Proc. Natd. Acad. Sci. USAVol. 91, pp. 3388-3392, April 1994Medical Sciences

Three-dimensional spectral-spatial EPR imaging of free radicals inthe heart: A technique for imaging tissue metabolismand oxygenation

(aorta/oximetry)

PERIANNAN KUPPUSAMY, MICHAEL CHZHAN, KAMAL VIJ, MICHAEL SHTEYNBUK, DAVID J. LEFER,ELIANA GIANNELLA, AND JAY L. ZWEIERThe EPR Laboratories and Department of Medicine, The Johns Hopkins University School of Medicine, Francis Scott Key Medical Center, 5501 HopkinsBayview Circle, Baltimore, MD 21224

Communicated by William A. Hagins, December 10, 1993

ABSTRACT It has been hypothesized that free radicalmetabolism and oxygenation in living organs and tissues suchas the heart may vary over the spatially defined tissue struc-ture. In an effort to study these spatially defined differences, wehave developed electron paramagnetic resonance imaging in-strumentation enabling the performance of threedimensionalspectralpatial images of free radicals infufised into the heartand large vessels. Using this instrumentation, high-qualitythree-dimensional spectralpatial images of isolated perfusedrat hearts and rabbit aortas are obtained. In the isolated aorta,it is shown that spatially and spectrally accurate images of thevessel lumen and wall could be obtained in this living vasculartissue. In the isolated rat heart, imaging experiments wereperformed to determine the kinetics of radical clearance atdifferent spatial locations within the heart during myocardialischemla. The kinetic data show the existence of regional andtransmural differences in myocardlal free radical clearance. Itis further demonstrated that EPR imaging can be used tononinvasively measure spatially localized oxygen concentra-tions in the heart. Thus, the technique of spectra-patial EPRimaging is shown to be a powerful tool in providing spatialinformation regarding the free radical distribution, metabo-lism, and tissue oxygenation in living biological organs andtissues.

Electron paramagnetic resonance (EPR) spectroscopy hasbeen widely applied to study free radicals and paramagneticmetal ions in chemical and biological systems. It is the directand definitive technique for measuring and characterizingmolecules with unpaired electron spin. However, EPR stud-ies ofbiological organs or tissues have been hampered by thelossy nature of these samples and the need for speciallydesigned resonators to accommodate these large samples.Over the last decade EPR has been widely applied to measurethe mechanisms offree radical generation in isolated cells andtissues. Recently, instruments have been developed thatenable noninvasive in vivo EPR measurements of free radi-cals in whole animals and functioning whole organs (1-5).These instruments have generally utilized low-frequencymicrowaves of 1-2 GHz or below to enable tissue penetra-tion. In addition, lumped circuit resonator technology hasenabled optimization of sensitivity for in vivo applications.

Free radicals have been suggested to be key mediators ofthe injury that occurs upon reperfusion of the ischemic heart(6, 7). It has also been suggested that ischemia may alter theability of the heart to metabolize reactive oxygen free radi-cals. There are also fundamental questions regarding oxygenmetabolism in the heart and its role in regulating contractile

function and energy metabolism. In addition, there are ques-tions regarding the presence of spatially defined alterations incellular metabolism in the heart and large arterial vessels. Inthe heart, the endocardium, the inner layer of the cardiacchambers, is more sensitive to ischemic injury than theepicardium, and it has been hypothesized that there may betransmural variations in myocardial oxygenation and metab-olism (8, 9). However, there has been no previous techniquethat could determine if these transmural variations exist. Inlarge arterial vessels it has been shown that occlusion with alack of flow results in injury with altered vascular reactivity(8, 9). This injury has been difficult to explain since it occursin the face ofnormal intra-arterial oxygen tensions, and it hasnot been previously possible to measure transmural metab-olism and the transmural concentrations of oxygen in thesevessels. Thus, there has been a great need for a techniquecapable of measuring the spatial distribution of free radicalsand oxygen in organs and tissues such as the heart and largevessels.EPR spectroscopy can be applied to obtain spatial infor-

mation by utilizing magnetic field gradients (10), in a mannersimilar to that of NMR imaging. EPR imaging, however, isfaced with a number of technical problems, which make thistechnique more difficult to achieve in practice than that ofNMR. The linewidths of EPR signals are 3 orders of magni-tude larger than those of NMR signals, and hence EPRimaging requires 100-1000 times more powerful gradients.The paramagnetic centers to be studied are present in sub-millimolar concentrations compared to more than 100 Mconcentrations of water protons utilized in NMR imaging.Since spectral information is required to characterize theproperties and structure offree radicals, as well as to performoximetry measurements, spectral-spatial imaging must beperformed to address the important problems discussedabove.Over the last decade, EPR imaging experiments have been

performed by a number of groups around the world on avariety of samples (10-21). With biological organs and livingsamples, these studies, in general, have provided only low-resolution spatial images or one-dimensional spectral-one-dimensional spatial images offree radicals. Recently methodshave been developed enabling localized EPR spectroscopyby implanting oxygen-sensitive paramagnetic probes (22). Toperform oximetry studies on asymmetric biological samples,such as the heart, using imaging, it is necessary to include aspectral dimension along with two or three spatial dimen-sions. The experiment must be performed and the image must

Abbreviations: 2D, two dimensional; 3D, three dimensional; PC,personal computer; GPIB, general purpose interface bus; TEMPO,2,2,6,6-tetramethylpiperidine-1-oxyl; PDT, 4-oxo-2,2,6,6-tetrameth-ylpiperidine-d16-1-oxyl.

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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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be obtained on a fast time scale so as to capture the temporalvariations in radical metabolism or oxygen consumption. Inaddition, reasonably good submillimeter image resolutionmust be obtained in order to make meaningful interpreta-tions.

In view of the important cardiovascular pathophysiologicquestions that cannot be addressed with other existing tech-niques, we have developed instrumentation enabling theperformance of three-dimensional (3D) spectral-spatial EPRimages of the heart and large vessels. This instrumentation isapplied to obtain images of free radical metabolism andoxygen concentrations in the heart. Assessment of regionaland transmural differences in vascular and myocardial freeradical metabolism and oxygenation are performed.

METHODSL-Band Resonator and Spectrometer. EPR spectra were

recorded using an L-band bridge similar in design to thatdescribed previously (4). A new ceramic three-loop two-gapreentrant resonator design was developed to enable the highsensitivity and high stability required for EPR imaging (23).This resonator design has the advantage over previous single-loop, loop-gap designs (4), in that its thickness is much lessand can be made almost as thin as the sample. This isparticularly important for EPR imaging where two or threesets of powerful water-cooled gradient coils must be accom-modated, in addition to the sample, sample cavity, andmodulation coils. The resonator dimensions were as follows:radius, 10 mm; capacitive plate length, 6 mm; capacitive platewidth, 17 mm; and capacitive gap, 0.23 mm (23). Thisresonator provides a relatively selective slice ofmaximum Hifield. The resonant frequency was 1.319 GHz with an un-loaded Q factor of 2930.EPR measurements were performed using a Bruker signal

channel (ER 023M) and field controller (ER 032M) interfacedto a personal computer (PC). The microwave frequency wasmeasured over the general purpose interface bus (GPIB)using an EIP 975 microwave counter.

Gradient Coils. EPR imaging measurements were per-formed using a modified Varian XL-100 NMR magnet inwhich the pole faces were remachined to a gap of 4.5 inchesand appropriate ring shims were placed to achieve an opti-mized homogeneity of better than 1 part in 105 over a samplevolume of 1 cm3 at the center of the gap.Three sets of water-cooled gradient coil assemblies were

attached to the pole faces of the magnet. A modified Helm-holtz pair was used to generate the gradient along the z axis(the direction ofthe static magnetic field), and two sets ofpaircoils were used to generate the x and y gradients. Each setwas capable of generating sustained field gradients of at least150 G/cm. Each set of coils was independently powered witha Hewlett-Packard power supply (model 6030A), interfacedto the PC via GPIB bus.Data Acquisition and Image Reconstruction. Projection data

acquisition and subsequent image reconstruction were per-formed using a 486-based PC equipped with an IEEE-488GPIB board. Software capable of 3D spectral-spatial dataacquisition, reduction, image reconstruction, image analysis,and simulation was developed. The PC was programmed tocontrol the signal channel, field controller, and the gradientpower supplies (Fig. 1). The software controlled the threegradients, enabling the user to select any arbitrary spatialplane of interest for image acquisition and reconstruction.

Projection data was then collected forN orientations in thespatial-spatial plane and N orientations in the spectral-spatial plane for each spatial-spatial orientation, thus for atotal ofN x N orientations for each image. TheN projectionsin a plane were obtained over 1800 with equal angular

I 38 mm125 mm[]38 mm |GRADIENTS GRADIENTS

FIG. 1. Block diagram of EPR imaging instrument.

increments. For each projection, the magnitude of the gra-dient, G, for a projection angle a, was calculated as [24]

G = (AH/AX) tan(a), [1]

where AH and AX are the values of spectral and spatialwindows defining the pseudo-plane from which data areacquired. The scan range was varied from the base value AHaccording to the relationship [24]:

scan range = AH/cos(a). [2]

The scan time for each projection data collection was keptproportional to the scan range, such that the resultant sweeprate (gauss/second) was constant. Projections involving rel-atively large gradients, and hence longer scan times or largerscan ranges, were measured with increased receiver gain andtime constant, and the data was normalized accordingly forbase value (zero-gradient) settings. The microwave fre-quency was measured at the end of each projection and usedto correct any shift in the projection due to drift in thefrequency. A delay time of 500 msec was given betweenacquisitions to allow stabilization of each new gradient field.Where required, the order of projections was scrambled tominimize heating of the gradient coils.A total of 256 points per projection were collected by

integrative digitization of the analog output from the signalchannel. During data reduction, the 256 points were subsam-pled to 64 points and normalized for constant intensity (24).The data was then integrated, baseline corrected, andsmoothed using a Gaussian filter, all in the Fourier space. Athree-point (second difference) filter was then applied to thedata. A two-stage backprojection algorithm was used toreconstruct the image of the object (25). The first stageproduced N pseudo two-dimensional (2D) spectral-spatialimage slices (N x 64 x 64), while the second stage producedthe 3D spectral-spatial-spatial image (64 x 64 x 64).

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In the case of imaging with 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) or 4-oxo-2,2,6,6-tetramethyl[1-15N]piperi-dine-d16-1-oxyl ([15N]PDT), only the low-field peak was cen-tered in the spectral window. The overall data acquisitiontime was about 6 min for 64 projections and 16 min for 144projections, based on a 2-sec scan time (scan rate, 3 G/sec)for zero-gradient projection. The data reduction and back-projection required about 1-2 min with a 486/50-MHz PC.Thus, the combined acquisition and reconstruction programallowed rapid acquisition and visualization of EPR imagesrequired for efficient performance of physiological experi-ments on the heart.

Isolated Heart and Vessel Perfusion. Isolated rat hearts andrabbit aorta were perfused at 370C in a constant pressuremode with Krebs bicarbonate-buffered perfusate consistingof 117 mM NaCl, 24.6 mM NaHCO3, 5.9 mM KCl, 1.2 mMMgCl2, 2.0 mM CaCl2, and 16.7 mM glucose bubbled with95% 02/5% CO2. The cardioplegic solution used consisted of110 mM NaCl, 10 mM NaHCO3, 16 mM KCl, 16 mM MgCl2,and 1.2 mM CaCl2.

Aortas were obtained from New Zealand White rabbitsweighing 5.5-6.5 kg. The entire length of the thoracic aortawas removed and placed in ice-cold perfusate. A 3.0-cmsegment was then cannulated at both ends and secured ontoa specially designed perfusion stage (Fig. 2). The vesselswere perfused at a constant pressure of 60 mmHg, andproximal and distal perfusion pressures were measured withpressure transducers and regulated by perfusate columns ofset height. Flow was determined by collection of the effluent.Hearts were obtained from female Sprague-Dawley rats

(300-400 g) and perfused retrogradely with a constant pres-sure of80 mmHg by the method of Langendorff. A fluid-filledballoon was placed within the left ventricle and connected viaa hydraulic line td a pressure transducer to enable continuousmeasurement of contractile function (4) (Fig. 2). Spin-labelconcentrations of 0.5 mM or 1.0 mM were used, which weresufficiently low as to produce no alterations in contractilefunction and negligable concentration-dependent broadeningof the EPR signal.

RESULTSImaging of Phantoms. To check the quality of spectral-

spatial data obtained on large lossy aqueous samples, phan-toms were constructed consisting ofeither single or concentrictubes containing free radical solutions of TEMPO or[15N]PDT. 3D spectral spatial imaging of a 9-mm i.d. tubecontaining an aqueous solution of 0.5 mM [15N]PDT wasperformed perpendicular to the longitudinal axis of the tube.As shown in Fig. 3A, a solid uniform disk was obtained. Thespatial dimension measured from the image was in good

PERFUSION CANNULA-

I PERFUSATE DRAIN-

TRANSDUCERCATHETER I

CENTERING RING

FIG. 3. 3D spectral-spatial EPR image of phantoms. (A) 2Dspatial cross-sectional (14 x 14 mm2) EPR intensity image of a 9-mm(i.d.) cylindrical tube containing 0.5 mM aqueous [15N]PDT (projec-tions, 256; spectral window, 8.0 G; spatial window, 14 mm; maxi-mum gradient, 57.7 G/cm). (B) 2D spatial cross-sectional (20 x 20mm2) image of EPR intensity of 1 mM TEMPO ring (14-mm o.d,10-mm i.d.) phantom (projections, 64; spectral window, 6.0 G; spatialwindow, 20 mm; maximum gradient, 24.65 Gauss/cm).

agreement with the sample size. The spectral data ofthe imageshowed a uniform linewidth of 1.5 G (peak-to-peak width ofthe derivative line) over the entire sample. Fig. 3B shows thefree radical image of a ring phantom of a 1 mM aqueousTEMPO solution filled in the annular space between twoconcentric tubes of 14mm (i.d.) and 10mm (o.d.). The spectraldata of the image showed a uniform linewidth of 1.7 G.Imaging of the Aorta. The isolated perfused rabbit aorta

preparation was studied as described above. Control EPRspectra were recorded, and then an infusion of the TEMPOfree radical was started with a concentration of 1.0 mM in theperfusate solution. Serial 10-sec zero-gradient EPR spectrawere obtained to monitor the development of the TEMPOsignal. After about 10 min of infusion, a steady-state magni-tude of the EPR signal intensity was reached. With theperfusion continued, 3D spectral-spatial EPR images wereacquired. A total of 64 projections were acquired in 12 minwith a base projection acquisition time of 4 sec. Fig. 4A showsthe spatial map ofTEMPO signal intensity projected across thelongitudinal axis of the aorta. Subsequently, the aorta wasperfused with perfusate containing 40 mM ferricyanide, aparamagnetic contrast agent. After steady state was reachedwith ferricyanide, the image acquisition was repeated. As canbe seen from Fig. 4B, in the presence offerricyanide, the signalarising from the radical pool within the lumen ofthe vessel wasgreatly attenuated and the vessel wall could be clearly seen.Imaging of the Heart. Hearts were infused with TEMPO for

10 min to reach steady state, and EPR acquisitions of 64 or144 projections were performed every 12 or 16 min, respec-tively. A series of 10 images was obtained from the loadedheart. These images corresponded well with cross-sectional

SUPPORT RODAORTA

HEART AORTA

FIG. 2. Diagram of isolated rat heart and rabbit aorta preparationused for EPR imaging experiment.

FIG. 4. EPR images (8 x 8 mm2) rabbit aorta obtained from a 3Dspectral-spatial data reconstructed from 64 projections. (A) Aortaperfused with 1.0 mM TEMPO. (B) Aorta perfused with 1.0 mMTEMPO and 40 mM ferricyanide. The data acquisition parameterswere as follows: acquisition time, 12 min; spectral window, 7.0 G;spatial window, 8 mm; maximum gradient, 49.3 G/cm.

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FIG. 5. Cross-sectional transverse 2D spatial EPR image (22 X 22mm2) of the rat heart obtained from a 3D spectral-spatial datareconstructed from 144 projections. The intensity data was obtainedby selective integration of spectral slices 20-44. The structure of theleft ventricle (LV) and right ventricle (RV) are observed in the EPRimage to be similar to that of a short axis histologic tissue slice. Thedata acquisition parameters were as follows: acquisition time, 16min; spectral window, 8.0 G; spatial window, 22 mm; maximumgradient, 27.4 G/cm.

short axis sections through the rat heart as would have beenexpected from the orientation of the heart in the center of theresonator. The left ventricular cavity, right ventricular cav-ity, and left ventricular and right ventricular myocardiumwere visualized (Fig. 5). In another heart after loading andacquisition oftwo control images, the heart was cardioplegedand subjected to a global ischemia at 240C. During cardiople-gia, a series of four additional images were obtained followedby two images during reperfusion (Fig. 6). The intensity oftheimages decreased as a function of duration of ischemia as theradical label was metabolized. The label cleared more rapidlyfrom the outer myocardial walls (epicardium) than from theendocardium or from the center of the left ventricle. Theepicardial contour corresponded to the outer rim of theimage. The endocardial contour was defined as the darkcircle at the center of the image and corresponded to the site

of the balloon placed in the left ventricle. The ballooncontained a volume of 0.2 ml and had a diameter of =5 mm,which served to confirm the endocardial location. At fourselected regions, 12, 3, 6, and 9 o'clock positions, the kineticsof radical clearance were measured in the epicardium, mid-myocardium, and endocardium (Fig. 7). The kinetics could bemodeled as first-order exponential processes with rate con-stants of 0.12 min-, 0.056 min-, and 0.03 min', respec-tively. As shown in Fig. 7, a slower rate of radical clearancewas observed at the location of the endocardium and the leftventricular chamber.

Myocardial Oxygenation. Measurements ofmyocardial ox-ygen concentrations were performed from the changes inEPR linewidth, which were observed as a function of theduration of ischemia as was reported previously for simple invivo EPR spectroscopy (4). Examination of the spectral dataof the images of the cardiopleged heart showed a gradualdecrease in the linewidth at each spatial location (Fig. 8).Over a 60-min duration of ischemia, the linewidth decreasedby 0.45 G and approached the linewidth observed in theabsence of oxygen. As reported previously, this would cor-respond to a drop in myocardial oxygen concentration from625 pM to near 0 juM (4). The linewidth data obtained fromthe 3D images corresponded closely to those measured fromsingle scan, zero-gradient EPR spectra.

DISCUSSIONAfter a decade of development and experimentation, thefields ofEPR spectroscopy and EPR imaging have advancedto the point of enabling useful physiological and biochemicalinformation to be obtained from living tissues. The develop-ment of low-frequency EPR instrumentation at L-band, 1-2GHz, as well as lumped circuit resonators have made itpossible to perform high-quality measurements on these largelossy aqueous samples. Previous studies focused on globalmeasurements of free radical metabolism and measurementsof myocardial oxygenation by EPR oximetry techniques (4).

In this manuscript we have reported the development ofinstrumentation suitable for performing 3D spectral-spatialEPR imaging experiments on large biological samples such asthe rat heart. Three-dimensional spectral-spatial imageswere obtained from isolated perfused rabbit aortas and rathearts. In the isolated aorta it was observed that spatially andspectrally accurate images could be obtained. In the presenceofaparamagnetic contrast agent, the vascularlumen could beclearly resolved. In the isolated rat heart, experiments thatimaged the kinetics of radical metabolism during myocardialischemia were performed. It was observed that there were

FIG. 6. Series of 2D cross-sectional (22 x 22 mm2)EPR intensity maps of TEMPO radicals in the heartduring normal perfusion (A, 0 min), global ischemia (B,12-24 min; C, 24-36 min; D, 36-48 min; E, 50-62 min),and reperfusion (F). These images were obtained froma 3D spectral-spatial data reconstructed from 64 pro-jections. Radical clearance of the TEMPO label isobserved during ischemia resulting in a decrease in theEPR signal intensity. On reperfusion with TEMPO, theimage intensity largely returns.

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>- 20001.

z

z 1000

0

3000

LEFT RIGHT

>- 2000

z

z 1000

0 10 20 30 40 50 60 0 10 20 30 40 50 60

TIME (min) TIME (min)

FiG. 7. Graph of the decrease in EPR intensity in

(m), midmyocardium(o), and endocardium (e)

duration of ischemia. The Top, Right, Bottom,

represent 12, 3, 6, and 9 o'clock positions

respectively. The solid line through each data

first-order exponential decay curve corresponding

spatially defined differences in the rate of radical

with the free radical pool present in the area of

dium and left ventricular cavity cleared at a slower

that within the epi- or midmyocardium. From

data, information was obtained regarding the concentration

of oxygen at different spatial locations within

It has been previously demonstrated that the

myocardial infarction progresses as a wave front

non from the endocardial surface to the epicardial

9). It has been clearly shown that the endocardium

susceptible to ischemic injury; however, the

phenomenon is unknown. It has been demonstrated

reactive oxygen free radicals are generated in

reperfused hearts, and it has been suggested that

0.

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0o

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2L

0 10 20 30 40 50 60

TIME (MIN)

FIG. 8. Graph of the changes in EPR linewidth (o) observed

within the heart images (Fig. 6) as a function

ischemia. The vertical axis shows the value

caused by molecular oxygen and is calculated

between the observed linewidth from the

and the intrinsic width of the signal observed

conditions (4). The open circles show the

zero-gradient spectra at the corresponding

first-order exponential fit (k = 0.07 min')

(Inset) Semilogarithmic plot of the width

of ischemia could deplete antioxidative defenses

ability of the heart to metabolize these radicals (8, 9). As such

the results obtained in the present study showing transmu-

ral gradient in the rate of myocardial radical clearance

slower clearance in the endocardium could serve

the sensitivity of the endocardium to ischemic

We observed that myocardial oxygen concentrations

ually decreased in cardiopleged hearts subjected

temperature ischemia. In these hearts no evidence

ortransmural differences in myocardial oxygenation

The rate of decrease in oxygen concentration was observed to

be considerably slower than that we have previously

in noncardiopleged hearts (4). This would be expected

the cardiopleged hearts contractile function is arrested result-

ing in a much lower rate of oxygen consumption.

Thus, we have demonstrated that the technique of

spatial EPR imaging of free radicals can be

living biological organs and tissue. This technique

ability to address a number of important biomedical

including localized free radical metabolism and

We thank Dr. H. M. Swartz, Dr. R. K. Woods,

Dr. A. Sotgiu, Dr. U. Ewert, and Dr. M. L. Weisfeldt

discussions and advice. This work was supported

Institutes of Health Grants HL-38324 and HL-17655 Amer-

ican Heart Association Established Investigator

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