site with dental
cement and held in place by custom made metal bars attached with
pins on either side of the frame (Yoder and Kleinfeld, 2002). The
bars were secured to the stereotaxic stage by the ear bar holders
and adjusted until the cranial window was oriented horizontally.
The well over the cranial window was then filled with 1% wt./vol.
agarose and covered with a custom cut Corning glass #1 cover slip.
The animal and sample stage were then moved and fixed to the
microscope stage. Anesthesia was switched from isoflurane to an
intravenous solution of a-chloralose (80 mg/kg BW initial bolus,
followed by a constant infusion of 27 mg/kg BW/h) supplemented
with pancuronium bromide as needed (2 mg/kg BW/90 min) to
minimize motion artifacts. To visualize vessels, blood plasma was
fluorescently labeled by an intravenous injection of 150–300 Al of0.5 mg/ml rhodamine-labeled dextran (70,000 MW) in phosphate-
buffered saline.
Blood gases and hypercapnia
Blood gases were carefully monitored and adjusted throughout
experiments to ensure accurate normocapnic and hypercapnic
conditions. Ventilation parameters (rate, tidal-volume and oxygen
concentration in gas mixture) were adjusted to correct PaO2 and
PaCO2. Whenever necessary, sodium bicarbonate was injected
intravenously to correct for acidic blood pH. Once the blood gases
were in physiological range (normocapnic condition), the imaging
commenced. Blood gases were periodically sampled to ensure
physiological stability throughout imaging. Hypercapnia was induced
by adding 2–3% CO2 to the inhaled air mixture. A few minutes were
allowed before blood gases were sampled again to confirm
equilibration under hypercapnic conditions. After the last image
during the hypercapniawas acquired, the last blood samplewas drawn
to verify the preservation of the hypercapnic condition. Typical
experimental duration was 40 min (20 min for each of normo- and
hypercapnia). The trials in which either normocapnia or hypercapnia
could not be maintained were excluded from the analysis.
E.B. Hutchinson et al. / NeuroImage 32 (2006) 520–530522
Optical microscopy and vessel diameter measurement
Imaging was performed with a Bio-Rad Radiance 2100 MP
two-photon laser-scanning microscope. The pump laser used was a
Tsunami Ti:S fs pulsed laser, which was tuned to 805 nm and
operated at 8.25 or 8.5 W. The lens used was a Nikon 10� water
immersion objective with a working distance of 2 mm and 0.3
numerical aperture. Emitted fluorescence was detected with a PMT
using a 620/100 nm bandpass filter.
High spatial resolution (matrix size of 512 � 512), Kalman
averaged, horizontal planar images were acquired at 0.8� 0.8 Am2 in
plane resolution and stepped in 1 Am increments from the pial surface
down to a cortical depth of 200 Am. The maximal pixel intensities in
these imageswere then projected onto a single plane to yield a 2Dmap
of the 3D vasculature. From this map, line profiles across vessels of
interest were created, from which the full-width at half-maximum
(FWHM) values were computed to yield the vessel diameter. This
method of measurement was employed due to the relatively large (¨3
Am in the z axis) focal volume of the objective, which exceeded the
step size and ensured imaging of the maximal diameter in the x –y
plane. This procedure was followed for each vessel under both normal
and hypercapnic conditions so that diameter estimates of each vessel
could be paired and changes quantified.
Bolus passage
As a measure of the changes in both blood volume and blood
flow under hypercapnia, the transit of a bolus of dye through the
vasculature was imaged. A reasonable imaging plane containing
both arterial and venous vessels was selected from within the stack
volume, and a 30-s time course recorded at a rate of 1 frame every
222 ms. A 150-Al bolus of the dextran-rhodamine dye was rapidly
injected (within less than 1 s, using a 1-cm3 syringe) via the femoral
vein catheter. The dextran-rhodamine dye sequentially filled the
appropriate vessels in the subsequently acquired frames. This bolus
method was repeated several times before saturation of the dye.
For the purposes of the analysis, the vessel that was both largest
in diameter and first to enhance was chosen as the arterial input
function to which the rest of the vascular responses were
referenced. The latest onset arteriole before the capillary bed, the
first venule after the capillary bed and the largest and latest onset
venous vessel were also identified. Time courses of the passage of
the bolus of dye were plotted for each of the selected vessels. The
time point at which the signal intensity first reached one-half of its
maximum value (t1/2) was determined and compared to the t1/2 of
the arterial input vessel to estimate the transit time from the arterial
input to the other three vessels types in both normocapnic and
hypercapnic states. A method similar to the one described above
was employed to measure the transit of a bolus of dye through the
capillary network. The temporal resolution was improved to 140
ms per frame by reducing the field of view. In these trials, the onset
times of capillaries of several different sizes, as well as venules,
were referenced to a feeding arteriole.
RBC velocity measurement
Very high temporal resolution measurements of RBC velocities
were obtained by 1.333 ms line scans along the axis of selected
capillaries of different diameters in the direction of flow as
described previously (Kleinfeld et al., 1998). The RBCs appeared
as dark segments in a line of brightly labeled plasma. With forward
motion over line scans taken through time, the RBCs created dark
tracks angled at a slope given by the length of the capillary divided
by the time to clear the capillary. From these scans, velocity
measurements were obtained for individual RBCs over a period of
30 s and averages over all RBCs in each capillary computed.
Data in text and figures are presented as mean T standard
deviation. Student’s t tests were performed to test for statistical
significance. Unless otherwise specified, a P value of <0.05 was
considered indicative of a statistically significant difference.
Results
Blood physiology
Rats were kept under stable conditions at all times throughout
the experiments. Rectal temperature and end-tidal CO2 were
monitored continuously, and arterial blood gases were sampled
periodically. Under normocapnic conditions, the across-subject
mean values for PaO2, PaCO2 and pH were 105.2 T 6.2 mm Hg,
33.0 T 3.2 mm Hg and 7.42 T 0.03, respectively, n = 20. During
hypercapnia, these values changed to 98.5 T 10.0 mm Hg (P <
0.002 compared to normocapnia), 45.4 T 6.8 mm Hg (P < 0.001)
and 7.31 T 0.04 (P < 0.001), respectively. Because the data were
paired (that is, values were sampled from each rat before and after
introducing CO2 to the breathing mixture), the three blood gases
parameters were significantly different after CO2 compared to
normocapnia (P < 0.001), showing that the addition of 2–3% CO2
to the breathing mixture was a gentle, yet effective way to raise
PaCO2 to cause moderate increases in CBF.
Measurement of large vessel diameters
Fig. 1 shows typical two-photon images of the rat cerebral
microcirculation. The experimental setup allowed visualization of
vessels located up to 200 Am below the pial surface (Fig. 1A).
However, most experiments described herein were performed at
depths ranging from 50 to 150 Ambelow the pial surface.Within this
volume, the typical structure of the rat cortical vasculature was
observed. The pial surface contained large arteries and veins running
along the cortical surface (Fig. 1B). Anastomoses were visible and
predominant on the arterial side. Penetrating arteries and arterioles
were also seen, together with venules and veins, running perpen-
dicular to the pial surface (Fig. 1B). Capillaries were scarce at the
pial surface, but much denser deeper in the cortex.
To measure vessel diameters independently of their orientation
and depth, a maximum intensity projection (MIP) stack of the 3D
vasculature onto a single horizontal plane was created, such as the
one displayed in Fig. 1C. This image shows a wide range of vessel
diameters, with capillary vessels as narrow as 2 Am and veins wider
than 90 Am in diameter. In general, capillaries were randomly
oriented below the pial surface and exhibited high interconnectivity,
with several anastomoses clearly visible in the MIP. Large arteries as
well as veins were oriented more regularly, usually running from
side to the middle of the brain. Penetrating arteries and arterioles can
also be seen in Fig. 1C, along with surface venules and veins. Based
on the diameter estimates (U) obtained from the stack MIPs, each
vessel was assigned to one of three classes (Nakagawa et al., 1995):
& the arterial network, made up of arteries (U > 50 Am) and
arterioles (U < 50 Am);
Fig. 1. Typical two-photon images of the rat cerebral microcirculation. (A) Single planar images at six different depths spaced at 25-Am increments throughout the
175-Am depth of the imaged space. (B) Reconstructed 3D views of the XZ (top) and YZ (bottom planes) show large vessels (arteries and veins) running along the
pial surface and smaller vessels below the pial surface. Penetrating arteries and arterioles, as well as draining venules and veins, run perpendicular to the pial
surface. (C) Stack projection of the 3D vasculature onto a single horizontal plane shows a wide range of vessel diameters. Scale bars = 100 Am.
E.B. Hutchinson et al. / NeuroImage 32 (2006) 520–530 523
& the capillary network (U < 10 Am); or
& the venous network, consisting of venules (U < 60 Am) and
veins (U > 75 Am).
No veins or venules were found in the 60–75 Am diameter
range. In addition, the identity of arterial and venous structures
was verified using the bolus data, as described below in the
following subsection.
Large vessel diameters were computed for each condition and
their changes due to mild hypercapnia compared between the
arterial and the venous networks. Arterial and arteriole diameters
increased by 11.2 T 14.9% (4 rats, 28 arteries and arterioles, P <
0.004 for paired t test, P < 0.002 for Wilcoxon rank-sum test),
while venous and venule diameters increased by 7.3 T 13.6% (4
rats, 44 venules and veins, P < 0.002 for paired t test, P < 0.0002
for Wilcoxon rank-sum test). The large standard deviations
resulted from the heterogeneity in the sign of individual vessel
diameter changes: some vessels dilated; others did not react to the
presence of CO2; and others still were constricted by hypercapnia.
Nevertheless, both the arterial as well as the venous vasodilatation
E.B. Hutchinson et al. / NeuroImage 32 (2006) 520–530524
elicited by hypercapnia were significant (P < 0.05), but not
different from each other (P > 0.1).
Large vessel bolus transit time changes
Vessel-specific dynamics of cerebral blood flow were probed by
following the passage of a bolus of contrast agent through the
cerebral circulation. Fig. 2 shows typical time courses of a bolus
passage through an artery, arteriole, venule and vein. The dynamics
of the passage of a bolus through each of the four vessel types
show a clear distinction in onset times, times-to-peak and
dispersion (full-width at half-maximum). Recirculation of the
contrast was clearly visible in the form of an elevated baseline
after the passage of the bolus, as well as by secondary and tertiary
increases in signal intensity following the first passage. An
Fig. 2. The transit of a bolus of dye through the rat cerebral vasculature: (A)
passage of bolus from artery (top left) to arteriole (top right) to venule
(bottom left) to vein (bottom right), with regions of interest indicating each
of the four vessel types. (B) Time courses obtained from each ROI indicated
in panel A show a clear distinction in onsets, times-to-peak and full-widths
at half-maximum between the different blood vessels. Recirculation of the
bolus can also be seen in the form of an elevated baseline and secondary
and tertiary passages.
example of this phenomenon can be seen in Fig. 2B. The arterial
time course shows a second passage of bolus well before
conclusion of the venous washout of the first passage.
To avoid possible confounds introduced by recirculation, only
the onset to the first passage of bolus was used to probe vessel
reactivity to CO2. Fig. 3A shows averaged (n = 6) time courses
of the first passage of dextran-rhodamine in each of the four
vessel types, under normocapnic and hypercapnic conditions. The
onset (defined as the time point at which the signal intensity first
reached one-half of its maximum value) – t1/2 – was determined
for each vessel type, and compared to the onset time of the
arterial input vessel, to yield the bolus transit time for that vessel.
Under hypercapnic conditions, the average transit time of a bolus
from the artery to the vein was 1.77 T 0.62 s, 27% shorter (P <
0.05) than the corresponding transit time during normocapnia,
2.43 T 0.66 s. Venules showed a smaller yet significant (P <
0.05) decrease in transit time due to CO2. The transit time from
artery to venule was 1.66 T 0.50 s during normocapnia,
decreasing (P < 0.05) by 14% to 1.43 T 0.50 s during
hypercapnia. There was no detectable difference in the transit
times to arteriole during either condition (0.21 T 0.23 s during
normocapnia and 0.18 T 0.20 s during hypercapnia, P > 0.7).
However, these measurements were probably limited by the low
frame rate (222 ms) employed. Fig. 3B shows plots of the transit
times for arterioles, venules and veins during normocapnic and
hypercapnic conditions.
Capillary diameter changes
Once the larger vasculature reactivity to mild CO2 administra-
tion was characterized, as described above, the focus of the study
turned to the microcirculation. The size distribution of 393
capillaries sampled from 13 different animals was obtained from
stack MIPs of the microcirculation and plotted in Fig. 4A for
normocapnic and hypercapnic conditions. Capillaries with appar-
ent mean diameters as low as 1.6 Am were found, although their
incidence was rare, about 1% of the total number of capillaries
sampled. Hypercapnia induced vasodilatation of the cerebral
microvasculature, as noticed by a right shift of the histogram
obtained during hypercapnia, and manifested by a significant
decrease in the number of capillaries smaller than 3.2 Am which
was accompanied by a significant increase in the number of
capillaries larger than 5.2 Am. The mean capillary diameter during
hypercapnia was 4.1 T 1.1 Am, significantly larger than the average
capillary diameter during normocapnia, 3.7 T 1.1 Am (P < 0.005).
There was a pronounced heterogeneity in the hypercapnia-induced
changes in capillary diameter. As shown in Fig. 4B, the largest
changes in diameter (5–77%; mean T SD: 25 T 34%, P < 0.001)
induced by hypercapnia occurred in capillaries with small resting
diameters (�4.0 Am, n = 271, 69% of the total number of
capillaries), while no significant changes (P > 0.07) were observed
in capillaries of larger resting diameter (4.4 Am–6.8 Am, n = 122,
31% of the population), with the exception of capillaries that were
5.6 Am (4% of the capillary population) in diameter. These 5.6 Amdiameter capillaries showed a significant decrease (�14% T 19%,
n = 17, P < 0.004) in resting diameter with hypercapnia.
RBC velocity changes
Fig. 5 shows line scans of three different capillaries, circled in
Fig. 5A, used to measure the reactivity of RBC velocities to
E.B. Hutchinson et al. / NeuroImage 32 (2006) 520–530 525
hypercapnia. The horizontal axis in Fig. 5C represents the length of
the capillary vessel in the direction of blood flow (indicated by the
green arrow in Fig. 5B), and the vertical axis represents time. Red
blood cells appear as dark streaks, with slopes that indicate their
velocities as they traverse the capillaries. Steeper slopes correspond
Fig. 4. Histograms of capillaries according to diameter (A) and percent
changes in diameter (B) induced by hypercapnia. Significant overall
vasodilatation is seen, with the largest changes occurring in the smallest
capillaries (*P < 0.03; error bars correspond to 1 standard deviation).
to slower RBC velocities. Hypercapnia elicited changes in RBC
velocities, measured by a change in the slope of the dark streaks in
the line scans of the different capillaries. In the example given in
Fig. 5, RBC velocity increased during hypercapnia in the medium
and large capillaries, but decreased in the small capillary.
Mild hypercapnia induced an increase in RBC velocities in
most capillaries. During normocapnia, the mean RBC velocity
across all capillaries was 1.12 T 0.89 mm/s, significantly lower
than the mean RBC velocity during mild hypercapnia, of 1.81 T1.60 mm/s (P < 0.0002, n = 46). The RBC velocity across the
capillary network thus increased by an average 75 T 114%. The
distribution of the capillary RBC velocity changes induced by mild
hypercapnia is shown in Fig. 6. As seen in Fig. 6A, a significant
increase in RBC velocities was observed in capillaries with
diameters from 2.8 to 6.0 Am. However, as shown in Fig. 6B,
relative changes in RBC velocities were more pronounced in the
medium and larger capillaries (resting diameters >4 Am, 107 T
Fig. 3. Transit of a bolus of dye through the vasculature during
normocapnic and hypercapnic conditions. (A) Normalized mean intensity
time courses from 6 animals. The onset (t1/2) of the bolus in each vessel was
computed and referenced to the leading artery. The vertical lines indicate a
clear reduction in transit time for venules and veins during hypercapnia. (B)
Plot of the mean (top) and individual (bottom) transit times for arterioles,
venules and veins at normocapnia and hypercapnia. There was a significant
reduction in transit times in venules and veins during hypercapnia (*P <
0.05, error bars correspond to 1 standard deviation).
Fig. 5. Typical line scan data for determining RBC velocities and their
reactivity to hypercapnia. (A) MIP showing the location of the capillary
vessels. (B) High-resolution image of a single capillary, indicating the
direction of flow (bottom). (C) Line scans of a small (left, U = 2.4 Am),
medium (middle, U = 5.6 Am) and large (U = 8.0 Am) capillary under
normocapnic and hypercapnic conditions. The horizontal axis represents the
length of the capillary vessel in the direction of blood flow, while time is
shown along the vertical axis. RBCs appear as diagonal dark streaks, with
slopes that indicate their velocities as they traverse the capillaries. Steeper
slopes correspond to slower velocities.
Fig. 6. (A) Mean RBC velocity distribution during normocapnia and
hypercapnia as a function of capillary diameter shows increases in RBC
velocity in all capillaries. (B) Mean RBC velocity percent changes as a
function of the resting capillary diameter (*P < 0.05).
E.B. Hutchinson et al. / NeuroImage 32 (2006) 520–530526
142%, P < 0.003, n = 24) than in smaller capillaries (resting
diameters = 4 Am, 39 T 51%, P < 0.004, n = 22). This result is in
contrast to the changes in capillary diameters of Fig. 4B, indicating
a spatial dissociation between increases in microvascular RBC
velocities and blood volume.
Discussion
In the present study, spatial and temporal patterns of cortical
microcirculatory response to mild hypercapnia were investigated
via two-photon laser-scanning microscopy of rodent somatosen-
sory cortex. Carbon dioxide was chosen to produce robust global,
yet gentle vasodilatation, manifested as an increase in the diameter
of arteries, arterioles, venules and veins, as well as a decrease in
transit time of the fluorescent dextrans from the arterial to venous
side of the vasculature. Complimentary to most previous studies of
the reactivity of the cerebral vasculature to CO2, special attention
was given to the capillary vessels (resting diameter <10 Am)
located at least 50 Am and as deep as 200 Am below the pial
surface. Histograms of capillary diameters were obtained during
normocapnia and following CO2-induced vasodilatation. RBC
velocity profiles were also obtained during both conditions. Most
notably, the data illustrate a pronounced spatial dissociation
between volume and velocity changes in the cortical microcircu-
latory response to hypercapnia. In the following, the significance
of the present results is discussed in detail.
E.B. Hutchinson et al. / NeuroImage 32 (2006) 520–530 527
Hypercapnic modulation
Carbon dioxide, a very widely studied vasodilator, was used to
produce a robust and rapid rise in global CBF (Poulin et al., 1996).
While acetazolamide – an erythrocyte carbonic anhydrase
inhibitor – has cerebrovascular effects that are very similar to
those of CO2 (Ehrenreich et al., 1961; Vorstrup et al., 1984), CO2
administration has distinct advantages of easy dosage adjustments,
prompt agent removal and low cost. Addition of 2–3% CO2 to the
inspired air produced an average increase of 12.4 T 7.5 mm Hg in
PaCO2. This was a gentle effect, just enough to produce mild yet
robustly detectable changes in CBF. Carbon dioxide readily reacts
with water to form carbonic acid, which dissociates into carbonate,
bicarbonate and hydrogen ions. The slight drop in pH by 0.11 T0.05 mm Hg following the gentle increase in PaCO2 was expected
and is consistent with the mild hypercapnic conditions, under
which the vasodilatory effects of CO2 cannot be completely
disentangled from H+-induced hyperemia. It is important to point
out, however, that the purpose of this study was not to take the
cerebral vasculature to a point of maximal vasodilatation, but only
to instigate robust and detectable changes in CBF, without running
into physiological complications of acute hypercapnia, such as
acidosis, vascular adaptation, etc.
Bolus tracking
In this work, blood plasma was labeled by intravenous
injections of dextran-rhodamine, a fluorescent intravascular mark-
er. This approach enabled visualization of vessels of all sizes (Fig.
1). To distinguish the arterial from the venous size of the
vasculature, images were acquired during the administration of a
bolus of dye (Fig. 2) and transit times were computed (Fig. 3). The
use of bolus tracking techniques is well established for imaging
blood flow both by MRI and PET (Ostergaard et al., 1998;
Calamante et al., 1999). To the best of our knowledge, this is the
first time such a strategy has been employed in conjunction with
two-photon microscopy. The volumetric increases of arteries,
arterioles, venules and veins along with the decrease in the
arterial–venous transit times were clear indication that mild
hypercapnia produced a robust vascular response. However, no
attempts were made at quantification of regional blood flow or
volume as is typically done in other neuroimaging techniques
(Ostergaard et al., 1998; Calamante et al., 1999) since this would
require careful quantification of the length of each vessel. In future
studies, the length and diameters of specific vessels will be
measured and used to quantify blood flow and volume from bolus
tracking data. That approach will be complementary to the RBC
velocity measurements presented here and could be used as an
alternative method to quantify flow in vessels that are too large to
allow velocity measurements of individual RBCs.
Capillary dynamics
In the present study, capillary diameters ranged from 2 to 6.8
Am, in agreement with a recent two-photon microscopy study of
the rat olfactory bulb glomeruli which considered vessels of
diameters below 6 Am as capillaries (Chaigneau et al., 2003). The
observed distribution of capillary sizes also agrees well with
another study (Hudetz et al., 1993). However, the presently
observed microvessels are of considerably smaller caliber than
those studied in a number of earlier experiments (Morii et al.,
1986). In particular, all capillary diameters in an earlier two-photon
microscopy experiment exceeded 5 Am (Kleinfeld et al., 1998).
This discrepancy may well be a result of an anesthesia-induced
arterial PaCO2 elevation in the latter study (no information about
resting arterial pCO2 was given therein). In the present work,
careful steps were undertaken to maintain normal arterial PaCO2
(of about 35 mm Hg) throughout the experiments. Indeed, the high
sensitivity of the resting vessel diameter to PaCO2 was demon-
strated by detection of capillaries with diameters below 2 Amduring hypocapnic conditions (data not shown). Nonetheless, the
use of maximum intensity projections (MIP) for vessel diameter
measurements may have caused a systematic underestimation of
vessel sizes, depending on the signal to noise ratio of the
measurements. In all cases, however, the underestimation of vessel
diameter could not have been any bigger than one pixel width (i.e.,
0.8 Am). To investigate whether any such bias was present, we
performed line scans at higher sensitivity in a subset of subjects but
found no significant differences with respect to the data presented.
The average hypercapnia-elicited change in diameter, taken
across all capillaries, was small but significantly above zero,
consistent with perfused brain studies (Duelli and Kuschinsky,
1993) and in vivo confocal microscopy study of surface vessels
(Villringer et al., 1994). Notably, most – if not all – of the observed
capillary bed dilatation was concentrated in the smallest capillaries
imaged (with vessel diameters �4 Am). There was no significant
change in diameter of medium or larger capillaries (4.4 Am–6.8 Amdiameters). Some of the larger capillaries exhibited vasoconstriction
(Fig. 4B). The width of the distribution of capillary vessel diameters
decreased during hypercapnia as the smallest vessels dilated and the
larger ones did not respond, thus producing amore uniform capillary
bed flow in the hypercapnic condition relative to the normocapnic
condition. This is consistent with previous results derived from
confocal microscopy (Villringer et al., 1994) and also from
microscopical analysis of in-vivo-marked plasma perfusion
(Bereczki et al., 1993; Abounader et al., 1995).
RBC velocities
To determine whether the pattern of capillary dilatation led to a
corresponding pattern in capillary RBC velocity changes, two-
photon microscopy was used in line scan mode to measure RBC
velocities (Fig. 5). The range of RBC velocities measured agrees
quite well with previously reported values as does the large
variation in RBC velocities across individual capillaries (Villringer
et al., 1994; Hudetz, 1997; Kleinfeld et al., 1998; Seylaz et al.,
1999; Pinard et al., 2000; Helmchen et al., 2001; Chaigneau et al.,
2003). The variation in the mean capillary bed RBC velocity is
likely influenced by the number and size of the capillaries
considered in addition to the choice and level of anesthesia (Wei
et al., 1993; Hirase et al., 2004). Alternatively, such variations
could come from spatial and temporal heterogeneities in capillary
flow derived from the network architecture (branching order) or
from random and periodic fluctuations caused by pressure differ-
ences, by arteriolar vasomotion or by plasma skimming (for a
review, see Hudetz, 1997).
Consistent with previous confocal scanning microscopy studies
(Villringer et al., 1994), all vessels were filled with fluorescent
dextrans under normocapnic conditions. There was thus no
evidence for recruitment (i.e., ‘‘all or none’’ opening) of capillaries,
in agreement with earlier studies (Kuschinsky and Paulson, 1992;
E.B. Hutchinson et al. / NeuroImage 32 (2006) 520–530528
Villringer et al., 1994; Seylaz et al., 1999; Pinard et al., 2000).
However, some capillaries were very poorly perfused with RBCs
and some showed periods when RBCs stopped moving (data not
shown). In accordance with earlier work (Villringer et al., 1994;
Hudetz, 1997), these findings support the existence of functional
reserve capillaries and hence allow for functional recruitment of
capillaries under increased flow conditions (Kuschinsky and
Paulson, 1992; Akgoren and Lauritzen, 1999). Moreover, the
variability in flow pattern illustrates the highly dynamic nature of
the microcirculatory system and points out the challenges in
characterizing the microcirculatory network via measurements of
individual capillaries.
Spatial dissociation between microvascular blood volume and
RBC velocities
The changes in capillary RBC velocity induced by hypercapnia
were not coupled to the observed capillary diameter changes. The
overall increase in RBC velocity across all capillaries and a
broadening of the RBC velocity distribution with hypercapnia
found here are consistent with findings of previous studies
(Villringer et al., 1994; Hudetz, 1997). The average RBC velocity
increase of 75% across the capillary bed agrees well with earlier
hypercapnia studies in rats (Villringer et al., 1994; Barfod et al.,
1997). On the other hand, the fact that most of the changes in RBC
velocity occurred in the larger capillaries suggests a spatial
dissociation between capillary volume and RBC velocity changes.
The increase in RBC velocity in the larger capillaries without any
change in volume indicates that the major resistance to flow is
upstream of the capillary, at the level of arteriole or at the pre-
capillary sphincter (Nakai et al., 1981; Jokelainen et al., 1982). The
change in diameter of the smaller capillaries that is accompanied
by a change in RBC velocity suggests that there may be some
resistance to flow at the level of these capillaries. While such
distention may be passive (due to a resistance drop upstream), there
is anatomical evidence – namely the existence of vascular
pericytes on the abluminal surface of the capillary wall or close
to the capillary branching points as well as the presence of actin-
and myosin-like filaments in the cytoplasm of endothelial cells and
pericytes (Owman et al., 1977; Le Beux and Willemot, 1978; Ehler
et al., 1995; Rodriguez-Baeza et al., 1998; Harrison et al., 2002;
Hirase et al., 2004) – that supports active control of the capillary
diameter. Further studies are needed to address this issue.
It is unclear whether the observed dissociation between
increases in RBC velocities and capillary dilatation in response
to mild hypercapnia would also be present during increased
hyperemia induced by neuronal activation. A number of recent
studies have used two-photon microscopy to observe vasodilata-
tion and changes in RBC velocities during somatosensory
stimulation (Kleinfeld et al., 1998) or odor stimulation (Chaigneau
et al., 2003) in rats and during bicuculline-induced focal
epileptiform activity in mice (Hirase et al., 2004). It will be
interesting to investigate the changes in capillary volume and RBC
velocities elicited by somatosensory stimulation, and this is the
subject of ongoing experiments in our laboratory.
Implications for mechanism of CBF regulation
It is tempting to speculate on the relative blood flow increase that
occurs in different capillaries in response to mild hypercapnia. Using
the Central Volume Principle (Stewart, 1894), which relates the flow
F to the volume V and the transit time t, one can write the flow along
a vessel of diameterD and length L as F ¼ Vt¼ AL
t¼ p
4D2v, where
v is the mean velocity over the vessel. Thus, if an increase in flow
resulted from an increase in velocity alone, one would expect a linear
relation between velocity and flow. On the other hand, if flow
increases were solely due to a rise in cross-sectional area of the
vessel, then the flow increases should be a quadratic function of the
vessel diameter changes. Macroscopically, both vasodilatation and
RBC velocity increase take place, so that the exponent of the
empirically attained power law, describing the dependence of flow
changes on volume changes, was reported to be on the range of
2.63–3.45 for hypercapnic perturbations in rodents, rhesus
monkeys and humans (Grubb et al., 1974; Ito et al., 2003; Rostrup
et al., 2005; Jones et al., 2005). In the current study, we observed an
¨107% increase in velocity (and hence in flow) in capillaries larger
than 4 Am without any dilatation (Fig. 6B). On the other hand, the
¨39% increase in velocity observed in capillaries smaller than 4 Am(Fig. 6B) was accompanied by¨25% increase in their diameter (Fig.
4B). Thus, in these capillaries,
FCO2¼ p
4D2
CO2vCO2
¼ p4
1:25D0ð Þ21:39v0 ¼ 2:17p4D2
0v0
��
¼ 2:17F0:
This flow increase is thus similar to the estimated flow change
in the larger capillaries. Such uniformity in the flow change across
the capillaries – despite the heterogeneity in the microvascular
architecture – is indicative of a heterogeneous microcirculatory
CBF regulation and, in particular, heterogeneity in the CO2
reactivity of the microvessels that act to raise the blood-flow-
carrying capacity of the entire capillary network as a whole. The
fact that both diameter and RBC velocity increase in small
capillaries makes them a much more efficient target for microcir-
culatory CBF changes.
Acknowledgments
The authors would like to acknowledge Ruperto Villadiego for
excellent machinery services. This research was supported by the
Intramural Research Program of the NIH, NINDS (Eugene Major
and Henry McFarland, Acting Scientific Directors).
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