Functional reactivity of cerebral capillaries

Functional reactivity of cerebral capillaries

Bojana Stefanovic1*, Elizabeth Hutchinson1, Victoria Yakovleva1, Vincent Schram2,James T Russell2, Leonardo Belluscio1, Alan P Koretsky1 and Afonso C Silva1

1Laboratory of Functional and Molecular Imaging, National Institute of Neurological Disorders and Stroke,Bethesda, Maryland, USA; 2National Institute of Child Health and Human Development, Bethesda,Maryland, USA

The spatiotemporal evolution of cerebral microcirculatory adjustments to functional brainstimulation is the fundamental determinant of the functional specificity of hemodynamicallyweighted neuroimaging signals. Very little data, however, exist on the functional reactivity ofcapillaries, the vessels most proximal to the activated neuronal population. Here, we used two-photon laser scanning microscopy, in combination with intracranial electrophysiology and intravitalvideo microscopy, to explore the changes in cortical hemodynamics, at the level of individualcapillaries, in response to steady-state forepaw stimulation in an anesthetized rodent model.Overall, the microcirculatory response to functional stimulation was characterized by a pronounceddecrease in vascular transit times (20%±8%), a dilatation of the capillary bed (10.9%±1.2%), andsignificant increases in red blood cell speed (33.0%±7.7%) and flux (19.5%±6.2%). Capillariesdilated more than the medium-caliber vessels, indicating a decreased heterogeneity in vesselvolumes and increased blood flow-carrying capacity during neuronal activation relative to baseline.Capillary dilatation accounted for an estimated B18% of the total change in the focal cerebral bloodvolume. In support of a capacity for focal redistribution of microvascular flow and volume,significant, though less frequent, local stimulation-induced decreases in capillary volume anderythrocyte speed and flux also occurred. The present findings provide further evidence of a strongfunctional reactivity of cerebral capillaries and underscore the importance of changes in thecapillary geometry in the hemodynamic response to neuronal activation.Journal of Cerebral Blood Flow & Metabolism (2008) 28, 961–972; doi:10.1038/sj.jcbfm.9600590; published online5 December 2007

Keywords: brain; cerebral blood volume; functional activation; red blood cell flow; two-photon laser scanningmicroscopy

Introduction

On the mesoscopic scale accessible by currentneuroimaging techniques, such as positron emissiontomography, functional magnetic resonance imaging(fMRI), and optical imaging of intrinsic signals, thevascular response to functional stimulation isspatially restricted and temporally locked to neuro-nal activity under a wide range of conditions(Kuschinsky and Wahl, 1978). Such remarkablecoupling between neuronal and hemodynamicresponses to stimulation enables brain functionmapping via hemodynamically weighted signals.Nonetheless, the mechanism underlying this phe-

nomenon, the range of spatial scales across whichthe neurovascular coupling is preserved, and hencea detailed understanding of fundamental limits onthe spatial, temporal, and amplitude resolution ofpresent neuroimaging methods are still unclear(Villringer and Dirnagl, 1995; Attwell and Iadecola,2002; Lauritzen, 2005; Berwick et al, 2005). Speci-fically, little is known about the functional responseof capillaries—the vessels most proximal to theactivated neurons and the site of dilatory signalgeneration (Iadecola et al, 1997).

The superficial cerebral microvessels have tradi-tionally been imaged using intravital (Hudetz, 1997;Schulte et al, 2003) or confocal (Villringer et al,1994; Seylaz et al, 1999) fluorescence microscopy.Two-photon microscopy has more recently beenemployed to extend the imaged volume hundreds ofmicrons deeper into the highly scattering braintissue, thereby enabling the imaging of vessels closerto the epicenter of neuronal activation (Kleinfeld etal, 1998; Chaigneau et al, 2003; Hillman et al,2007; Chaigneau et al, 2007). The assessment of the

Received 5 October 2007; revised 26 October 2007; accepted 30October 2007; published online 5 December 2007

*Correspondence: Dr B Stefanovic, Cerebral MicrocirculationUnit, National Institute of Neurological Disorders and Stroke,National Institutes of Health, 10 Center Drive, Building 10, RoomB1D109, Bethesda, MD 20892-1065, USA.E-mail: [email protected]

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capillary response to functional stimulation in theseinvestigations has been restricted to line scanning-derived measurements of RBC flux and velocity.Although line scanning necessarily results in under-sampling of the capillary network, statisticallysignificant changes in RBC flux and velocity insome capillaries have invariably been observed.

On the other hand, stimulation-induced capillaryvolume changes have not been examined, althoughcapillary dilatation in response to hypercapnia hasbeen reported (Atkinson et al, 1990; Duelli andKuschinsky, 1993; Villringer et al, 1994; Hutchinsonet al, 2006). In these studies, the capillary caliberchanges have been assessed via changes in theapparent distance between the vessel boundaries ineither single slice or maximum intensity projectionimages at a single point along the vessel underbaseline and hypercapnic conditions. However,these estimates are clearly confounded by the vesselarchitecture and the geometry of the evoked volumechanges. Indeed, morphological data suggest pro-nounced heterogeneity of vessel diameter changesalong the longitudinal microvessel axis, evidencedby the discontinuous perivascular strips formed bysmooth muscles on small arterioles, the dependenceof the perivascular strip distribution and size onvessel caliber, and the discontinuities in the pericy-tic processes along the capillary walls (Rodriguez-Baeza et al, 1998; Peppiatt et al, 2006). Furthermore,of particular importance for studies of functionalreactivity of the microvasculature, stimulation-in-duced changes in capillary diameter have exhibitedstrong heterogeneity along the vessel length in situ(Peppiatt et al, 2006).

In the present work, we sought to investigate themicrovascular response to functional brain stimula-tion in further detail. Given the aforementionedwork and the recent evidence for stimulation-induced capillary flow regulation (Peppiatt et al,2006), we postulated that cerebral capillaries exhibita strong response to functional stimulation in vivo.Our prior study of capillary reactivity to a CO2

challenge indicated a large heterogeneity in capil-lary perfusion that was decreased during mildhypercapnia, with smaller capillaries dilating morethan larger capillaries (Hutchinson et al, 2006).Here, we hypothesized that neuronal activation,likewise, reduces the resting heterogeneity in micro-vascular calibers. To allow the interpretation of thecurrent data in the broader context of neuroimagingstudies, we used an electrophysiologically well-described paradigm (Matsuura and Kanno, 2001;Ureshi et al, 2005) that is frequently employed in fMRIand optical imaging (Hyder et al, 1994; Spenger et al,2000; Ances et al, 2001; Sheth et al, 2003) and focusedon steady-state rather than transient responses. Weemployed intracranial electrophysiology and bolustracking for region of interest validation. Furthermore,bolus tracking enabled estimation of the total changein the regional blood volume. We measured thestimulation-induced changes in the volume of

individual vessels, in addition to RBC speed andflux, to extend the characterization of the micro-vascular hemodynamics, allow the assessment ofthe capillary contribution to the total blood volumechange, and overcome the potential limitations ofthe point diameter estimates.

Materials and methods

Animal Preparation

Experiments were conducted on 15 male adult Sprague–Dawley rats, weighing 120 to 250 g. During the surgicalprocedures, the animals were anesthetized with isoflurane(5% for initial induction and 2% for maintenance) inO2-enriched medical air, orally intubated, and mechanicallyventilated for the remainder of the experiments. The rightfemoral artery and the right femoral veins were cannulatedusing PE-50 catheters for blood gas analysis and intrave-nous administration of anesthesia and fluorescent agents,respectively. Stereotaxic surgery was performed to preparea small (B5 mm diameter), closed (1% agarose) cranialwindow over the forelimb representation in the primarysomatosensory cortex (B3.5 mm lateral to bregma). Rectaltemperature was monitored via thermistor rectal probeand maintained at 37.01C±1.01C via a low-voltage DCproportional controller using a resistive heating blanket(CWE Inc., Ardmore, PA, USA). Isoflurane was discon-tinued and anesthesia switched to a-chloralose (80 mg/kginitial bolus, followed by a constant infusion of 26.7 mgper kg per h) for the remainder of the experiment.Pancuronium bromide was administered as a 2 mg/kgbolus (1 mL/kg) every 45 mins to minimize residualmotion. Respiratory parameters, including end-tidal CO2

levels, were monitored via a capnograph (BCI 300Capnocheck, BCI Inc., Waukesha, WI, USA). Blood gaseswere periodically sampled and adjusted, as needed, toensure physiological stability throughout the experiments.Rectal temperature, tidal pressure of ventilation, arterialblood pressure, and heart rate were monitored andrecorded using a BIOPAC MP system (Biopac SystemsInc., Goleta, CA, USA) throughout the experiments: datarecorded when temperature, blood pressure, or heart ratewas outside of the physiological range were excludedfrom the analysis. The functional paradigm comprisedelectrical stimulation of the forepaw in 90 secs off/60 secson/90 secs off blocks, with each 60-sec stimulationinterval made up of 180 0.3-ms, 2-mA pulses, given afrequency of 3 Hz. All measurements were commenced30 secs after the change in the stimulation state to ensuresteady-state conditions.

Electrophysiology

To provide electrophysiological description of the functionalresponse with high sensitivity and specificity but withoutcompromising the local vasculature, we recorded somatosen-sory evoked potentials (SEPs) intracranially. Specifically,three burr holes, 0.3mm in diameter and <1mm in extentbelow the skull, were made via a hand-driven drill, over left

Functional reactivity of cerebral capillariesB Stefanovic et al

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Journal of Cerebral Blood Flow & Metabolism (2008) 28, 961–972

and right forelimb areas of the primary somatosensorycortices (S1FL), ±3.5 mm lateral to bregma, as well as atthe midline of the cerebellum, B10 mm posterior tobregma (for the reference electrode). A 100-mm-diameterplatinum electrode was inserted into each of these holes(tripolar recording electrode, Plastics 1, Roanoke, VA,USA), with the tip of each wire in the epidural space, sothat no damage was made to either dura or the cortex. Theuse of tripolar electrodes allowed the recording ofdifferential voltage between left and right S1FL areas,with the cerebellum as the reference. Using a BIOPACMP150 system (Biopac Systems Inc., Goleta, CA, USA),the SEPs were amplified 20,000-fold, band-pass filteredbetween 0.5 and 500 Hz, digitized at a sampling rate of2.5 kHz, and transferred to a computer.

In each subject, the SEP recording over the latter 30 secsof the 60-sec stimulus on period was averaged, locked tothe time of each 2-mA, 333-ms pulse presentation, to arriveat the mean SEP trace describing the neuronal response toforepaw stimulation at steady state. The standard devia-tion on this average trace was used to gauge the stability ofthe electrophysiological response to stimulation.

Two-Photon Laser Scanning Microscopy

Two-photon microscopy was performed using a BioRadRadiance 2100 MP TPLSM (BioRad Cell Science,Hercules, CA) or Zeiss LSM Meta 510 microscope (CarlZeiss MicroImaging, Thornwood, NY) (Ti:Sapphire fspulsed laser; lex = 805 nm; using 10� 0.3NA or 20�0.5NA water immersion objectives). Emitted fluorescencewas detected with a PMT using a 620/100 nm band-passfilter (BioRad) or 560 nm long-pass filter (Zeiss). Two150 to 300 mL boluses (5 mg/mL in phosphate-bufferedsaline) of rhodamine-labeled dextran (70,000 MW) wereadministered intravenously.

Three types of acquisitions were performed. To measurethe vascular transit time, the bolus passage was tracked byacquiring a time series of a single 1,024� 1,024 mm2

imaging slice, about 150mm below the cortical surface, at225 ms per frame and with a spatial resolution of8.0� 8.0mm. To estimate vessel volume, we obtained astack of high-resolution images: 60 to 70 slices at 1 to 2mmnominal lateral resolution and 1.5 to 3 mm axial step.Finally, to track RBC flow, line scans were acquired alongthe longitudinal axis of selected capillaries at 1.5 to 2 msper line. Each recording was performed during bothbaseline and stimulation (in randomized order) andcommenced after at least 30 secs of a given functionalstate, to ensure steady-state conditions.

Two-Photon Data Analysis

Transit Time Measurements: The bolus data series werelow-pass filtered (with a Gaussian kernel, sx,y = 2dx,y) andcoarsely segmented into extra- and intravascular spaces.Voxel-wise nonlinear least squares optimization (based onthe interior reflective Newton method) was performed inthe intravascular voxels (subsampled by a factor of 3 toreduce the computation time) to fit a standard gamma

variate function, while modeling the baseline drift. Thesignal intensity in each voxel was thus fit to

SðtÞ ¼ Aðt=TTPÞa expð�ðt � TTP=bÞ;with a ¼ TTP2=FWHM2 � 8 log 2; and

b ¼ FWHM2=TTP=8= log 2;

where A is a scaling constant, TTP is the time to peak,FWHM is the full-width at half-maximum, and t is thetime. The time to peak parameter estimate from thisanalysis was normalized to bolus time arrival in theimaged slice so as to produce estimates of vascular transittimes.

Vessel Blood Volume Measurements: The z-stacksunderwent exponential correction for depth intensityattenuation and were low-pass filtered using a 3DGaussian kernel (sx,y,z = 3dx,y,z). Semiautomated segmenta-tion of both baseline and activation stacks was performedbased on the region growing from manually defined seedsin each vessel that was fully enclosed in the acquiredstack under both resting and activation conditions(i.e., vessels for which branches at both ends were clearlyidentified during both baseline and stimulation). Giventhe limited extent of the imaged stack, this criterionnecessarily led to elimination of all macrovessels. Toenable comparisons with literature data and rough vesselcategorization at rest, we also performed single pointdiameter estimates on the segmented vessels. Briefly, thesignal profile along the normal to a local tangent to eachsegmented vessel in the maximum intensity projectionimage was fit to a Gaussian function, and the correspond-ing estimate of full-width at half-maximum was used as ameasure of the vessel diameter.

RBC Velocity Measurements: The line scan data werefiltered using a 5� 5 median filter followed by low-passGaussian filtering. Thereafter, the average RBC speedduring each 150 to 200 ms of line scan data was estimatedaccording to a previously described algorithm (Kleinfeldet al, 1998). In short, the line scan data sections wereincrementally rotated and singular value decompositionperformed on each rotated image until the rotation angle(amax) resulting in the maximum first singular value hadbeen identified. The average RBC speed for the given linescan data was then estimated as

vRBC ¼ X=ðt tan jamaxjÞwhere X is the length of the line scan along the capillary

(typically B30 to 50 mm) and t is the temporal extent of thecurrent window (i.e., 150 to 200 ms).

RBC Labeling

Because line scanning as a way of estimating RBC speedrequires that a single vessel be interrogated at a time, thedata acquisition efficiency is poor, limiting the number ofrepeated measurements that may be made in a givenvessel. To investigate the RBC flow in a network ofcapillaries at a time, we performed standard intravitalfluorescence microscopy of the superficial vasculature


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using ex vivo-labeled erythrocytes. Specifically, B1 mL ofwhole blood was obtained from a donor rat and theerythrocytes were labeled using the PKH67 Green Fluor-escent Cell Linker kit (Sigma-Aldrich, Saint Louis, MO,USA) according to the manufacturer’s instructions,suspended in phosphate-buffered saline, and injectedthe next day. In preliminary in vitro experiments, we haveobserved fairly uniform labeling of more than 90% oferythrocytes, with minimal dye leakage for injectionsperformed at least 12 h after labeling. The fluorescentlylabeled RBC (FRBC) suspension was injected intrave-nously in tracer quantities to produce B4% to 5% labeledcell population.

Intravital Fluorescence Microscopy

A 100-W mercury vapor short-arc lamp was used forexcitation, and a 20� 0.5NA objective was employed toimage the labeled RBC passage through a network ofsuperficial capillaries (within B40 mm of the surface) atboth rest and during forepaw stimulation, employing thesame functional paradigm as described above. The 10242

,

512� 512-mm2 images were acquired at 30 to 60 frames/secusing a CCD camera (DALSA Pantera TF 1M60, DALSA,Waterloo, ON, Canada) mounted on the LSM Meta 510microscope.

For all vessels exhibiting a single file FRBC flow andhence labeled as capillaries, line profiles were drawnperpendicular to the local vessel orientation. From theseprofiles, the fluorescent cell passage times were identifiedand FRBC flux (defined as the number of FRBC passingthrough the capillary per unit time) was estimated duringboth rest and activation. The FRBC flux data weresubsequently binned into 1-sec intervals and temporalvariation in the flux examined during each condition.Finally, the changes in the FRBC flux between the twoconditions were estimated for all capillaries imaged.

Results

We observed no statistically significant changes(P > 0.05) in any of the physiological parametersmonitored with either stimulation or fluorescentdextran administration and found no evidence foran anaphylactoid reaction to the fluorescent dextranadministration in any subject. The somatosensoryevoked potentials were composed of three majorcomponents: an early small positive peak (P1),followed by a strong negative peak (N1), and anotherpositive peak (P2). The SEP recording over the latter30 secs of the 60-sec stimulation period, in thetypical subject, is displayed in Figure 1A. Thecorresponding mean SEP trace, along with thestandard deviation, is shown in Figure 1B. Whilethe signal-to-noise ratio of the raw EEG recordingafter a single 333-ms pulse prevented reliable pulse-wise estimation of peak amplitudes, the SEPrecording clearly indicated a sustained neuronalresponse throughout the stimulation period.

We observed a consistent decrease in the transittime across the imaged vasculature in each subject,validating the location of the cranial window andhence the imaged ROI. Across all subjects, two-wayANOVA revealed a statistically significant effect ofstimulation on transit time when controlling forintersubject variability (P < 0.05). Across subjects,the transit time decreased from 1.6±0.3 secs at restto 1.2±0.2 secs during stimulation. Normalizing thetransit time at activation by the transit time at rest ineach subject, and then averaging across all subjects,the stimulation induced an average change of20%±8% or 0.5±0.1 secs in the transit time. Aframe of the bolus tracking series in a samplesubject, with the corresponding intravascular map,and the gamma variate fit to the representativevoxel’s signal time course are shown in Figures 2Ato 2C. The time-to-peak maps resulting from thevoxel-wise fitting of the bolus passage data to thegamma variate function in this subject are shown

0 5 10 15 20 25 30-500

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Figure 1 SEP recording over the latter 30-s period of the 60-sstimulation interval (containing 90 333-ms pulses) in a typicalsubject (A). The corresponding average SEP trace (B).


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in Figure 3. The map of transit time estimates(i.e., time-to-peak estimates of the bolus passage,normalized to bolus arrival time) in this subject aredisplayed at rest (A) and during activation (B). Thehistograms of transit time estimates across allintravascular voxels in the same subject are shownin Figures 3C and 3D. The across-voxel mean transittime in this subject decreased from 1.34±0.4 secs atrest to 0.78±0.02 secs during activation.

The stimulation condition produced a statisticallysignificant effect (P < 0.05) on the vessel volume ofthe 120 vessels (average diameter = 8.2±0.6 mm)analyzed. On stimulation, the cerebral blood volume(CBV) of these vessels increased by an average9.9%±1.0%. Figure 4 displays the results ofsemiautomatic segmentation of all vessels identi-fied, on close visual inspection, as fully enclosed inthe imaged volume (i.e., found to both originate andterminate within the imaged stack) in a samplesubject at rest. As observed earlier for the hyper-capnic challenge (Hutchinson et al, 2006), thedegree of stimulation-induced dilatation exhibiteda strong dependence on the resting vessel caliber.Following the literature data on the microvascular

diameters (Pawlik et al, 1981), the 120 analyzedvessels were categorized into small vessels or capil-laries, with diameter below 10 mm, and medium-sizevessels, with diameter above 10 mm and below30 mm. Given this ad hoc data segmentation, thesmaller vessels or capillaries (mean d = 4.8±0.2 mm;range 1.5 to 9.9 mm; N = 87) dilated by 10.9%±1.2%on average (P < 0.05), a figure that was significantly(P < 0.05) larger than the average 6.4%±1.0% dila-tion (P < 0.05) of the medium-size vessels (meand = 17.1±1.6 mm; range 10.2 to 28.9 mm; N = 33). Interms of absolute CBV changes, the capillariesdilated by an average (1.4±0.2)� 103 mm3, whereasthe volume of medium-caliber vessels increased, onaverage, by (11.8±2.3)� 103mm3. These findingssuggest a larger functional reactivity of capillarieswhen compared with medium vessels (on thiscaliber range) and a decreased heterogeneity ofvessel calibers at activation with regard to restingcondition. The decreased CBV heterogeneity is alsoindicated by the statistically significant (P < 0.05)stimulation-induced decrease in the coefficient ofvariation of the vessel volume of 3.4%±1.5%.Figure 5 shows the individual vessel CBV changeas a function of the resting vessel diameter (Figure5A) and as a function of the resting vessel volume(Figure 5B). The histograms of relative volumechanges in capillaries and medium-caliber vesselsare displayed in Figures 5C and 5D, respectively.

It is instructive to note that whereas the abovediscussion segments the data using an ad hocliterature-based threshold of 10 mm to define capil-laries, a very similar clustering may also be arrivedat via data-driven segmentation. In particular,performing k-means clustering of the CBV changeas a function of resting CBV data, a robust segrega-tion (with mean silhouette value of 0.94) isachieved, with two clusters having the boundaryroughly at the resting volume of 2� 105 mm3,indicated by the dashed line in Figure 5B. Takingthe resulting two groups as those representing small(capillary) and medium vessels, in turn, results in acapillary DCBV estimate of 10.0%±1.0% and amedium vessel DCBV change of 6.0%±1.3%, inclose concordance with the preceding results.

When controlling for intervessel variability,forepaw stimulation produced a statistically signifi-cant effect on the RBC speed across the imagedcapillaries (P < 0.05, N = 25). The histograms of meanRBC speed during baseline and stimulation areshown in Figures 6A and 6B. Across the entirepopulation, the mean change in the RBC speed was12.3%±7.2%. Of these 25 capillaries, the change inthe RBC speed between baseline and activationacquisitions was individually statistically signifi-cant (P < 0.05) in 16 vessels, with an averageresponse of 16.3%±10.1%: the mean changes inthe RBC speed in the responding capillaries areplotted in Figure 6C. Twelve of these vesselsexhibited an increase in RBC speed on stimulation(33.0%±7.7%) while the remaining four capillaries

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Figure 2 A 1,024� l,024-mm2 frame in the bolus trackingseries of a typical subject at rest (A). The corresponding coarseintravascular map (B). (Note that the absence of smaller vesselsfrom this map does not affect the estimate of the parameter ofinterest, namely the overall shortening in the transit time acrossthe imaged vasculature.) Gamma variate fit (solid curve withstandard error of the fit shown by dashed dotted lines) to thenormalized time-course data (shown as ‘x’) of a sample voxel inthis data set (C).


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showed a decrease in RBC speed (�33.8%±16.8%)during forepaw stimulation relative to their baselinevalues. We observed no significant correlationbetween the stimulation-induced change in theRBC speed and the resting RBC speed (P > 0.9).

The mean fluorescent RBC flux across the super-ficial capillaries visualized in intravital fluores-cence microscopy experiments increased by19.5%±6.2% (P < 0.05) after functional stimulation.The histogram of FRBC flux estimates, normalizedto the total number of FRBCs that passed througheach vessel during the acquisition period, is shownin Figure 7 at rest (A) and during activation (B). Thestimulation-induced changes in the average FRBCflux as a function of average baseline FRBC flux aredisplayed in Figure 7C.

To investigate the temporal variation of the FRBCflux, the standard deviation and the coefficient ofvariation of the 1-sec binned FRBC flux estimateswere plotted against mean flux, following earlierwork (Hirase et al, 2004). There was a positivecorrelation between the standard deviation of FRBCflux and mean FRBC flux under both baseline (rankcorrelation = 0.94, P < 0.05) and activation (rankcorrelation = 0.95, P < 0.05) conditions. The inverseof coefficient of variation and the mean FRBC flux

A B

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Figure 3 Maps of transit time estimates, overlaid on a single frame of the bolus tracking time series in the subject of Figure 2 atbaseline (A) and during activation (B). Note the dramatic decrease in transit time at stimulation (B), relative to baseline (A). Thecorresponding histogram of intravascular voxel-wise transit times at rest (C) and during activation (D). The mean across-voxel transittime for this subject decreased from 1.34±0.04 s at rest to 0.78±0.02 s during activation.

Figure 4 Results of semiautomatic segmentation of thosevessels identified as being strictly included in the imaged stack(i.e., those found to both originate and terminate within theimaged volume) overlaid on the maximum intensity projectionimage (along axial direction) in a sample subject.


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exhibited linear correlation (baseline: r = 0.77,P < 0.05, slope = 0.0040 sec/FRBC, offset = 0.0068; ac-tivation: r = 0.73, P < 0.05, slope = 0.0033 sec/FRBC,offset = 0.0082). Inthe correlation analysis of the1-sec binned FRBC flux and time, we did notobserve time-dependent changes of FRBC flux inany capillary under resting condition (P > 0.05); onlyone capillary showed time-dependent increase inFRBC during the activation period (P < 0.05).

Discussion

The present study investigated spatial and temporalcharacteristics of cortical microcirculatory changesduring steady-state functional stimulation. Two-photon microscopy afforded imaging of individualvessels in the supragranular layers of the primarysomatosensory cortex of anesthetized rats. For thefirst time, vessel volume rather than point measure-ments of the vessel diameter were employed toestimate the stimulation-induced changes in thevessel caliber. Intracranial SEP recordings wereemployed to show a sustained neuronal responsethroughout the stimulation period. Independentvalidation of the region of interest was afforded via

tracking of the fluorescent bolus and subsequentestimation of transit times under baseline andstimulation conditions. Combined, the findingstestify to (1) decreased vascular transit times, (2)increased microvessel blood volume with largerdilatation of the smaller vessels and hence de-creased vascular heterogeneity, and (3) significantincreases in RBC speed and flux with a smallproportion of capillaries exhibiting RBC speed/fluxdecreases, suggestive of focal redistribution ofcapillary flow during stimulation relative to rest.

Forepaw stimulation induced an average decreaseof 0.5±0.1 secs in the transit time across the imagedvasculature, indicating a robust vascular response inthe imaged region to forepaw stimulation. Thetransit time measurement provided independentvalidation of the mesoscopic region of interest. Inaddition, maps of transit times were produced forboth baseline and stimulation periods, providing aview of the spatial distribution of the vasculartransit time shortening. Combining these measure-ments with vessel volume measurements, althoughbeyond the scope of the present study, willallow estimation of stimulation-induced plasmaflow changes in smaller vessels (where it can becontrasted to patterns of RBC flow adjustments

A

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Figure 5 The vessel-wise blood volume change as a function of a point estimate of the resting vessel diameter (A) or vessel bloodvolume (B) (N = 120 vessels; with 14 to 35 vessels/subject). The small vessels (green) are defined as having a resting diameterbelow 10 mm, and medium vessels (red) a resting diameter above 10mm. Histograms of stimulation-induced volume changes ofsmall vessels (N = 87) (C) and medium vessels (N = 33) (D). The mean CBV increase was 10.9%±1.2% in small vessels and6.4%±1.0% in the medium-caliber vessels. Data-driven clustering of the DCBV versus resting CBV data produces an effectiveborder between small and medium vessel clusters at about 2� l05mm3 (dashed line shown in (B)), producing DCBV estimates verysimilar to those quoted above.


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A B

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rest act

Figure 6 The histogram of RBC speed during baseline (A) and during activation (B) across all capillaries. Sample 1.5-s segments ofline scans during rest and activation in a capillary (showing average vRBC increase of 17.0%±3.9% upon activation) are shown in(C). The average stimulation-induced change in the RBC speed as a function of average baseline RBC speed in the responding vessels(D). The mean activation-induced change in RBC speed across all imaged capillaries (N = 25) was 12.3%±7.2% (P < 10�3). Theaverage change observed in the responding capillaries (N = 16) was 16.3% + 10.1%, with 12 capillaries showing increases(33.0%±7.7%) and 4 capillaries exhibiting RBC speed decreases (�33.8%±16.8%) during forepaw stimulation.

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Figure 7 The histogram of normalized fluorescent RBC flux during baseline (A) and during activation (B). The stimulation-inducedchange in the average FRBC flux as a function of average baseline fluorescent RBC flux (C). The mean activation-induced change inFRBC flux was 19.5%±6.2% (P < 10�6).


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measured via line scanning) as well as in largervessels (where the speed and geometry of RBC flowpreclude vRBC measurements via line scanning).

The mean stimulation-induced increase in bloodvolume was achieved by larger dilatation of thesmaller vessels or capillaries (10.9%±1.2% ofcapillaries versus 6.4%±1.0% of medium-calibervessels), suggesting decreased vascular heterogene-ity at activation relative to rest, in agreement withour earlier study of the microcirculatory response tomild hypercapnia (Hutchinson et al, 2006). Whilecapillary caliber changes have not been the focus ofprevious studies of functional activation, capillarydilatation in response to hypercapnia has beenreported by a number of groups, including ourown (Atkinson et al, 1990; Duelli and Kuschinsky,1993; Villringer et al, 1994; Hutchinson et al, 2006).

More than 60% of the studied capillaries showeda statistically significant change in RBC speed withstimulation. The erythrocyte speed increased in themajority of these capillaries (DvRBC = 33.0%±7.7%,N = 12). Stimulation-induced increases in RBCspeed have been reported in prior two-photonmicroscopy studies after brief (1 to 5 secs) vibrissal(DvRBCr20%; 5 of 14 capillaries) and hindlimbstimulation (DvRBCr15%; 3 of 9 capillaries) (Klein-feld et al, 1998) and 2-sec odor stimulation(DvRBC = 23%±10%) in rats (Chaigneau et al, 2003).The larger amplitude changes observed here likelyresulted from the sustained stimulation and steady-state conditions.

In contrast to earlier studies of direct electricalstimulation of the somatosensory cortex (Schulte etal, 2003), we observed no significant correlationbetween basal RBC speed and stimulation-inducedchanges in RBC speed. As the level of hyperemiawas therein found to significantly affect the degreeof DvRBC dependence on vRBC

rest (Schulte et al, 2003),this difference likely arises from the lower level ofhyperemia produced by the present 0.3-ms, 2-mAforepaw stimulation with respect to 3 ms, 3- to 5-mAdirect cortical stimulation used by Schulte et al(2003). The limited set of caliber measurementscollected in combination with line scans precludedan investigation of DvRBC changes on vessel size.

The CCD recordings of fluorescently labeled RBCsafforded an independent measure of erythrocyteflow changes after functional stimulation. The meanfluorescent RBC flux across the superficial capil-laries thus visualized increased by 19.5%±6.2%(P < 0.05) after functional stimulation, well in therange of previous investigations (Schulte et al,2003). (The poor spatial resolution of the images incombination with limited contrast between bloodand vascular wall under epiillumination preventsreliable vessel caliber estimation.) Consistent withvRBC measurements, we observed no significantdependence of RBC flux change on baseline RBCflux.

It is of note that while functional stimulationprincipally resulted in capillary dilatation and RBC

speed and flux increases, we also observed stimula-tion-induced capillary constriction, as well asdecreases in erythrocyte speed and flux, albeit atmuch lower frequency. Indeed, brief (1 sec) forepawstimulation has recently been reported to result invasodilatation followed by vasoconstriction of cor-tical vessels mostly less than 30 mm in diameter,with vasoconstriction dominating the responsedistant from the center of the neuronal activation(Devor et al, 2007). Capillary constriction has alsobeen observed in the cerebral cortex of mice(Fernandez-Klett et al, 2007). A small portion ofthe responding capillaries (3 out of 32) in the studyof rat olfactory bulb glomeruli exhibited odor-induced decreases in RBC speed (Chaigneau et al,2003). Similar observations of a combination ofincreased and decreased erythrocyte speed havebeen reported in capillaries closer to the corticalsurface when applying a low current amplitudecortical stimulation (Schulte et al, 2003). Thecombination of increases and decreases in capillaryvolume, RBC speeds, and RBC flux observedsuggests a focal redistribution of capillary flow/volume after functional stimulation and a capacityfor local flow/volume changes in the absence of netchanges in cerebral blood flow or volume, insupport of earlier findings in the olfactory bulb(Chaigneau et al, 2003).

Given the present data, it is tempting to speculateon the contribution of the capillaries to the totalstimulation-induced CBV changes. Under the cen-tral volume principle, CBV = CBF*TT, so that theB20% shortening in the transit time, in combina-tion with our earlier CASL fMRI measurements of anB50% average increase in flow using the samefunctional paradigm (Stefanovic et al, 2007), yields a20% predicted increase in the total blood volume.This figure is to be compared with the averageB14% total CBV increase measured via MION fMRIin the aforementioned study (Stefanovic et al, 2007)and an B17% total CBV increase predicted by theempirically derived power law dependence ofvolume on flow (Grubb et al, 1974). Assuming thatcapillary compartment comprises 33% of the totalvascular volume (Sharan et al, 1989) and given theB11% dilatation in the capillary volume, the totalCBV at rest is given by

CBV0tot ¼ CBV0

macro þ CBV0micro ð1Þ

where CBV0micro ¼ 0:33CBV0

tot ð2ÞDuring stimulation, in turn, CBV is given by

CBV tot ¼CBVmacro þ CBVmicro

¼wmacro CBV0macro þ 1:11CBV0

micro

¼1:2CBV0tot

ð3Þ

Solving equations for the noncapillary bloodvolume fraction at activation relative to rest, wmacro,


969


we estimate a B24% increase in the non-capillary (i.e.,arterial, arteriolar, venular, and venous) blood volumein response to functional stimulation. At the same time,

DCBVmicro ¼ 0:11CBV0micro ð4Þ

DCBV tot ¼ 0:2CBV0tot ð5Þ

so that combining (2) and (4) and dividing by (5),B18% of the total blood volume increase isoccurring on the level of capillaries (DCBVtot/DCBVmicroB0.18) and the remaining B82% on thelevel of larger vessels.

It is at present not clear whether the observedcapillary dilatation is a passive consequence ofarteriolar dilatation or a result of an active regula-tion of the capillary diameter (for a review of thepossible mechanisms, see Hudetz, 1997). Directinvestigation of this mechanism in vivo poses verystringent requirements on both spatial and temporalresolution. Recently, electrical stimulation- andneurotransmitter release- induced capillary con-striction via pericytes have been shown in bothwhole retina and cerebellar slices, with the localizedconstriction propagating so as to cause constriction ofdistant capillaries (Peppiatt et al, 2006). On themesoscopic scale, functional activation has beenfound to result in the production of a variety ofvasoactive agents by both neurons and astrocytes(Villringer and Dirnagl, 1995; Attwell and Iadecola,2002; Iadecola, 2004; Lauritzen, 2005; Takano et al,2006). In an fMRI study employing the currentfunctional paradigm, we have recently reported thatcyclooxygenase-2 (COX-2) inhibition causes maximalattenuation of the stimulation-induced blood flowresponse in layer IV (Stefanovic et al, 2006), consistentwith COX-2 expression in the axonal terminals ofexcitatory neurons in the capillary-rich layer IV (Wanget al, 2005), which receive direct input from thethalamus (Brecht and Sakmann, 2002). We thuspostulate that COX-2-mediated production of vasodi-latory prostanoids affords a fast, afferent-drivenresponse of the microvasculature (Iadecola et al,1997) to the early increases in neuronal activity(Armstrong-James et al, 1992). Future studies invol-ving a reconstruction of vascular architecture andanalysis of transient changes in hemodynamics acrossthe vascular compartments in relation to the spatio-temporal pattern of neuronal activity are needed torefine our understanding of microvascular hemody-namics and hemodynamic response generation.

On the methodological note, a number of factorsought to be considered when translating the presentfindings to awake, behaving animals. The closedcranial window preparation, while offering a power-ful method for in vivo observation of cerebralmicrocirculation, is at least ‘semiinvasive,’ andleads to progressive degradation of the prepara-tion. The capillary hemodynamics is particularlysensitive to arterial CO2 tension, necessitating closemonitoring of arterial blood gases. Furthermore, the

amount of heating of the capillaries in the course ofline scans (the recordings having the smallest targetvolume and yet the highest scanning rate), whilenondestructive, may affect the hemodynamics ofinterest. Every effort has been made in the course ofthis study to minimize the data acquisition time,rigorously monitor the animal physiology, andensure normal systemic physiological conditionsthroughout the experiments. In addition, closeattention has been paid to the reproducibility ofthe reference responses from a subset of capillariesidentified in each preparation. The currently em-ployed functional paradigm has been optimized,with regard to the hemodynamic response ampli-tude, in extensive fMRI studies by our and othergroups doing functional neuroimaging of anesthe-tized rodents (Silva et al, 1999; Ureshi et al, 2005).The agreement between the nature of the micro-vascular response in the present study and earlierstudies of mild hypercapnia (Atkinson et al, 1990;Duelli and Kuschinsky, 1993; Villringer et al, 1994;Hutchinson et al, 2006) further suggests general-izability of the current findings. In the light of thepreserved neurometabolic and neurovascular cou-pling under a-chloralose (Ueki et al, 1992; Lindaueret al, 1993), this anesthetic is routinely used forbrain function investigations, with a wealth of datashowing strong, focal neuronal, metabolic, andhemodynamic responses under its influence (Hyderet al, 1997; Ureshi et al, 2005; Devor et al, 2007).

In conclusion, the microcirculatory response tofunctional stimulation is characterized by a pro-nounced decrease in vascular transit times; micro-vascular blood volume increase, with largerdilatation of the smaller vessels and thus decreasedheterogeneity of microvessel calibers at stimulationrelative to rest; and increases in both RBC speed andflux. Significant, though less frequent, stimulation-induced decreases in microvessel volume as well aserythrocytic flow suggest a capacity for focal redis-tribution of microcirculatory flow and volumeduring functional stimulation. The present findingstestify to a strong capillary response to functionalstimulation, suggest an B18% contribution of thecapillary compartment to the total CBV increase,and underscore the importance of capillaries in thehemodynamic response to neuronal activation.

Acknowledgements

The authors would like to thank Ruperto Villadiego formachinery services and Sachy Orr-Gonzalez for animalsurgery and preparation. This work was supported bythe Intramural Research Program of NIH, NINDS.

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Functional reactivity of cerebral capillaries