Proc. NatI. Acad. Sci. USAVol. 87, pp. 9868-9872, December 1990Biophysics

Brain magnetic resonance imaging with contrast dependent onblood oxygenation

(cerebral blood flow/brain metabolism/oxygenation)

S. OGAWA, T. M. LEE, A. R. KAY, AND D. W. TANKBiophysics Research Department, AT&T Bell Laboratories, Murray Hill, NJ 07974

Communicated by Frank H. Stillinger, September 24, 1990 (received for review August 1, 1990)

ABSTRACT Paramagnetic deoxyhemoglobin in venousblood is a naturally occurring contrast agent for magneticresonance imaging (MRI). By accentuating the effects of thisagent through the use of gradient-echo techniques in highfields, we demonstrate in vivo images of brain microvasculaturewith image contrast reflecting the blood oxygen level. Thisblood oxygenation level-dependent (BOLD) contrast followsblood oxygen changes induced by anesthetics, by insulin-induced hypoglycemia, and by inhaled gas mixtures that altermetabolic demand or blood flow. The results suggest thatBOLD contrast can be used to provide in vivo real-time mapsof blood oxygenation in the brain under normal physiologicalconditions. BOLD contrast adds an additional feature to mag-netic resonance imaging and complements other techniquesthat are attempting to provide positron emission tomography-like measurements related to regional neural activity.

Magnetic resonance imaging (MRI) is a widely acceptedmodality for providing anatomical information. Current re-search (1) involves extending MRI methods to provide in-formation about biological function, in addition to the con-comitant anatomical information. In addition to localizedspectroscopy (2) and chemical shift imaging (3) that areapplicable to many chemical species, MRI of water protonshas been functionally extended to NMR angiography (4),perfusion imaging (5, 6), and perfusion imaging enhanced byexogenous contrast agents (7). Since water is by far thepredominant molecule in tissue, and since its signal domi-nates the information content in proton images, one wouldideally like to exploit changes in the water signal that arisefrom physiological events. Except for cases of water move-ment, such as blood flow, these changes are normally verysmall.

It has previously been demonstrated (8, 9) that the pres-ence of deoxyhemoglobin in blood changes the proton signalfrom water molecules surrounding a blood vessel in gradient-echo MRI, producing blood oxygenation level-dependent(BOLD) contrast. BOLD contrast has its origin in the factthat when normally diamagnetic oxyhemoglobin gives up itsoxygen, the resulting deoxyhemoglobin is paramagnetic. Thepresence of paramagnetic molecules in blood produces adifference in magnetic susceptibility between the blood ves-sel and the surrounding tissue. This susceptibility differenceis "felt" both by the water molecules in the blood and bythose in the surrounding tissue, the effect extending signifi-cantly beyond the vessel wall. This increase in the number ofspins affected by deoxyhemoglobin is a form of amplification.When the susceptibility-induced local field differences existwithin an imaging voxel, there is a resultant distribution ofshifts in water resonance frequencies. In the gradient-echomethod, a phase dispersion of water proton signals is pro-

duced at the echo time. This dispersion reduces the signalintensity and the voxel appears dark in the image. Theseintensity losses, which at high magnetic fields (-4 T) extendsignificantly beyond the boundary of the blood vessel, are thesource of BOLD contrast. This form of contrast is notobserved in spin-echo images. Through simulations (9), wehave shown that vessels as small as 50 ,um in diameter can bedetected in images with a pixel size of 100 pum. We have alsodemonstrated that the size of the susceptibility-induced localfield depends on (i) the concentration of paramagnetic deox-yhemoglobin and (ii) the orientation of the vessel relative tothe main magnetic field (8, 9).

Since BOLD contrast depends on the state of bloodoxygenation, physiological events that change the oxy/deoxyhemoglobin ratio should lend themselves to noninva-sive detection through the accentuation of BOLD contrast ingradient-echo proton images at high magnetic fields. Wereport here that this is indeed the case and demonstratechanges in BOLD-contrast microimages ofbrain produced bychanges in inhaled gas mixture under urethane anesthesia, byinsulin-induced hypoglycemia under diazepam sedation, andby changes in the level of halothane anesthesia. The observedchanges in BOLD contrast correlate with the anticipatedchanges in blood oxygen level produced by altered metabolicload or blood flow (10).

MATERIALS AND METHODSPhysiology and Anesthetics. Sprague-Dawley rats (female,

250 ± 25 g) were anesthetized with the described anestheticsand ventilated without assistance. Animal care and the ad-ministration of anesthetics were in accordance with NationalInstitutes of Health guidelines. For MRI experiments, theanimal was placed in a thermally regulated compartmentwithin the magnet and rectal temperature and electrocardio-gram were monitored. Blood was periodically sampled fromthe femoral artery and blood gas concentrations were mea-sured with a Radiometer model ABL330 gas analyzer. Bloodglucose was estimated using a Boehringer-Mannheim Accu-Check Ilm. To measure the electroencephalogram (EEG)during MRI experiments, small tubular salt bridges (0.8 mmi.d.) were implanted in small holes through the skull that werelocated inside the area encircled by the surface coil used forNMR signal detection. Care was taken not to break the dura.Magnetic Resonance Microimaging. Gradient-echo images

were obtained with a 7-T horizontal magnet at 300 MHz(Spectroscopy Imaging Systems, Fremont, CA). Surfacecoils of 20 to 25-mm diameter were positioned on the surfaceof the cranium and used for both excitation and detection ofsignals. The maximum field gradient used to produce imageswas 4 G/cm (1 G = 0.1 mT). The slice width of the imagesshown was 500 ,4m and the in-plane pixel size was 117 ,um X

Abbreviations: MRI, magnetic resonance imaging; BOLD, bloodoxygenation level-dependent; EEG, electroencephalogram.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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FIG. 1. Effect of blood CO2 level on BOLD contrast. (a) Coronalslice brain image showing BOLD contrast from a rat anesthetizedwith urethane. The gas inspired was 100% 02- (b) The same brain butwith 90% 02/10% CO2 as the gas inspired. BOLD contrast is greatlyreduced. The arrow points to the sagittal sinus, showing increasedsignal intensity, indicative of increased blood oxygenation. Themagnetic field was perpendicular to the slice plane. The imaging timewas 9 min with a repetition time of 0.26 sec and an echo time of 12msec. The pixel size of the images was 117 Am x 117 ,um with a slicethickness of 550 Aum. The scale represents linear dimension in cm.Core body temperature was maintained at 35-36.50C.

117 Am. The echo time ofthe imaging was 12-18 msec and theimage acquisition time was 6-15 min.Measurement of Blood Flow Velocity and Effect of Blood

Oxygenation on Signal Intensity at the Sagittal Sinus. Thevenous blood flow velocity (cm/sec) at the sagittal sinus wasmeasured by the method of presaturation recovery by flow(11). Proton spins within a 1-mm- or 2-mm-thick slice wereselectively presaturated and after a specified delay time a0.5-mm-thick slice at the same location was selected forimage observation by a gradient-echo pulse sequence with12-msec echo time. The major direction of the blood flow inthe sagittal sinus was normal to the slice plane. The venousblood signal recovered with increasing delay time, providinga measure of blood flow velocity. In this presaturation-recovery-by-flow method, the contribution to the flow signalby the venous blood drainage into the sagittal sinus within thepresaturation slice was minimized because of the slow lon-gitudinal relaxation time (T1) recovery of these spins.

In the standard gradient-echo images shown in the figures(no presaturation), the signal intensity of the venous bloodwater at the sagittal sinus depends on the blood oxygenation.This is because the transverse relaxation time (T2) of bloodwater is strongly dependent on blood oxygenation (12). At the7-T field strength used in this study, T2 varies from 50 msec

FIG. 2. Effect of insulin-induced hypoglycemia on BOLD con-trast. (a) After injection ofbovine insulin, BOLD contrast is apparentat blood glucose levels ¢20 mg/dl. (b) Loss of contrast is observedat blood glucose levels

Proc. Nati. Acad. Sci. USA 87 (1990)

perpendicular to the applied magnetic field and having avolume magnetic susceptibility difference of Ax relative tothe surrounding tissue. The frequency shift (o) as a functionof position used in the simulation was then

outside the cylinder: wz/wo = 2,irA(r/a)2[(2cos26 - 1)]

inside the cylinder: wS/w0 = -(2T/3)AX,where r (r, 6) is the position vector relative to the center ofthe cylinder with angle 6 to the magnetic field. The resonancefrequency without the local field is o". The field inside thecylinder was assumed to be homogeneous, but the T2 decayrate of blood water had a quadratic dependence on theoxygenation level (12). The voxel size in the simulation was100 ,um x 100 Atm x 500 ,m and the pixel size in frequencyalong the read gradient was 100 Hz. The maximum frequencyshift at the surface ofthe vessel (2rcoAX) was 200 Hz, similarto that measured in in vitro experiments with deoxygenatedblood (9). The echo time was 10 msec.

RESULTSEffects of Anesthetics and Blood Flow on BOLD Contrast.

Coronal slice images of rat brains with BOLD contrast

demonstrating that image contrast is reduced as blood flow isincreased are shown in Fig. 1. Rats under urethane (850mg/kg i.p. with atropine at 0.04 mg/kg s.c.) anesthesia with100% 02 as the gas inspired had strong BOLD contrast (Fig.la), manifested as numerous dark lines in the vicinity ofvenous blood vessels. In neocortex these vessels lie perpen-dicular to the cortical surface. This BOLD contrast wascompletely lost (Fig. lb) when the gas inhaled was switchedto 10% C02/90% 02. The addition of CO2 to the inhalant gasincreased blood CO2 levels from 50 to 80 mmHg (1 mmHg =133 kPa). In general, increased blood CO2 produces increasedblood flow. This increased blood flow provides a greatersupply of oxygen to the brain and, in the absence of a changein the metabolic load, should increase venous blood oxygen-ation, consistent with our observed loss of BOLD contrastwith change in blood CO2 level. By using a presaturation-recovery-by-flow MRI method, we could directly measurethe increase in blood flow produced in the sagittal sinus. Themeasured flow velocity (cm/sec) increased from 0.4 to 1.7cm/sec. Note that there was a concomitant signal intensityincrease at the sagittal sinus (Fig. lb, arrow) in the gradient-echo image. This increase also indicates a higher level of

'Av~~4#A&FM:~~Y#Mp

200 uV2 sec

FIG. 3. Dependence of BOLD image contrast halothane dose. Brain images of a rat inspiring 0.75% halothane in oxygen (a), 3% halothanein oxygen (b), or 100% N2 (c) are shown. Also shown are EEG measurements at each halothane level shortly before the images were acquired.

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oxygenation in the venous blood, because of the knowndependence of water T2 on blood oxygenation. Thus the lossof BOLD contrast and the high venous blood oxygenationlevel measured at the sagittal sinus were consistent with thelarge increase in blood flow velocity induced by the increasedsystemic CO2.

Insulin-Induced Hypoglycemia Reduces BOLD Contrast. Asshown in Fig. 2, under diazepam sedation (10 mg/kg) andwith 50% 02/50% N2 as the inhalant gas, a rat brain imagedemonstrated a BOLD contrast slightly more pronouncedthan that in Fig. la. (The signal intensity at the sagittal sinuswas less than that in Fig. la, suggesting a lower level of bloodoxygenation under these conditions.) After injection of in-sulin (bovine, 16 units/kg; Sigma), BOLD contrast did notchange while blood glucose remained >20 mg/dl (Fig. 2a).When blood glucose dropped to well below 20 mg/dl, BOLDcontrast was greatly reduced (Fig. 2b). Addition ofglucose ormannose (400 mg/kg i.p.) reversed this change, restoring thecontrast (Fig. 2c).The reduction in BOLD contrast during hypoglycemia was

correlated with a modest increase in blood velocity at thesagittal sinus. Paco2 levels in blood before insulin-inducedhypoglycemia were 35-40 mmHg with blood velocity at thesagittal sinus of 0.4-0.6 cm/sec. When the loss of BOLDcontrast occurred, Paco2 was 30-38 mmHg and blood ve-locity was 0.7-1.1 cm/sec. However, it seems likely that theloss ofBOLD contrast produced by strong hypoglycemia wasnot entirely produced by the increase in blood flow but alsofrom a reduced metabolism, since, at normal glucose levels,modest increases in blood flow produced by other methodsdid not cause a complete elimination of BOLD contrast (datanot shown).BOLD-Contrast Reduction Without Increased Blood Flow.

When the inhalation anesthetic halothane is used, it is pos-sible to vary the state of anesthesia of the animal by varyingthe concentration of halothane and to demonstrate a changein BOLD contrast not produced by changes in blood flowvelocity. We have compared a series of brain images from thesame animal produced under identical imaging but differentdegrees of anesthesia. To obtain an estimate of overallneuronal activity, the EEG signal was measured in situ beforeor after image acquisition from electrodes positioned on thedura near the imaged coronal section. The dose dependenceof the BOLD contrast is shown in Fig. 3, along with thecorresponding EEG signals. With 0.75% halothane in oxygenas the inhalant gas, radiating venous blood vessels wereobserved in the neocortex, demonstrating BOLD contrast(Fig. 3a). Increasing the halothane content to 3% led todepression of the EEG (brief periods of isoelectric signal areevident) and concomitant loss of contrast in the MRI image(Fig. 3b). Finally, to exhibit maximum possible contrast, theanesthetized animal was ventilated with 100%o nitrogen, lead-ing to an isoelectric EEG. The highly deoxygenated blood inthe brain under this condition gave very high BOLD imagecontrast (Fig. 3c). Unlike the urethane experiments describedabove, it is unlikely that the decreased image contrast ob-served with increasing halothane content can be explained asan effect of halothane on cerebral blood flow or volume. Inseparate experiments the venous blood flow velocity wasmeasured at various halothane levels. Flow velocity actuallydecreased from 0.25 cm/sec at 0.75% halothane to 0.12cm/sec at 3% halothane. The decreased BOLD contrast inFig. 3b is therefore probably due to the lowered oxygenconsumption at 3% halothane, where the animal reached astate of low brain activity as judged by the EEG.Image Simulation. As a step toward quantifying expected

changes in BOLD contrast, we have used image simulation toestimate its dependence on blood oxygenation. Image simu-lations were performed for a cylindrical blood vessel perpen-dicular to the main magnetic field, using experimentally

determined values for the magnetic parameters of the bloodand the surrounding tissue. The relation between BOLDcontrast and blood oxygenation that resulted from the sim-ulations is shown in Fig. 4a for a variety of blood vessel sizes.The normalized BOLD contrast, (IO - ])/Io, changed in therange of blood oxygenation between 90% and 50%, theanticipated range of venous blood oxygenation. The apparentwidth of the vessel in the image is also blood oxygenationdependent for those with diameters that exceed the pixel size(9). An anoxic brain image is shown with a correspondingsignal intensity profile in Fig. 4b. An evaluation of thesensitivity of BOLD contrast to the change in the oxygentension in the blood can be made for this image from thesimulation results. If the minimum detectable change in thecontrast/noise ratio (Al/N) is assumed to be 2 in an imagewith a signal/noise ratio (IO/N) of 40, the correspondingchange in the blood oxygenation level (Y) at Y 0.5 in a50-tim size vessel would be 0.15. Taking account of thecooperative oxygen binding to hemoglobin in blood, thischange in Y relates to a change of 20%o in the blood oxygentension. The sensitivity of the method will be reduced forvessels that are not orthogonal to the magnetic field direction.In this regard, the vasculature of the cortex is advantageousas it runs predominantly in the coronal plane.

a 1.0

0.8-

0.60

0.4

0.2

Diameter, ,m

200

100>t 4 M~~~~~~~oo

0.50 0.25Blood oxygenation

FIG. 4. (a) Relationship between BOLD contrast and bloodoxygenation. The image simulation was performed for a blood vesselwith its longitudinal axis perpendicular to the magnetic field and alsoperpendicular to the read-gradient axis in the slice plane. (b) Signalintensity profile along the line shown in the accompanying image ofan anoxic brain (ventilation with 100%o N2).

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In order to use BOLD contrast to quantitatively determineregional venous blood oxygenation, in vivo calibration will benecessary. For example, images obtained at two differentaverage blood oxygenation levels, produced by different 02compositions in the inhalant gas, provide sufficient informa-tion so that local contrast changes in subsequent images ofthe same area can be related to changes in local bloodoxygenation quantitatively.

DISCUSSIONWe have demonstrated that BOLD-contrast MRI of brain cantrack the changes in blood oxygenation expected from anes-thetic inhaled gases and from insulin-induced hypoglycemiathat change the physiological state of the animal. Since thecontrast is entirely dependent on blood oxygenation, it isdetermined by the balance of supply (blood flow) and demand(extraction by tissue) of oxygen. Under normoxic conditionsarterial blood is fully oxygenated and does not contribute toBOLD contrast, while venous blood vessels containing de-oxygenated blood contribute dark lines to the image. Theresults shown here indicate that BOLD contrast can be usedto noninvasively monitor in real time the blood oxygenationlevels of brain areas in response to central nervous systemdrugs that affect basal metabolism or blood flow. AlthoughBOLD-image contrast is enhanced at high magnetic fields,the effect is observed at 4.7 T*, a field strength that is closeto the highest field strength (4 T) presently available forhuman subjects.Many MRI methods are being explored as alternative

approaches to positron emission tomography (PET) in thestudy of regional brain activity, including methods thatmeasure regional blood flow and volume using exogenouscontrast agents (7). PET imaging relies on a family of tracermethods for measuring different physiological quantities in-

cluding blood volume, blood flow, and regional oxygenextraction (13). BOLD contrast adds to a similar, emergingset of functional MRI methodologies that are likely to becomplementary to PET imaging in the study of regional brainactivity. Unlike most tracer methods, BOLD contrast relieson an intrinsic contrast agent and image acquisition can beprecisely synchronized to external stimuli with good timeresolution. Even transient changes in blood oxygenation (14)can, in principle, be measured.The underlying mechanism of BOLD contrast is the ac-

centuation by gradient-echo imaging at high magnetic fieldsof effects produced by magnetic susceptibility variation thatis caused by an endogenous paramagnetic agent. The samemechanism may also be useful in the study of other iron-containing systems, including the spleen, liver, and bonemarrow.

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