Changes in cerebral arterial, tissue and venousoxygenation with evoked neural stimulation:implications for hemoglobin-based functionalneuroimaging

Alberto L Vazquez1, Mitsuhiro Fukuda1, Michelle L Tasker1, Kazuto Masamoto2

and Seong-Gi Kim1,3

1Department of Radiology, University of Pittsburgh, Pittsburgh, Pennsylvania, USA; 2Molecular ImagingCenter, National Institute of Radiological Sciences, Chiba, Japan; 3Department of Neurobiology, University ofPittsburgh, Pittsburgh, Pennsylvania, USA

Little is known regarding the changes in blood oxygen tension (PO2) with changes in brain function.This work aimed to measure the blood PO2 in surface arteries and veins as well as tissue with evokedsomato-sensory stimulation in the anesthetized rat. Electrical stimulation of the forepaw inducedaverage increases in blood flow of 44% as well as increases in the tissue PO2 of 28%. More impor-tantly, increases in PO2 throughout pial arteries (resting diameters = 59 to 129 lm) and pial veins(resting diameters = 62 to 361 lm) were observed. The largest increases in vascular PO2 were obser-ved in the small veins (from 33 to 40 mm Hg) and small arteries (from 78 to 88 mm Hg). The changes inoxygen saturation (SO2) were calculated and the largest increases were observed in small veins(D = + 11%) while its increase in small arteries was small (D = + 4%). The average diameter of arterialvessels was observed to increase by 4 to 6% while that of veins was not observed to change with evokedstimulation. These findings show that the increases in arterial PO2 contribute to the hyper-oxy-genation of tissue and, mostly likely, also to the signal changes in hemoglobin-based functionalimaging methods (e.g. BOLD fMRI).Journal of Cerebral Blood Flow & Metabolism (2010) 30, 428–439; doi:10.1038/jcbfm.2009.213; published online21 October 2009

Keywords: BOLD fMRI; CBF; CMRO2; Oxygen; Hemoglobin; OIS

Introduction

It is well known that the brain requires oxygen tomaintain its normal baseline function; however, therole of oxygen to satisfy changes in neural activity isless clear. A thorough understanding of the dynamicsof oxygen supply and consumption is important notonly because neuronal vitality relies on oxygenavailability, but also because a large number offunctional imaging techniques (e.g. functional mag-netic resonance imaging (fMRI) and optical imagingof intrinsic signal (OIS)) rely on the concomitantchanges in blood oxygenation to image brain func-tion. Physiologically, changes in cerebral blood flow(CBF) and cerebral oxygen consumption (CMRO2)have been measured with evoked neural stimulation

(Kwong et al, 1992; Kim, 1995; Kim et al, 1999; Daviset al, 1998; Mayhew, et al., 2000; Boas et al, 2003;Shulman et al, 2001). Both of these processesmodulate the oxygen concentration in both vesselsand tissue. In the vessels, increases in blood flowdecrease the longitudinal gradient of oxygen alongthe vascular tree, effectively increasing the venousand tissue oxygenation (Davis et al, 1998; Kim et al,1999; Berwick et al, 2005). In the tissue, increases inoxygen consumption decrease the tissue and venousoxygen concentration (Fukuda et al, 2006). Undernormal conditions, both of these processes take placeand the overall tissue oxygenation also increases dueto an enhanced transport of oxygen from vessel totissue at the capillary level (i.e. a larger transmuraloxygen gradient). One might imagine that the func-tional role of blood flow is to maintain a constanttissue oxygen tension, however, the increase intissue oxygenation has been reported to exceed thetissue oxygen consumption by more than a factor oftwo (Weiss et al, 1983; Vazquez et al, 2008). As aresult, an in-depth understanding of the delivery ofoxygen, including the more general mechanism(s)

Received 5 March 2009; revised 20 August 2009; accepted 15September 2009; published online 21 October 2009

Correspondence: Dr AL Vazquez, Department of Radiology,University of Pittsburgh, 3025 E Carson St, McGowan Center BIRCRm 159, Pittsburgh, PA, 15203, USA.E-mail: alv15@pitt.edu

Journal of Cerebral Blood Flow & Metabolism (2010) 30, 428–439& 2010 ISCBFM All rights reserved 0271-678X/10 $32.00

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driving the changes in oxygen transport to neurallyactive brain tissue, has remained elusive. Manyfunctional imaging methods rely on these changesin blood oxygenation to image and also quantifybrain function. Quantitative fMRI studies have reliedon measurements of evoked physiological changeslike CBF and also on the normalization of BOLDsignal changes which have been collectively used tocalculate CMRO2. However, the quantification offunctional changes in blood oxygenation suffers fromassumptions of the oxygen transport properties (e.g.no functional changes in arterial oxygenation), manyof which have not been verified.

Optical methods have long been used for oximetrywhere the oxygen saturation of blood hemoglobin iscalculated by measuring its absorption of light atdifferent wavelengths (Severinghaus, 1993; Boaset al, 2001). However, quantification from spatiallyspecific locations using optical imaging suffers fromseveral setbacks, including the lack of depth selec-tivity which results in unwanted signal contribu-tions from beyond the cortical surface. Thisdrawback has long been advantageously used inoptical imaging studies to gain intra-cortical sensi-tivity from this largely surface-biased technique. As aresult, less complex, single-wavelength optical ima-ging experiments are commonly performed wherethe sensitivity to oxygen saturation is preservedthough not readily quantifiable, similar to BOLDfMRI. To quantify the dynamics of oxygen transportfrom blood to tissue more selective techniques aredesired. Alternatively, Clark-type oxygen sensorshave been classically used to measure oxygentension in tissue and blood vessels (Ances et al,2001; Masamoto et al, 2003; Thompson et al, 2003;Vovenko, 1999; Tsai et al, 2003). Although they onlyrecord a point measurement, their spatial resolutiontypically span tens to a few hundred microns. Inaddition, their temporal resolution has been shownto be sufficient to see transient decreases andincreases in tissue oxygen tension.

In a previous report by our group, the dynamicproperties of oxygen delivery and consumption wereinvestigated using a model of the transport of oxygenfrom blood to tissue along with CBF and tissueoxygen tension (PO2) data (Vazquez et al, 2008). Thechanges in oxygen delivery to tissue were estimatedto be about 2.7�larger than the tissue oxygenconsumption. While this work explored much ofthe dynamics of oxygen transport to tissue, largelyunanswered questions that remain include: (1) Howdoes the longitudinal gradient from arteries to veinschange with brain function? In particular, where dothe changes in oxygenation take place, to whatextent, and, quantitatively, by how much? (2) Howdo the changes in regional cerebral arterial oxygena-tion, if any, contribute to the delivery of oxygen intissue? (3) How are these changes represented indifferent imaging/measurement methods? Theprimary objective of this work was to measure thechanges in cerebral oxygenation of surface arteries,

arterioles, venules, veins and tissue producedby oxygen delivery and oxygen consumption inorder to investigate these questions. Simultaneousmeasurements of the arterial, tissue and venous PO2

were obtained using Clark-type oxygen sensorsduring evoked stimulation of the rat somatosensorycortex and CBF was measured using laser Dopplerflowmetry.

Materials and methods

Animal Preparation

A total of nine male Sprague-Dawley rats (330 to 480 g)were used in this work following an experimental protocolapproved by the University of Pittsburgh InstitutionalAnimal Care and Use Committee. The animals wereinitially anesthetized using isoflurane (5% for induction,2% for surgery), nitrous oxide (50 to 65%) and oxygen (35to 50%) for intubation and placement of catheters in thefemoral artery and femoral vein. The respiration rate andvolume were controlled using a ventilator (TOPO, KentScientific, Torrington, CT). After intubation, the animalswere placed in a stereotaxic frame (Narishige, Tokyo,Japan) and the skull was exposed over the somato-sensoryarea. A well was made using dental acrylic surrounding anarea 5 mm� 7 mm on the left side of the skull, centered3.5 mm lateral and 1.5 mm rostral from Bregma (Hall et al,1974). The skull in this area was then removed using adental drill. The dura matter was resected and the CSFfluid was released around the fourth ventricle area tominimize herniation. The well and the CSF release areaswere then filled with 1.0% agarose gel at body temperature.Two needle electrodes were placed in the right forepawbetween digits 2 and 4 for electrical stimulation. Theanesthesia and breathing mixture were then changed toisoflurane (1.5%), oxygen (B10%) and air (B90%). Rectaltemperature was maintained at 37.0 1C throughout with aDC temperature control module (40-90-8C, FHC Inc.,Bowdoinham, ME). Arterial blood sampling was periodi-cally performed to measure systemic arterial blood oxygentension (PaO2), arterial carbon dioxide tension (PaCO2), pH,hemoglobin concentration ([Hb]) and hematocrit (Hct)using a blood gas analyzer (Stat Profile, Nova MedicalCorp., Waltham, MA). The arterial blood pressure, respira-tion rate, heart rate, rectal temperature, end-tidal CO2

tension and isoflurane level were monitored and recordedusing a polygraph data acquisition software (Acknowledge,Biopac Systems Inc., Goleta, CA).

Experimental Design

The general experimental procedure was similar to ourprevious study (Masamoto et al, 2008). Initially, a shortstimulation experiment was performed to locate theactivation area using optical imaging of intrinsic signal(OIS at 620 nm with bandwidth of 10 nm). At thiswavelength, deoxygenated hemoglobin absorbs about4�more light than oxygenated hemoglobin (Horecker,1943). Then, Clark-type oxygen sensors (Unisense,

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Aarhaus, Denmark) and a needle-type laser Doppler probe(diameter of 450mm and operating wavelength of 780 nm;Perimed, Stockholm,Sweden) were positioned over thetargeted locations to simultaneously measure PO2 and CBF.OIS was also simultaneously recorded in most experi-ments. The PO2 in tissue and blood were measured usingsensors with tip diameters of 30 and 4 mm, respectively.The spatial sensitivity of the oxygen sensors spans at most10� their tip diameter (Fatt, 1976), and these were selectedto sample a relatively large volume of tissue (30 mm probe)and restrict the vascular PO2 measurements to the vascularspace (4mm probes) since the vessels sampled were at least40 mm in diameter. The spatial sensitivity of the LDF probeused spans a volume of 1 mm3. The oxygen sensors werecalibrated before and after each experiment with 0%, 21%and 100% oxygen in saline solution at 37 1C.

Pre-mapping and Probes Placement

To map the activation area, OIS images were obtainedduring 4 secs of electrical forepaw stimulation deliveredusing a pulse generator and isolator (Master 8 and ISO-Flex, A.M.P.I., Jerusalem, Israel). The stimulus consisted of48 pulses with duration of 1.0 ms, amplitude of 1.6 mA andfrequency of 12 Hz (4 secs) repeated every 16 secs for165 secs. The OIS images were obtained using an analogCCD camera mounted on a macroscope (Olympus MVX-10,Olympus Inc., Tokyo, Japan). Images were captured at 30frames per second using a frame grabbing board andcustom software with resolution of 640� 480 pixels andfield-of-view between 4.6� 3.5 and 9.3� 7.0 mm2 depend-ing on the magnification. A differential analysis wasperformed where the average image obtained 1 sec priorto stimulation onset in each trial was subtracted from eachimage in the trial; all trials were then averaged (a samplepre-mapping image series is presented in Figure 5A). Theresulting data were used to calculate the centroid of theactivation area based on the magnitude of the negativechange in the reflected OIS signal (i.e. increase inabsorption) observed 1 to 2 secs after stimulus onset.

To simultaneously record tissue and blood PO2 as well asCBF responses, three oxygen sensors and a LDF probe werecarefully placed in close proximity over the activation area(see Figure 1). A Clark-type oxygen sensor was first placedin the tissue near the centroid of the active region at adepth of 300mm. Often this sensor was placed lateral toboth the targeted arterial and venous vessel locations tomaintain accessibility. Another Clark-type sensor was thenplaced on the superior surface of a selected arterysupplying blood to the active area. Preliminary testing ofthe spatial sensitivity of the probe for intra-luminal vssurface recordings determined that this placement issensitive to the vessel oxygen tension, in agreement withsimilar studies (Vovenko, 1999; Sharan, et al, 2008). Threepositions were sampled along the selected artery: one justprior to its intra-cortical penetrating point (referred to assmall artery, SmArt or SA) and two upstream parentbranches (referred to as medium and large artery, orMedArt and LarArt, or MA and LA, respectively). Thelocation of the probe on the artery was verified visually and

also by the relatively high PO2 reading. Then, anotherClark-type oxygen tension probe was placed on the super-ior surface of a selected vein in close proximity to theselected artery and draining blood from within theactivation area. Similarly, three positions were sampledalong the selected vein: one just after its emerging intra-cortical location (referred to as small vein or SmVen or SV)and two downstream parent branches (referred to asmedium and large vein, or MedVen and LarVen, or MVand LV, respectively). Lastly, the LDF was positioned suchthat it spanned the small artery, tissue and small vein areasas best as possible.

Measurements of the intra-luminal vessel diameters at thesampled locations were made from a surface image acquiredat a wavelength of 570 nm, which is near equally sensitive tooxy- and deoxy-hemoglobin. The vascular distance betweenthe sampled locations was also calculated.

Simultaneous PO2, CBF and OIS measurements

Experiments were performed while simultaneous measure-ments of arterial PO2, tissue PO2, venous PO2 and LDF wererecorded from their selected locations during 20 secs ofelectrical forepaw stimulation. The stimulus consisted of60 pulses (1.0 ms in duration and amplitude of 1.6 mA)delivered at a frequency of 3 Hz every 80 secs for 650 secs.In all animals tested, the location of the tissue PO2 and LDF

Figure 1 Sample diagram of the oxygen sensors (PO2) and laserDoppler flowmeter (LDF) probe placement. The blood vesselpattern depicted in this figure was obtained by tracing an imageof the surface vessels recorded from this subject. For eachexperiment in a subject, the arterial and venous PO2 probes wereplaced on a small pre-penetrating surface artery (SA or SmArt)and a small emerging surface vein (SV or SmVen), respectively,and also in their larger parent vessels; namely, medium artery(MA or MedArt) and medium vein (MV or MedVen), and largeartery (LA or LarArt) and large vein (LV or LarVen). The otherlocations sampled are represented by open circles with linesleading to the appropriate probe that sampled that location. Theinset shows the actual average OIS image from which thecentroid of activity was calculated.

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probes was fixed after placement. In three animals, onlythe small artery and small vein locations were sampled. Inall other animals, small, medium and large vessel locationswere sampled in separate experiments and in pseudo-random order. The PO2 and LDF signals were recorded at 1kHz using the polygraph recording software. In moststudies (8 of 9), OIS was also recorded as described above(see Pre-mapping section).

Data Analysis

All the data were analyzed using Matlab (Natick, MA) andall the population data are reported as mean±s.d., unlessotherwise specified. After recording the LDF and PO2 data,the various trials within each experimental condition wereaveraged. The PO2 data were then converted to absolute PO2

using the calibration curves determined for each animal.The PO2 measurement lag was also corrected (measuredindependently to be 1.0, 0.6 and 0.6 secs to 90% of the finalamplitude for the tissue, arterial and venous oxygen sensorused) by deconvolution with an exponential function. Theresulting data were then low-pass filtered with a 5 Hzrectangular cut-off. The average oxygen saturation fractiontime series were calculated for each sampled vessellocation using the Hill equation with exponent of 2.73and P50 of 38 mm Hg (Gray and Steadman 1964). Thiscalculation assumes that the concentration of oxygendissolved in plasma (measured by the PO2 probe) rapidlyassociates/dissociates with hemoglobin in red blood cells.Indeed, this process has been measured to be rapid, takingaround tens of milliseconds under normal conditions(Popel, 1989; Gibson et al, 1955). The baseline PO2 andLDF values were calculated by averaging the data over5 secs prior to stimulation onset. The activation valueswere calculated by the average of the data between 10 and20 secs after stimulation onset. Student’s t-tests wereperformed to test the significance of the increases in PO2

and LDF with stimulation. The significance of the oxygentension gradient was determined by paired t-tests betweenthe large artery location and all the other locations.

The optical imaging of intrinsic signal data (620 nmlight) were analyzed as follows. A total of eight ROIs wereoutlined, one for each vessel location sampled, positionedon the blood vessel adjacent to the respective probe’ssampling location (for a total of six ROIs), another centeredon the penetration point of the tissue PO2 sensor spanning adiameter of 100mm (excluding the probe), and anothercovering a 500mm diameter region over the centroidlocation. A sample location of the vessel ROIs in onesubject is presented in Figure 5B (bottom-right panel). Intwo OIS studies, the medium and large arteries and veinslocations were not sampled by PO2; nonetheless, the OISROIs were positioned on the parent branches of the vesselssampled. The temporal series from these ROIs weretemporally averaged to a nominal resolution of 10 Hz andbaseline normalized as done above for the PO2 and LDFdata. Lastly, the changes in vessel diameter as a function oftime were calculated for the large artery, medium artery,medium and large vein locations by placing a four-pixelwidth ROI perpendicular to the vessel direction. The image

within the ROI was linearly interpolated and the intensityalong the four-pixel direction was then summed in order toobtain projected intensity profiles. This yielded the intra-luminal vessel profile and its full-width-at-half-minimum(FWHM) was measured. Assuming the vessel is cylindri-cal, the actual diameter corresponds to 15.5% over theFWHM value. This method is illustrated in SupplementaryFigure S1 in the Supplementary Material.

Estimates of the onset time were obtained from the PO2

and OIS time series by measuring the time-to-20%-peakamplitude (t20) and time-to-50%-dip amplitude (t50�dip).Paired t-tests were performed to test for significance. Theaverage temporal latencies between the different PO2 timeseries were investigated by generating scatter plots of theaverage PO2 time series.

Results

Baseline Measurements

The physiological parameters of all the animalstested were maintained within normal physiologicallevels. The mean arterial blood pressure (MABP) wasmeasured to be, on average, 89 mm Hg at the femoralartery, and average blood gas measurements from thesame location yielded a pH of 7.45, PCO2 of37.6 mm Hg, PO2 of 133.3 mm Hg, [Hb] of 11.1 g/dL,and Hct of 33.3% (for further details see Supple-mentary Table S1).

Six vessel locations were targeted for oxygentension measurements (PO2) and their average intra-luminal diameters vessels were: 128.9 mm (largeartery), 84.0 mm (medium artery), 52.7 mm (smallartery), 61.9 mm (small vein), 144.6 mm (medium vein)and 361.0 mm (large vein; see Table 1). The averagevascular path between the sampled large andmedium artery locations and the medium and smallartery locations were measured to be1914.5±1683.2 mm and 783.1±788.0mm, respec-tively (n = 6). The average distance following thevascular tree between the sampled small andmedium vein locations and the sampled mediumand large vein locations were measured to be548.1±223.2 mm and 2645.8±2681.0mm, respec-tively (n = 6). The average distance between thesampled locations of the small artery and the smallvein was 437.4±319.5 mm, while the average dis-tance between the sampled small artery and tissueand the sampled small vein and tissue was969.2±542.9 mm (n = 9). The average oxygen tensionsand oxygen saturations at the sampled locations arereported in Table 1.

Stimulation-induced Po2 and CBF Responses

Forepaw stimulation for 20 secs induced significantchanges in CBF and PO2 in all the locations measured(see Figure 2 and Table 1). On average, increases of43.9% were observed in CBF over the last 10 secs ofthe stimulation period. Similarly, average increases

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in PO2 of 4.3%, 5.4%, 13.5%, 27.7%, 21.2%, 19.2%and 12.9% were observed in the sampled large artery,medium artery, small artery, tissue, small vein,medium vein and large vein locations, respectively.The data showed the largest changes in PO2 in the

tissue, small vein and medium vein locations;however, all of the increases were determined to besignificant based on t-tests with P < 0.05. Among thesampled arterial locations, small arteries showed thelargest increases in PO2 with stimulation. The PO2

Table 1 Vessel diameter, resting and active PO2 values, and calculated resting and active SO2 values at the targeted sampledlocations

Location VesselDiameter

(mm)

Resting PO2

(mm Hg)Active PO2

(mm Hg)PO2 Change

(mm Hg)RestingSO2 %

ActiveSO2 %

SO2 Change %

Femoral artery (n = 9) N/A 133.3±8.7 N/A N/A 96.9±0.6 N/A N/ALarge Artery (n = 6) 128.9±36.3 88.1±20.5 91.9±18.5 3.8±3.1 88.3±9.9 89.8±6.9 1.6±3.1Medium Artery (n = 6) 84.0±37.3 89.2±29.7 94.1±28.4 4.8±2.8 86.8±13.1 88.9±10.6 2.1±2.5Small Artery (n = 9) 52.7±17.6 77.9±24.9 88.4±21.8 10.5±5.9 81.6±10.9 85.9±10.6 4.3±9.6Tissue (n = 9) N/A 38.0±15.1 48.5±14.1 10.5±4.4 N/A N/A N/ASmall Vein (n = 9) 61.9±15.5 33.3±7.2 40.4±8.0 7.1±6.6 40.5±14.5 51.1±14.4 10.6±7.7Medium Vein (n = 6) 144.6±51.7 38.6±9.3 46.1±6.3 7.4±5.4 49.9±16.9 59.6±9.7 9.7±10.4Large Vein (n = 6) 361.0±116.9 36.9±8.0 41.6±9.5 4.8±4.4 46.8±14.4 54.0±14.0 7.2±6.7

N/A, not applicable.

Figure 2 Average baseline-normalized CBF (top-left), arterial oxygen tension (middle-left), venous oxygen tension (bottom-left),tissue oxygen tension (top-right), and calculated arterial oxygen saturation (middle-right) and venous oxygen saturation (bottom-right)responses to somato-sensory stimulation in the seven locations sampled. The particular location and its corresponding resting PO2

and SO2 level are indicated in the figure legend. The stimulation period is indicated in gray in each panel.

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changes closely resembled the changes in CBF,although temporal delays were observed. A transientdrop below baseline with stimulation onset wasobserved in the average tissue PO2 response with anamplitude of �2.1% that peaked 1.0 sec after stimu-lus onset. No detectable transient decreases belowbaseline were observed in the venous PO2 responses.Small post-stimulation undershoots were observedin the small artery and tissue location average timeseries. Since OIS and BOLD fMRI data are sensitiveto the changes in blood oxygen saturation, these werealso calculated from the PO2 data (see Figure 2). Thelargest difference between the measured PO2 timeseries and the calculated SO2 time series is theamplitude change (summarized in Table 1).

Temporally, the t20 of the PO2 time series wasmeasured in all the animals tested (see Figure 3A andSupplementary Table S2). Although there was not asignificant difference between the t20 values of thesmall and medium artery PO2, these are bothsignificantly earlier from the t20 of the small, mediumand large vein PO2 (t-tests with P < 0.05). The t20

values of the small and medium artery were also bothearlier than the t20 of the large artery PO2 withP < 0.10. The t20 values for the small, medium andlarge artery PO2 were not found to be significantlydifferent from the LDF t20. However, since the arterialPO2 changes are closely related to the changes inblood flow, the arterial t20 values suggest that, onaverage, the PO2 increase follows the CBF responseoriginating from the small arterial vasculature andpropagating upstream. These increases in PO2 alsopropagate into the venous vasculature, taking longerto reach the large vein. The scatter plot in Figures 3Band 3C illustrate the temporal differences between

the small artery and small vein PO2 responses. Thesmall artery PO2 leads the increase over the initial50% of the response onset. It is worth noting that thechanges in tissue (and venous) PO2 are slower thanarterial PO2 partly due to the opposite effect of tissueoxygen metabolism on the PO2 level.

Po2 Gradient Along the Arterial-Venous Vasculature

The baseline and active PO2 values were collected forall subjects tested and plotted as a function oflocation in Figure 4. Under baseline conditions, thesteepest gradients were observed on average betweenthe sampled small artery and small vein (as ex-pected), and also between the medium and smallartery locations. The standard deviation of theoxygen tension measurements was not small butmuch of this variability is expected due to variabilityin caliber of the vessels sampled as well as variationsin the physiological condition of the differentanimals tested (including the blood flow level). Asa result, no significant differences were foundbetween the different arterial locations, exceptbetween the arterial and venous locations (pairedt-tests with P < 0.05). Neural stimulation producedsignificant increases in PO2 in all of the sampledlocations (P < 0.05), although the change in the PO2

gradient was largest over the small artery, tissue andsmall vein locations. The corresponding SO2 wascalculated and its gradient along the vasculature wasobserved to be on average shallower within thearteries. Evoked stimulation produced significantincreases in the SO2 of all vein locations (P < 0.05),but not in the arterial locations; the largest increase

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Figure 3 (A) The average t20 measured from the PO2 data is plotted as a function of the sampled location (see Supplementary TableS2; error bars denote the standard error). The average t20 measured from the LDF data was found to be 1.24 secs, suggesting thatthe PO2 increase with function follows from the blood flow response and propagates from the small vasculature to the largervasculature. (B) Scatter plot of small artery and small vein against tissue oxygen tension (x-axis). This plot indicates that the changesin tissue PO2 lagged the changes in the small artery and small vein PO2 over 75% of the response onset. (C) Scatter plot of the smallvein oxygen tension (y-axis) against the small artery oxygen tension (x-axis). It is evident from this plot that the increase in the smallartery PO2 leads that of the small vein PO2 with stimulation onset. Direct proportionality lines have been included for each scatter pair(black line). Since the response offset is longer than the response onset, it contains a larger amount of point markers and can be usedto differentiate these two phases of the response in each plot.

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in SO2 was observed in the small vein locations. Theincrease in the SO2 gradient was significant betweenthe sampled arterial and venous locations, but notwithin the arterial or venous locations sampled.

Stimulation-induced OIS Responses

The ROI-averaged data recorded by OIS (620 nm)showed some differences in its temporal evolutioncompared to the PO2 measurements. The largeartery ROI showed a small but sustained decreasein signal that lasted over the stimulation period (seeFigure 5B, top-left panel). The magnitude of thedecrease initially peaked at 1.8 secs following stimu-lation onset (�0.20%). This observation was evidentin the difference image in large arteries and,

occasionally, also in medium and small arteries (asample image is presented in Figure 5A; although thestimulation parameters were different in this image,the overall features were the same). The medium andsmall artery ROIs also showed similar transientdecreases in signal of �0.18% and �0.21%, respec-tively, that peaked at 1.4 and 1.7 secs after stimula-tion onset, respectively. The signal in these ROIs,however, increased over pre-stimulation baselinelevels after 2.7 secs (Figure 5B, top-left panel). Bothtissue ROIs, one centered over the penetration pointof the tissue PO2 probe and another over the centroidof activation, also showed bi-phasic responses withstimulation onset (Figure 5B, bottom-left panel)which showed minimum peak decreases of �0.13%and �0.14%, respectively, occurring 1.3 secs afterstimulation onset, and also positive peak increases of

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g)Figure 4 Average longitudinal oxygen tension (PO2; A) and oxygen saturation (SO2; B) gradient sampled by oxygen sensors positionedon a large surface artery (LA), medium-size surface artery (MA), small-size surface artery (SA; pre-penetrating), tissue (T; 300 mmdepth), small-size surface vein (SV; post-emerging), medium-size surface vein (MV) and large surface vein (LV) on or adjacent to thesomato-sensory area. The bars indicate the standard error. The average diameters of the vessels sampled are reported in Table 1. Thetwo curves represent the average longitudinal gradient during the rest period (5 secs preceding stimulation; black line) and activatedperiod (between 10 and 20 secs after stimulation onset; blue line). The absolute differences in PO2 and SO2 induced by forepawstimulation are also plotted (red line). The systemic arterial oxygen tension and saturation were 133.3 mm Hg and 96.9%,respectively, measured at the femoral artery.

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+ 0.76% and + 0.50% for each tissue ROI, respec-tively. After stimulation offset, small decreasesbelow baseline also observed in both tissue ROIs.In the veins, the average magnitude of the early dipwas observed to decrease as the venous vessellocation increased (from �0.13% to �0.08% and0% in the small, medium and large vein ROIs,respectively; see Figure 5B; top-right panel); how-ever, its temporal location was about the same overall of the venous ROIs (B1.3 secs after stimulationonset). The OIS signal plateau increased withincreasing venous vessel size (from 0.69% to 1.37%and 2.44% in small, medium and large vein ROIs,respectively). The post-stimulation undershoot wasalso observed in all the venous ROIs and itsamplitude decreased as the venous vessel sizeincreased.

The t50�dip and t20 were also measured for the OISdata, (see Supplementary Table S2 in the Supple-mentary Material). The OIS dip has been hypothe-sized to be indicative of metabolic processes(Vanzetta et al, 2005), although it may also representincreases in blood volume. On average, the t50�dip

propagates from the tissue ROI to the small, mediumand large vein ROIs. Interestingly, the OIS t50�dip

measured from the tissue probe OIS ROI(0.64±0.35 secs) is similar to the t50�dip measured

from the tissue PO2 time series (0.53±0.31 secs;n = 9). All of the artery t50�dip values were found tobe larger than those in the veins, on average; only thelarge artery t50�dip was significantly different to allthe venous t50�dip with P < 0.05. While the OIS dip inthe venous ROIs may be representative of increasesin metabolism, the dip in the arteries is likely due toincreases in blood volume (and blood flow). In fact,there was sufficient contrast in the OIS images totrack the diameter of the targeted medium andlarge arteries and veins (Supplementary Figure S1).Detectable average increases of 4% and 6% wereevident in the large and medium arterial diameter,respectively, while none were detected in the largeand medium venous diameter (see Figure 6).

Discussion

The principal objective of this work was to measurethe changes in arterial, tissue and venous PO2 thattake place with neural stimulation. Stimulation ofthe rat forepaw induced measurable increases in PO2

throughout the vasculature. To our knowledge, this isthe first report of direct measurements of the cerebro-vascular PO2 with brain function. Of particularinterest is that the largest fractional increases in

Figure 5 (A) Sample optical imaging sequence of a preliminary mapping experiment in one subject (see Materials and Methodssection for experimental detail). Each image frame represents the average of 1 sec around the time indicated in each image. Thestimulus was delivered between t = 0 and t = 4 secs, and these images have been marked with a red dot to indicate they arestimulation images. The dark and bright vessels in the t = + 3 frame correspond to the arteries and veins, respectively. An extendedsequence has been included in Figure S2 in the Supplementary Material. (B) Average OIS time series obtained from 8 ROIs: (top-leftpanel) Large artery, medium artery and small artery ROIs; (bottom-left panel) tissue ROI centered around the tissue PO2 probe andtissue ROI centered on the centroid of activity as determined from an OIS pre-mapping experiment; (top-right panel) small vein,medium vein and large vein ROIs. These panels show the optical signal response to somato-sensory stimulation at those locationsnormalized relative to baseline. The stimulation period is indicated in gray in each panel. A sample of the location of the vessel ROIsis illustrated in the bottom-right panel.

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vascular PO2 were measured in the small vein and thesmall artery. Changes in SO2 showed the largestincreases in small veins while the increase in smallarteries was small. Temporally, the increases in thevascular PO2 were first observed in the small/mediumartery followed by the small vein. Not surprisingly,the most latent increase in PO2 was observed in thelarge vein. The ensuing discussion shows thefollowing: (1) These findings are in agreement withthe notion that BOLD fMRI is largely sensitive to thechanges in venous oxygenation, and (2) the increasesin arterial oxygen tension are significant, contributeto the hyper-oxygenation of tissue and, mostly likely,also to the BOLD fMRI signal.

The PO2 measurements of the large artery locationcorresponded to the largest middle cerebral arterybranch feeding the forepaw area. Similarly, the PO2

measurements of the large vein location corre-sponded to the largest vein draining blood from theforepaw area, typically the middle cerebral vein. Thebaseline PO2 gradient measured between these twophysiological end-points showed a noticeable stee-pening of the PO2 gradient from the medium arterylocation to the small vein location. Similar findingswere reported by Vovenko for his measurements ofthe baseline PO2 in cerebral vessels (Vovenko, 1999).A comparison of the absolute PO2 values show largerbaseline values for this work probably due to thesupplementary amount of oxygen added to thebreathing mixture (B10% O2). Nonetheless, ourresults are in good agreement with the averageoxygen gradient measured by Vovenko relative tothe largest artery measured. In this work, a relativelylarge amount of variability in the PO2 measurementswas observed (and expected) across animals. This

variability is due to the different vessel sizes targetedand to differences in the physiological condition ofthe animals during the experiment. Notwithstand-ing, the results obtained in each subject tested andthe targeted blood vessels were very consistent andreproducible. The vascular PO2 measurements weresampled from the surface of the vessel, not the intra-luminal space, and the vessel wall thickness isknown to vary with vessel diameter (i.e. ratios of25% and 10% and are commonly accepted forarteries and veins, respectively) (Burton, 1954).Hence, the real average intra-luminal PO2 is likelyto be higher, especially in arteries because of theirthicker vessel wall. The PO2 gradient in the radialdirection around the vessel wall has been measuredto be as high as 1.2 mm Hg/mm (Sharan, et al, 2008).This source of variability was minimized by theselection criteria of the tip diameter of the vascularPO2 probe (4 mm) to ensure its sensitivity of the intra-luminal space. While the vessel wall thickness of theveins is smaller, the venous PO2 gradient was alsosomewhat noisy. This was observed during theexperiment and repositioning the probe up or downthe same vessel was not observed to change themeasured PO2 level much. Vovenko also reportedsome variability in the venous PO2 gradient. It ispossible that this could be due to variations in thePO2 within the veins, especially near branchingpoints. In several experiments, the position of theprobes was not ideal, mostly because the centroid ofthe forepaw area in the somato-sensory cortex wasfrequently near the large artery and/or the large veins(these were excluded from the centroid calculation;for example, see darkening in Figure 5A). However,the final placement of the small artery, small vein

Figure 6 Estimated average changes in the large artery (top-left panel), medium artery (top-right), medium vein (bottom-left) andlarge vein (bottom-right) vessel diameter calculated from the OIS data (620 nm; n = 4). The image contrast was not sufficient tocalculate the vessel diameter in the small artery and small vein locations in all the subjects tested, and in the large and mediumartery locations in four of the subjects tested. In fact, the large amount of noise in the medium artery location reflects the decrease inthe image contrast at this location.

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and tissue PO2 probes was always verified to be wellwithin the forepaw area based on the preliminaryoptical imaging map.

Evoked brain stimulation was observed to increasethe PO2 throughout the targeted vasculature, indicat-ing a large span for the changes in CBF, including thelarge artery (a prominent branch of the middlecerebral artery). Another interesting finding was thatthe average increase in the small artery PO2 was veryclose to that obtained in tissue. This indicates adominant role for this size/type arterial vessel inblood flow regulation since the change in arterial PO2

is dominated by changes in CBF. A recent report fromour group concluded that increases in the capillaryPO2 supplied by increases in the upstream arterialoxygenation were necessary to explain the observedfunctional increases in tissue PO2 (Vazquez et al,2008). The results obtained in this work are in goodagreement with those findings. The largest increasein SO2 was calculated to take place in small surfaceveins with progressively smaller changes in thelarger venous vasculature, most likely due to thedilution of the oxygenation changes draining fromnon-activated cortical areas. In general, the resultsobtained are in agreement with optical imagingresults in the literature that show increases in Hbtand HbO2 in pre-penetrating arterial branches(Berwick et al, 2005; Hillman et al, 2007). The quan-tification of the absolute PO2 changes at the sampledvascular and tissue locations have provided sub-stance to these findings in light of the non-linearrelationship between SO2 and PO2. Temporally, theresults obtained suggest that the blood flow-drivenincreases in PO2 start at small and medium arteriesand propagate to the venous and larger arterialvasculature within 1.5 secs. These temporal changesare in agreement with optical imaging studies in theliterature (Vanzetta et al, 2005; Berwick et al, 2005;Hillman et al, 2007; Iadecola et al, 1997) wherearterial and venous signals were extracted to in-vestigate the temporal propagation of the vascularresponse. In those studies the vascular responseoriginated in arterioles and spread to capillaries andvenules; in addition, the magnitude of arterial res-ponse was smaller in larger caliber arterial vessels.Both of these trends are also evident in our results.Also in line with those studies was the observationthat the metabolic response onset (represented byt50�dip) preceded the blood flow response.

Optical imaging of intrinsic signal was alsoacquired in this study to generalize our findings tothe larger vascular network supplying oxygen to thesomato-sensory cortex. The temporal changes aver-aged over ROIs placed near the PO2 recording sitesshow two distinct features. First, the arterial OISchanges are of opposite polarity to the arterial PO2

changes. Second, while the magnitude of the venousPO2 increase was observed to decrease in larger veins,the average venous OIS signal was observed toincrease in larger veins. These features are evidentin the OIS difference images in Figure 5A. These two

features can be explained as follows. The absorptionof 620 nm light is dominated by deoxy-hemoglobincontent and, therefore, decreases in OIS signal implyincreases in deoxy-hemoglobin. In this fashion,although the arterial PO2 was observed to increase,a larger increase in arterial volume would reflect adecrease in OIS signal. The vessel diameter near thelarge and medium arterial locations was found toincrease (Figure 6), indicating that the decreases inarterial OIS signal are the result of dominantincreases in the arterial blood volume. In additionto optical imaging, there is a growing literaturedemonstrating significant arterial blood volumeincreases using MRI (Lee et al, 2001; Kim et al,2007). The larger increase in the OIS signal of largeveins is likely due to the increase in SO2 in thesevessels and also to the larger volume of the veins.Notwithstanding, the OIS difference images inFigure 5A show that the changes observed in arteriesand veins are fairly homogenous and that the PO2

findings can be generalizable across arteries andveins supplying the somato-sensory cortex.

Similar to the PO2 data, the temporal evolution ofthe OIS dip in the arterial ROIs shows a slightaverage progression of the blood volume (andblood flow) response that also originates in thesmaller arterial vasculature (t50�dip = 0.64 secs) andpropagates to the larger arterial vasculature(t50�dip = 0.80 secs; see Supplementary Table S2).However, the dip response observed in the venousOIS data was not evident in the venous PO2 data. It ispossible that this discrepancy is due to a lack insensitivity since small PO2 dips were present in thesmall vein location of two subjects and the mediumvein location of one subject (the larger diameter ofthe tissue PO2 probe provides better sensitivitycompared to the smaller probes used to measurethe vascular PO2). Considering dip measurements(t50�dip) from these PO2 data subsets, the overallobservation of the dip propagating from tissue tothe larger venous vasculature is maintained.

The results obtained here have several implica-tions for the quantification of BOLD fMRI signals;most notably, the measured baseline arterial PO2 inthe small arterial location shows that the intra-cortical arterial oxygen saturation in rodents is lessthan the assumed 100%, indicating that intra-corticalarteries likely contribute to the BOLD signal. Theextent of this contribution based solely on our PO2

measurements is confounded by PO2 contributionsfrom the vessel wall which would underestimate theintra-luminal arterial PO2 and SO2. Nonetheless, if theintra-cortical arterial oxygen saturation is less than100%, then the increases in arterial PO2 and arterialblood volume may also be significant enough toimpact the BOLD signal change. While it is possiblethat the net arterial oxygenation (deoxy-hemoglobin)may be negligible since the increases in PO2 maybalance the increases in blood volume, a bloodoxygenation effect was consistently observed in theOIS data. Therefore, the calculation of CMRO2 from

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BOLD fMRI data may require accounting for thebaseline arterial oxygen saturation, increases in thearterial oxygen saturation and increases in thearterial blood volume (usually assumed to notcontribute to the overall BOLD signal; not tomention, negligible increases in venous volume).Current models are still suitable to quantify theaverage CMRO2 change over large ROIs where theinput arterial oxygenation is closer to the systemicarterial SO2 and the increases in arterial SO2 are verysmall. However, these assumptions appear to breakdown over smaller intra-cortical brain regions esti-mated to be on order of 1 mm. An in-depthinvestigation of the impact of these results on thequantification of oxygen metabolism from hemoglo-bin-sensitive data (e.g. fMRI and OIS) is currentlyunder way. It is worth mentioning that the oxygensaturation results reported in this work (see Table 1)are for rodent hemoglobin; the oxygen saturation ofhuman hemoglobin will be higher assuming thesame PO2 values. For example, the SO2 of the largeand small arterial locations would be 95.6% (vs88.3% in Table 1) and 89.2% (vs 81.6%) using a P50of 26 mm Hg for human hemoglobin. Therefore, caremuse be taken if these results are extrapolated tohuman studies. It is also worth mentioning that onefundamental difference between the OIS signalchanges measured in this work and that of BOLDfMRI is that deoxy-hemoglobin-sensitive OIS (intra-vascular light absorbance) corresponds closely withthe extra-vascular BOLD fMRI signal. That is, thechange in the amount of hemoglobin in a bloodvessel is proportional to the amount of light absorbedby that vessel in OIS (e.g. 620 nm) and also to thechange in the extra-vascular BOLD signal. On theother hand, the intra-vascular BOLD fMRI signal willdepend on the blood oxygen saturation, not theamount, since the magnetic field differences experi-enced by intra-vascular water depend on the deoxy-hemoglobin concentration.

Acknowledgements

Sources of funding: This work was supported by NIHgrants F32-NS056682 and RO1-EB003375.

Conflict of interest

The authors declare no conflict of interest.

References

Ances BM, Buerk DG, Greenberg JH, Detre JA (2001)Temporal dynamics of the partial pressure of braintissue oxygen during functional forepaw stimulation inrats. Neurosci Lett 306:106–10

Berwick J, Johnston D, Jones M, Martindale J, Redgrave P,McLoughlin N et al. (2005) Neurovascular coupling

investigated with two-dimensional optical imagingspectroscopy in rat whisker barrel cortex. Eur J Neurosci22:1655–66

Burton AC (1954) Relation of structure to function ofthe tissues of the wall of blood vessels. Physiol Rev34:619–42

Boas DA, Strangman G, Culver JP, Hoge RD, Jasdzewski G,Poldrack RA et al. (2003) Can the cerebral metabolic rateof oxygen be estimated with near-infrared spectroscopy?Phys Med Biol 48:2405–18

Boas DA, Gaudette T, Strangman G, Cheng X, Marota JJ,Mandeville JB (2001) The accuracy of near infraredspectroscopy and imaging during focal changes incerebral hemodynamics. Neuroimage 13:76–90

Davis TL, Kwong KK, Weisskoff RM, Rosen BR (1998)Calibrated functional MRI: mapping the dynamics ofoxidative metabolism. Proc Natl Acad Sci USA95:1834–9

Fatt I (1976) The polarographic oxygen sensor: Its theory ofoperation and its application in biology, medicine, andtechnology. CRC Press

Fukuda M, Wang P, Moon CH, Tanifuji M, Kim SG (2006)Spatial specificity of the enhanced dip inherentlyinduced by prolonged oxygen consumption in cat visualcortex: implication for columnar resolution functionalMRI. Neuroimage 30:70–87

Gray LH, Steadman JM. (1964) Determination of theoxyhaemoglobin dissociation curves for mouse and ratblood. J Physiol 175:161–71

Gibson QH, Kreuzer F, Meda E, Roughton FJ (1955) Thekinetics of human haemoglobin in solution and in thered cell at 37 degrees C. J Physiol 129:65–89

Hall RD, Lindholm EP (1974) Organization of motor andsomatosensory neocortex in the albino rat. Brain Res66:23–8

Hillman EM, Devor A, Bouchard MB, Dunn AK,Krauss GW, Skoch J et al. (2007) Depth-resolved opticalimaging and microscopy of vascular compartmentdynamics during somatosensory stimulation. Neuro-image 35:89–104

Horecker BL (1943) The absorption spectra of hemoglobinand its derivatives in the visible and near infra-redregions. J Biol Chem 148:173–83

Iadecola C, Yang G, Ebner TJ, Chen G (1997) Local andpropagated vascular responses evoked by focal synapticactivity in cerebellar cortex. J Neurophysiol 78:651–9

Kim SG (1995) Quantification of relative cerebral bloodflow change by flow-sensitive alternating inversionrecovery (FAIR) technique: application to functionalmapping. Magn Reson Med 34:293–301

Kim SG, Rostrup E, Larsson HB, Ogawa S, Paulson OB (1999)Determination of relative CMRO2 from CBF and BOLDchanges: significant increase of oxygen consumption rateduring visual stimulation. Magn Reson Med 41:1152–61

Kim T, Hendrich KS, Masamoto K, Kim SG (2007) Arterialversus total blood volume changes during neural activity-induced cerebral blood flow change: implication for BOLDfMRI. J Cereb Blood Flow Metab 27:1235–1247

Kwong KK, Belliveau JW, Chesler DA, Goldberg IE,Weisskoff RM, Poncelet BP et al. (1992) Dynamicmagnetic resonance imaging of human brain activityduring primary sensory stimulation. Proc Natl Acad SciUSA 89:5675–9

Lee SP, Duong TQ, Yang G, Iadecola C, Kim SG (2001)Relative changes of cerebral arterial and venous bloodvolumes during increased cerebral blood flow: implica-tions for BOLD fMRI. Magn Reson Med 45:791–800

Vascular PO2 Changes with Neural StimulationAL Vazquez et al

438

Journal of Cerebral Blood Flow & Metabolism (2010) 30, 428–439

Masamoto K, Omura T, Takizawa N, Kobayashi H, Katura T,Maki A et al (2003) Biphasic changes in tissue partialpressure of oxygen closely related to localized neuralactivity in guinea pig auditory cortex. J Cereb Blood FlowMetab 23:1075–84

Masamoto K, Vazquez A, Wang P, Kim SG (2008) Trial-by-trial relationship between neural activity, oxygen con-sumption, and blood flow responses. Neuroimage40:442–50

Mayhew J, Johnston D, Berwick J, Jones M, Coffey P, Zheng Y(2000) Spectroscopic analysis of neural activity in brain:increased oxygen consumption following activation ofbarrel cortex. Neuroimage 12:664–75

Popel AS (1989) Theory of oxygen transport to tissue. CritRev Biomed Eng 17:257–321

Severinghaus JW (1993) History and recent developmentsin pulse oximetry. Scand J Clin Lab Invest Suppl214:105–11

Sharan M, Vovenko EP, Vadapalli A, Popel AS, Pittman RN(2008) Experimental and theoretical studies of oxygengradients in rat pial microvessels. J Cereb Blood FlowMetab 28:1597–604

Shulman RG, Hyder F, Rothman DL (2001) Lactate effluxand the neuroenergetic basis of brain function. NMRBiomed 14:389–96

Thompson JK, Peterson MR, Freeman RD. (2003) Single-neuron activity and tissue oxygenation in the cerebralcortex. Science 299:1070–2

Tsai AG, Johnson PC, Intaglietta M (2003) Oxygen gradientsin the microcirculation. Physiol Rev 83:933–63

Vanzetta I, Hildesheim R, Grinvald A (2005) Compartment-resolved imaging of activity-dependent dynamics of cor-tical blood volume and oximetry. J Neurosci 25:2233–44

Vazquez AL, Masamoto K, Kim SG (2008) Dynamics ofoxygen delivery and consumption during evoked neuralstimulation using a compartment model and CBF andtissue P(O2) measurements. Neuroimage 42:49–59

Vovenko E (1999) Distribution of oxygen tension on thesurface of arterioles, capillaries and venules of braincortex and in tissue in normoxia: an experimental studyon rats. Pflugers Arch 437:617–23

Weiss HR, Buchweitz E, Sinha AK (1983) Effect of hypoxic-hypocapnia on cerebral regional oxygen consumptionand supply. Microvasc Res 25:194–204

Supplementary Information accompanies the paper on the Journal of Cerebral Blood Flow & Metabolism website (http://www.nature.com/jcbfm)

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Journal of Cerebral Blood Flow & Metabolism (2010) 30, 428–439