PRECLINICAL ANDCLINICAL

SPECTROSCOPY -Rapid

Communication

Enabling Hyperpolarized 129Xe MR Spectroscopy andImaging of Pulmonary Gas Transfer to the Red BloodCells in Transgenic Mice Expressing Human Hemoglobin

Matthew S. Freeman,1,2y Zackary I. Cleveland,1y Yi Qi,1 and Bastiaan Driehuys1*

Purpose: Hyperpolarized (HP) 129Xe gas in the alveoli can bedetected separately from 129Xe dissolved in pulmonary barrier

tissues (blood plasma and parenchyma) and red blood cells(RBCs) of humans, allowing this isotope to probe impaired gasuptake. Unfortunately, mice, which are favored as lung disease

models, do not display a unique RBC resonance, thus limitingthe preclinical utility of 129Xe MR. Here we overcome this limi-

tation using a commercially available strain of transgenic micethat exclusively expresses human hemoglobin.Methods: Dynamic HP 129Xe MR spectroscopy, and three-

dimensional radial MRI of gaseous and dissolved 129Xe wereperformed in both wild-type (C57BL/6) and transgenic mice.

Results: Unlike wild-type animals, transgenic mice displayed twodissolved 129Xe NMR peaks at 198 and 217 ppm, correspondingto 129Xe dissolved in barrier tissues and RBCs, respectively. More-

over, signal from these resonances could be imaged separately,using a 1-point variant of the Dixon technique.Conclusion: It is now possible to examine the dynamics and

spatial distribution of pulmonary gas uptake by the RBCs ofmice using HP 129Xe MR spectroscopy and imaging. When

combined with ventilation imaging, this ability will enabletranslational “mouse-to-human” studies of impaired gasexchange in a variety of pulmonary diseases. Magn ResonMed 70:1192–1199, 2013. VC 2013 Wiley Periodicals, Inc.

Key words: hyperpolarized 129Xe; gas exchange; mousemodel; red blood cell; interstitial lung disease; chemical shift

saturation recovery

INTRODUCTION

Over the past several decades, the mouse has become themost commonly used animal in preclinical studies ofpulmonary disorders (1–4). In particular, mouse models

have been used extensively to characterize the cellularand molecular events that underlie lung disease andinjury. However, to elucidate how these disease mecha-nisms produce pathological conditions in humans, aswell as to determine the degree to which mouse modelsactually mimic human disease, it is necessary to deter-mine how these cellular and molecular events manifestas pathological changes in pulmonary physiology. Tothis end, a variety of specialized instruments and techni-ques have been developed to quantify lung function inmice. For instance, tools to measure respiratory mechan-ics, such as flexiVent (SCIREQ Scientific RespiratoryEquipment Inc. Tempe, AZ), are particularly well devel-oped (5–7). More recently, it has become possible tomove beyond global measures of respiratory mechanicsin mice and provide regional physiological informationby directly visualizing the distribution of pulmonaryventilation, using hyperpolarized (HP) 3He MRI (8,9).

While the ability to visualize pathological changes inventilation will be invaluable when working with mod-els of obstructive pulmonary disease (e.g., chronicobstructive pulmonary disease and asthma), additionaltools are required to fully assess the range of pathophysi-ology observed in human disease. In particular, impairedgas exchange is the primary pathological characteristic ofa variety of human illnesses, including pneumonitis andinterstitial lung disease. Unfortunately, compared withrespiratory mechanics, the ability to characterize gasexchange and quantify gas exchange impairment remainsrelatively underdeveloped. For instance, obtaining eventhe most commonly used clinical measure of gasexchange, the diffusing capacity for carbon monoxide(DLCO), has only recently become possible in mice (10).Moreover, DLCO, like respiratory mechanics, providesonly a global measure of lung function. Thus, it is neces-sary to develop tools that allow the spatial distributionof gas exchange to be observed in mouse models ofhuman disease.

A particularly promising candidate for providing thisinformation is HP 129Xe, which like HP 3He, can be usedto visualize ventilation in both humans (11–13) and ani-mals (14–17). However, unlike 3He, 129Xe is soluble intissues and possesses a large chemical shift range invivo. In humans inhaled, gaseous 129Xe (typically refer-enced to 0 ppm) can be detected separately from 129Xedissolved in the red blood cells (RBCs, 218 ppm) and129Xe dissolved in the adjacent parenchymal tissues andblood plasma (197 ppm) (18). These properties make

1Center for In Vivo Microscopy, Duke University Medical Center, Durham,North Carolina, USA.2Medical Physics Graduate Program, Duke University, Durham, NorthCarolina, USA.

Grant sponsor: NHLBI; Grant numbers: R01HL105643, 1K99-HL-111217-01A1; Grant sponsor: National Institutes of Health/National Institute of Bio-medical Imaging and Bioengineering (NIH / NIBIB) National BiomedicalTechnology Resource Center; Grant number: NIBIB P41 EB015897.

*Correspondence to: Bastiaan Driehuys, Ph.D., Center for In VivoMicroscopy, Box 3302, Duke University Medical Center, Durham, NC27710. E-mail: bastiaan.driehuys@duke.eduyThese authors contributed equally to this work.

Received 20 May 2013; revised 5 July 2013; accepted 17 July 2013

DOI 10.1002/mrm.24915Published online 4 September 2013 in Wiley Online Library(wileyonlinelibrary.com).

Magnetic Resonance in Medicine 70:1192–1199 (2013)

VC 2013 Wiley Periodicals, Inc. 1192

129Xe uniquely suited to probe pulmonary gas exchangeand, when deployed in rats (19,20), have allowed 129Xespectroscopy to probe impaired gas exchange in a ratmodel of acute lung injury (21). Subsequently, theseproperties were exploited to image impaired gas uptakeby the red blood cells in a rat model of pulmonary fibro-sis. This step was accomplished by using a 1-point vari-ant of the Dixon MRI technique (22) to separately imagethe 129Xe uptake in the barrier tissues (lung parenchymaand blood plasma) and RBC compartments (16).

Unfortunately, these properties have been impossibleto exploit in mouse models of disease, because mice dis-play only a single dissolved 129Xe resonance at 198 ppm(23) that originates from 129Xe dissolved in both barriertissues and RBCs. While it is possible to examine thebulk uptake of 129Xe by all pulmonary tissues (i.e., bloodand parenchymal tissues combined) in mice (24), theuptake of gas by the red blood cells is of primary physio-logical interest. Thus, the absence of a distinct RBC peakseverely limits the preclinical utility of 129Xe MRI forstudying gas exchange.

Here, we demonstrate that this obstacle can be over-come by performing 129Xe MR spectroscopy and imagingin a transgenic strain of mice that exclusively expressesboth the a and b subunits of human hemoglobin (25).Furthermore, we found that 129Xe MR data from thistransgenic strain display almost identical spectral prop-erties to those observed in humans, and thus provides afar richer level of information about diffusive gasexchange than can be obtained from comparable wild-type mice. Additionally, we show that it is possible toobtain high-resolution, three-dimensional (3D) images ofventilation from these same animals, thus allowing awide range of physiological information to be extractedfrom mice using a single contrast agent.

METHODS

Animal Preparation

Wild-type (C57BL/6, n ¼ 3, Jackson Laboratory, Bar Har-bor, ME) and transgenic mice (B6;129-Hbatm1(HBA)Tow

Hbbtm2(HBG1,HBB*)Tow/Hbbtm3(HBG1,HBB)Tow/J, n ¼ 3 Jack-son Laboratory) were prepared following proceduresapproved by the Duke University Institutional AnimalCare and Use Committee. It should be noted that thetransgenic strain was originally developed as a model ofsickle cell disease. However, only nonsickling animalsthat were homozygous for normal, rather than sickle cell,human hemoglobin were used in this work.

Before MR experiments, animals were anesthetized byintraperitoneal (IP) injection of 75-mg/kg sodium pento-barbital (Nembutal, Lundbeck, Inc., Deerfield, IL), andintubated by tracheostomy with an 18-G catheter (Abbo-cath, Hospira, Inc., Lake Forest, IL) to provide an airtightseal for mechanical ventilation. Anesthesia was main-tained with periodic injections of sodium pentobarbital(25 mg/kg) administered by means of a 22-G IP catheter.Animals were ventilated (75% N2 and 25% O2) at a rateof 100 breaths/min on an HP-gas compatible, constant-volume, mechanical ventilator (26) using a tidal volumeof 0.20 mL. During 129Xe MR acquisition, HP 129Xereplaced the N2 in the breathing gas mixture, while hold-

ing the O2 concentration constant. During MR acquisi-tions, the ventilation rate was reduced to 75 breaths/min(140-ms inhalation period, a 360-ms breath-hold, and a300-ms period of passive exhalation) to accommodate alonger mechanical breath-hold.

While in the MR magnet, the body temperature of themice was monitored by means of a rectal thermistor andmaintained at �35

�C by warm air flowing through the

magnet bore. Airway pressure was monitored by a pres-sure transducer attached to the ventilator gas deliverylines near the trachea of the mouse. Heart rate was moni-tored by means of electrocardiogram readings using cus-tom LabVIEW 8.1 software (National InstrumentsCorporation, Austin, TX). This software also controlledthe timing of the ventilator and triggered the MR acquisi-tion. Following imaging, mice were euthanized by injec-tion of sodium pentobarbitol overdose.

HP 129Xe Production and Delivery

Either natural abundance (26% 129Xe) or isotopicallyenriched (85% 129Xe) xenon was hyperpolarized in a 1%Xe, 10% N2, 89% He mixture (Linde Electronics andSpecialty Gases Inc., Stewartsville, NJ), by spin exchangeoptical pumping using a prototype commercial polarizer(model 9800, MITI, Durham, NC) and extracted from thebuffer gases in a liquid nitrogen trap (27). Followingcryogenic accumulation, HP 129Xe (polarization �10%)was thawed into 150-mL Tedlar bags (Jensen Inert Prod-ucts, Coral Springs, FL) housed inside a Plexiglas cylin-der. This cylinder was then pressurized to 3.5 psig, andHP 129Xe was delivered to the mice using the customventilator, as described previously (26).

MR Spectroscopy

All 129Xe MR spectra were acquired from natural abun-dance xenon at 2.0 Tesla (T) using a horizontal, 30-cmbore magnet (Oxford Instruments, Oxford, UK) equippedwith 180-mT/m shielded gradients and a GE Excite 12.0console (GE Healthcare, Milwaukee, WI). This consolewas interfaced with a dual-resonance 129Xe / 1H (23.66 /85.54 MHz), 4-cm long, quadrature birdcage coil (m2mImaging, Cleveland, OH) and operated at 23.66 / 85.54MHz instead of its intrinsic frequency (63.86 MHz) usinga frequency up-down converter (Cummings ElectronicsLabs, North Andover, MA).

Before data acquisition, the mouse was briefly venti-lated with HP 129Xe, and spectroscopy was performed todetermine the resonance frequencies of the dissolvedand gaseous 129Xe peaks, set the RF flip angle, and per-form in vivo shimming. Preliminary spectroscopy wasalso used to determine the nominal echo time (TE)required for the RBC and barrier tissue resonances toaccumulate the 90

�phase difference (TE90) needed to

perform 1-point Dixon imaging of 129Xe uptake (16).To examine the global dissolved 129Xe replenishment

dynamics, 129Xe spectra were acquired from the wholelung after applying 90

�RF pulses to the dissolved phase

peaks at repetition times (TRs) ranging from 20 to 200 ms(14 TRs in total). The RF pulse consisted of a 3-lobe, 1000ms sinc pulse with 4-kHz excitation bandwidth, which pri-marily excited the dissolved phase but also applied a flip

HP 129Xe MR of Transgenic Mice with Human Hemoglobin 1193

angle of �1�

to the gas-phase resonance. All spectra wereacquired with gating over the course of multiple breath-holds (TE ¼ 1132 ms, BW ¼ 15.625 kHz, and 512 pointsper FID). For each mouse, one set of spectra was acquiredduring the breath-hold and one set was acquired at endexpiration. To construct the dynamic signal intensitybuild-up curves, a varying number of acquisitions wereaveraged (51–201 frames) to yield sufficient signal inten-sity for data fitting, while limiting the total acquisitiontime per spectrum to �30 s, and the first FID obtainedfrom each breath was discarded before processing andaveraging with matNMR (28). The intensities of the dis-solved peaks were determined by Gaussian deconvolutionin MATLAB R2011b (MathWorks, Inc., Natick, MA). Inten-sities of the narrower gas-phase excitations were deter-mined using a Lorentzian fit.

Signal Dynamics

The signal dynamics of both dissolved HP 129Xe signalpeaks were fit to a slightly modified version of a recentlypublished model of xenon exchange, MOXE (29). Previ-ous efforts to model 129Xe uptake by the gas exchange

tissues either: (i) accounted for diffusive uptake by theRBC and barrier tissues, but assumed a static “slab” oftissues (16); or (ii) accounted for continued accumulationof dissolved 129Xe signal due to blood flow, but did notincorporate the spectral information available from thetwo dissolved 129Xe peaks (30). In contrast, the MOXEmodel expresses time-dependent signals from barrierpeak, Sbarrier, and RBC peak, SRBC, as:

SbarrierðTRÞ ¼ bbarrier2d

d� 8

p2

Xn¼odd

1

n21� cos

npd

d

� �e�n2TR=tEx

" #

þbbarrierð1� hÞ

2 1� 2d

d

� �TR

tTx� 8tEx

p2tTx

Xn¼odd

1

n4cos

npd

d

� �ð1� e�n2TR=tEx Þ

" #

þ 1� TR

tTx

� �1� 2d

d

� �� 8

p2

Xn¼odd

1

n2cos

npd

d

� �e�n2TR=tEx

" #

8>>>>>><>>>>>>:

9>>>>>>=>>>>>>;

[1]

and

SRBCðTRÞ ¼ bRBCh

2 1� 2d

d

� �TR

tTx� 8tEx

p2tTx

Xn¼odd

1

n4cos

npd

d

� �ð1� e�n2TR=tEx Þ

" #

þ 1� TR

tTx

� �� 1� 2d

d

� �� 8

p2

Xn¼odd

1

n2cos

npd

d

� �e�n2TR=tEx

" #8>>>>><>>>>>:

9>>>>>=>>>>>;

[2]

In Eqs. [1] and [2], TR is the repetition time, d is the bar-rier thickness, d is the septal thickness. The parameter h

is the fraction of xenon dissolved in the RBCs relative tototal xenon dissolved in the blood, which is given by:

h ¼ lRBC

lplasma=Hct þ lRBC -lplasma[3]

where Hct is the blood hematocrit, and kRBC and kplasma

are the solubility of xenon in the red blood cells andblood plasma, respectively. Literature values for theseconstants appear in Table 1. Additionally, the fittingparameters used in the model are the Xe-exchange timeconstant, tEx; pulmonary-capillary transit time, tTx; andscaling parameters bRBC and bbarrier. It should be notedthat the original MOXE model used a single scalingparameter b, whereas two scaling parameters were usedhere to allow the formulae to fit data with varying RBC /barrier ratios.

In the 3 transgenic animals, a total of 6 TR-dependentbuildup curves were acquired (2 curves each mouse).Signal intensites from each time point were then aver-aged across all 6 datasets and divided by their corre-sponding gas phase signals. Finally, both RBC andbarrier datasets were simultaneously fit to Eqs. [1] and[2] using a steepest descent gradient-fitting routine andthe shared set of fitting parameters. The errors calculatedfor each data point are the residuals (root mean squaredifference) between the Gaussian fits and the acquiredspectra, combined across data sets using standard errorpropagation. The 95% confidence intervals of the fitwere determined based on the x2 goodness-of-fit. Datafrom the three wild-type animals (2 curves each), whichdisplayed a single resonance, were averaged and fit tothe sum of Eqs. [1] and [2]. To reduce the chance thatthe steepest descent algorithm converged on a localrather than global minimum, the algorithm was run 1000times from randomly generated vectors with starting

Table 1Physiological Constants for Modeling Xenon Exchange

Constant Value Reference

d, barrier thickness 0.57 mm Mercer, 1994 (42)d, septal thickness 1.51 mm Mei, 2007 (43)

Hct, hematocrit 0.42 Ryan, 1997 (25)lRBC

a, Xe solubilityin RBCs

0.2710 Chen, 1980 (44)

lPlasmaa, Xe solubility

in plasma0.0939 Chen, 1980 (44)

g 0.676 Eq. [3]aOstwald solubilities were obtained from canine blood.

1194 Freeman et al.

values (tEx: 0.01–0.11 s, tTx: 0.2–0.7 s, with bRBC and bbar-

rier scaling randomly between 0 and 200% of the maxi-mum intensity for each resonance).

Imaging

All 129Xe MR images were acquired from isotopicallyenriched xenon. Ventilation imaging used a 3D radialsequence (views ¼ 2501, views per breath ¼ 4, TR ¼ 10ms, flip angle ¼ 30

�, matrix ¼ 64 � 64 � 64, FOV ¼ 2.0

cm, acquisition BW ¼ 8 kHz), which used a nonselec-tive, 500-ms, 3-lobe sinc pulse with 8-kHz excitationbandwidth. While this 500-ms pulse provided non-negligible off-resonance excitation of the dissolved-phasepool, the resulting dissolved-phase 129Xe signal wasinsignificant relative to the gaseous signal originatingfrom the 100-fold larger alveolar magnetization pool. Dis-solved 129Xe image acquisition used the same 3D radialsequence, but used a 1200-ms, 3-lobe sinc pulse (excita-tion BW ¼ 3 kHz) to selectively excite dissolved 129Xe.This pulse caused a �0.1

�flip to be applied to the gas

phase, which did not materially affect image qualityowing to a minimal effect on the source gas-phase mag-netization pool. Additional dissolved 129Xe acquisitionparameters included: radial views ¼ 1073, views perbreath ¼ 6, TR ¼ 50 ms, TE ¼ 858 ms, flip angle ¼ 90

�,

matrix ¼ 32�32�32, number of excitations (NEX) ¼ 16,FOV ¼ 2 cm, and BW ¼ 15.625 kHz. Note, the dissolved129Xe images were acquired with a longer TR, relative toventilation images, to permit diffusive replenishment ofthe dissolved magnetization from the alveolar spaces.Ventilation images benefit further from a reduction inTR in terms of reduced T1 relaxation, and reducedmotional blurring.

For transgenic mice exhibiting both barrier (parenchy-mal tissue and blood plasma) and RBC resonances,dissolved-phase images were acquired using the 1-pointDixon technique (22). For this acquisition, both the exci-tation and receiver frequencies were set on resonancewith the RBC peak, such that the barrier peak lagged inphase behind the RBC peak as the transverse magnetiza-tion evolved following excitation. Image acquisition wastimed such that the phase difference between the twopeaks reached 90

�when imaging encoding was initiated.

The nominal TE at which this phase separation occurredwas empirically determined to be 858 ms using whole-lung 129Xe spectroscopy (31).

RESULTS

HP 129Xe Spectroscopy

Consistent with previous observations (23), spectraobtained from the lungs of wild-type mice exhibited asingle NMR resonance at 198 ppm (Fig. 1a and 1b) thatoriginated from 129Xe dissolved in the pulmonary tis-sues, including the RBCs. Although a second peakappeared at 194 ppm when sufficiently long repetitiontimes (TR > 1 s) were used (see Figure 1c), this spectralfeature can be attributed to 129Xe dissolved in extrapul-monary fatty tissues (20). When the same experimentwas performed using a transgenic mouse strain, in whichthe wild-type mouse hemoglobin (both a and b subunits)

was knocked out and replaced by human hemoglobin,the NMR spectra displayed two dissolved peaks at 198and 217 ppm (see Figure 1d), which are almost identicalto the frequencies observed in humans. The 129Xe spec-trum from these transgenic animals also displayed athird peak at 194 ppm (Fig. 1e), which appeared at longTRs (>1 s), indicating that, like the similar peakobserved from wild-type animals, it arose from extrapul-monary fat tissues.

HP 129Xe Signal Dynamics

For wild-type mice, the 129Xe dynamic, signal build-upcurve (32) (see Figure 2a) was characterized by relativelyrapid signal increase up to a saturation time of �100 ms,followed by gradual increase from signal in the distal,extrapulmonary tissues. When the data were fit for boththe xenon exchange time and the pulmonary-capillarytransit time, values of tEx ¼ 300 6 20 ms and tTx ¼ 4606 20 ms were obtained. This value for tTx is plausiblyclose to the previously published value for thepulmonary-capillary transit time of 830 ms, found usingsynchrotron radiation angiography (33). However, the

FIG. 1. HP 129Xe spectroscopy in mice with 20-Hz line broaden-ing. a: Spectrum from a wild-type (C57/BL6) mouse at TR ¼ 360ms and NEX ¼ 101 referenced to gas-phase 129Xe at 0 ppm. b:Close-up of the same dissolved-phase peak from a wild-typemouse, showing only one peak. c: Dissolved-phase region of the

spectrum at TR ¼ 8 s and NEX ¼ 75, showing, a second peakassociated with 129Xe dissolved in fat outside the lung. d: Dis-solved 129Xe spectrum at TR ¼ 360 ms and NEX ¼ 201 from a

transgenic mouse, showing two peaks: one associated with theplasma and parenchymal tissues and one with red blood cells. e:Spectrum from the same transgenic animal as shown in 1d, atTR ¼ 8 s and NEX ¼ 75, showing the emergence of a third,fat-associated peak.

HP 129Xe MR of Transgenic Mice with Human Hemoglobin 1195

value for tEx is unphysiologically long. In contrast, theadditional spectral information provided by the trans-genic animals allowed signal recovery data from both thebarrier tissues and RBCs (Fig. 2b) to be analyzed, and theresulting fit yielded reasonable, population-averaged val-ues of tEx ¼ 56 6 6 ms and tTx ¼ 610 6 60 ms, both ofwhich are physiologically realistic.

Imaging

Ventilation images acquired in this work (see Figure 3)were obtained with a 3D, isotropic resolution of 312 mmand a relatively high parenchymal SNR of 21. The com-bination of resolution and SNR allowed structural fea-tures to be visualized that were not observable inpreviously published 2D, HP 129Xe images of mouselungs (34). For instance, the first two generations of theairways can be seen and are clearly delineated from129Xe in the more distal lung parenchyma. Also visiblein the image is a signal intensity void corresponding tothe expected anatomical location of the vena cava.

Similar structural features can also be observed in dis-solved 129Xe images obtained from a wild-type mouse(see Figure 4) with an isotropic 625-mm resolution and

an SNR of 12. That is, circular dark spots are observedin regions corresponding to the locations of the largerairways seen in the ventilation images, where no gasexchange occurs. Additionally, the signal intensity ofthis image displays a reasonably high coefficient of varia-tion (CV) of 0.20, indicating that there is significantregional heterogeneity in gas uptake, even in this normal,healthy animal. Interestingly, several circular brightspots can also be seen in the image. Although the sourceof these bright spots has not been investigated, they maycorrespond to regions of increased gas uptake or tissuedensity. Alternatively, it is possible that, at TR ¼ 50 ms,there was sufficient time for magnetization in theseregions to wash away from the gas-exchange tissues, andthat the bright spots thus correspond to dissolved 129Xesignal arising from the larger vasculature.

Dissolved 129Xe images with the same 625-mm iso-tropic resolution were also acquired from a transgenicanimal (see right two columns of Figure 4). However, byselecting the appropriate echo time (858 ms), the result-ing data could be separated into images resulting from129Xe gas uptake by the barrier tissues and RBCs. Thebarrier image was obtained with an SNR of 32 and a CVof 0.23, while the RBC image has an SNR of 22 and a CVof 0.25. These SNR values are in reasonable proportionto the fraction of the dissolved 129Xe spectrum attribut-able to both fractions. (Note, the higher dissolved-phaseSNR observed from the transgenic compared with thewild-type animal resulted from greater signal averaging,NEX¼16 versus NEX ¼ 8, and possibly varying polariza-tion levels.)

DISCUSSION

HP 129Xe Spectroscopy in Mice

The appearance of a single dissolved 129Xe resonancefrom the barrier tissues and RBCs observed in wild-typemice, a pattern which incidentally is also observed inrabbits (35), stands in stark contrast to spectra observedfrom most other species studied to date—includinghumans (13,18,36), rats (21,37), dogs (38), sheep (39),and pigs (40)—that display distinct barrier tissue- andRBC-specific peaks. Although the exact mechanism thatcauses this spectral difference remains unknown, it iswell established that the RBC-specific peaks result from129Xe interactions with hemoglobin within the RBCs(41). Thus, it is reasonable to speculate that the absenceof RBC-specific peaks in mice results from differingxenon-hemoglobin binding interactions relative to otherspecies. Indeed, we have now demonstrated that thisspeculation is correct by showing that transgenic miceexpressing human hemoglobin generate two dissolved129Xe peaks, which occur at almost identical frequenciesto the barrier (197 ppm) and RBC peaks (218 ppm) seenin human subjects (18,36). Moreover, the ability to sepa-rately detect the RBC and barrier components of the gas-exchange tissues potentially provides a means of extract-ing more accurate and useful physiological informationin mice.

For instance, curve fitting of our data from the wild-type mice did not yield physiologically reasonable val-ues for the exchange time. More specifically, the

FIG. 2. HP 129Xe signal dynamics in mice fit to the MOXE modelof 129Xe uptake (solid lines). Dotted lines represent 95% confi-

dence intervals. a: Signal build-up curve for wild-type mice (aver-age of all three). b: Signal build-up curve for the transgenic mice(average of all three) showing time-dependent HP 129Xe accumu-

lation by both the RBC and barrier tissues.

1196 Freeman et al.

resulting estimate of the pulmonary-capillary transit time(460 ms) was reasonable, but the xenon exchange timewas unphysiologically long. In contrast, having the abil-ity to fit the barrier and RBC signal recovery datatogether (Fig. 2b) permitted both the exchange and capil-lary transit time to be determined, and the result for thecapillary transit time of 610 ms was in reasonable agree-ment with the value of 830 6 30 ms (33) previouslypublished.

It should be noted that Imai, et al., (24) demonstratedthat physiologically sensible parameters could beextracted from dissolved-phase signal dynamics in wild-type mice, and Patz, et al., (30) demonstrated that sensi-ble results could also be obtained from humans at 0.2 T,where the two dissolved 129Xe resonances are collapsedinto a single peak. Both of these earlier studies used aconventional chemical shift saturation recovery (CSSR)sequence that used a saturation-excitation RF pair sepa-rated by a variable signal replenishment delay time. Inwild-type mice (24), sensible values were likelyobtained, because the CSSR sequence permitted the useof signal replenishment times as short as 1.4 ms, whichenabled the relatively rapid signal dynamics present inmice to be probed in sufficient detail. In the low-fieldhuman work, replenishment times did not extend below20 ms, but this was likely sufficiently short to capturethe slower physiology present in human subjects. Thus,in future work, it may be possible to extract even morerobust and accurate physiological information from thesetransgenic mice using a CSSR approach.

Of interest, while the two dissolved 129Xe peaksobserved in the transgenic mice display spectral frequen-cies similar to those observed in humans, the linewidthsof these peaks were quite broad. That is, both peaks dis-

played linewidths of 11 ppm, which exceeded by morethan 200% the 5-ppm linewidths observed from the sin-gle peak seen in wild-type mice. These broad lines can-not be trivially attributed to poor shimming, because gas-phase line widths obtained from whole-lung spectros-copy did not noticeably vary between wild-type andtransgenic mice. Additionally, the width of theseresonances did not increase at longer TR, indicatingblood flow to other, more distal tissues did not cause theobserved spectral broadening. Interestingly, the dissolved129Xe linewidths from the transgenic mice are alsoapproximately 40% broader than those observed fromrats, using identical MR equipment and spectral process-ing techniques (31). It should be noted that although thehemoglobin has been altered in these transgenic mice,they still retain their native cell structure, which is likelydifferent from humans and rats. Taken together, theseobservations suggest that the broadening observed in thetransgenic mice likely resulted from more rapid diffusive129Xe exchange between the mouse erythrocytes and theadjacent spectral compartments.

Imaging Lung Function

Previous images of HP 129Xe in the mouse lung havebeen limited to 2D and displayed a relatively low, 1.25mm � 0.8 mm in-plane resolution (34), which was insuf-ficient to distinguish finer anatomical details. The supe-rior SNR and image quality displayed by our images(Fig. 3) was made possible by acquiring image data dur-ing multiple inspiratory breaths, using a highly repro-ducible, hyperpolarized 129Xe compatable, mechanicalventilator (26). Indeed, using a similar ventilation andimaging strategy, we were also able to generate

FIG. 3. 129Xe ventilation imaging

with an isotropic resolution of312 mm in the transgenic mouse,showing regions of high signal

intensity in the first two genera-tions of airways (thin arrows), as

well as regions of lower intensityin the region corresponding tothe anatomical location of the

vena cava (thick arrow).

HP 129Xe MR of Transgenic Mice with Human Hemoglobin 1197

sufficiently high signal intensity to image 129Xe dis-solved in the pulmonary tissues. Furthermore, applying1-point Dixon imaging to dissolved-phase 129Xe in thetransgenic animal enabled the visualization of 129Xetransfer into the barrier and RBC compartments sepa-rately. In this case, the images illustrate the normal func-tion expected in a healthy mouse. That is, 129Xe signal isobserved from the RBCs wherever there is also uptake bythe barrier tissues. However, regional mismatching (i.e.,reduced RBC signal relative to barrier tissue signal) isexpected when diffusive gas exchange is impaired, forinstance as a result of barrier tissue thickening caused bypulmonary fibrosis (16,31).

CONCLUSIONS

Using a transgenic strain of mice that expresses humanhemoglobin, it is now possible to probe diffusive gasexchange between the alveolar spaces and the red bloodcells using both dynamic 129Xe spectroscopy and 3D iso-tropic imaging. This new capability will significantlyenhance the utility of 129Xe MRI in mouse models ofinterstitial lung disease. Furthermore, when combinedwith quantitative (11) ventilation imaging, these methodsmake possible translational “mouse-to-human” studies ofimpaired gas exchange in a variety of pulmonarydiseases.

ACKNOWLEDGMENTS

This study was funded by NHLBI and performed at theDuke Center for In Vivo Microscopy, a National Insti-tutes of Health/National Institute of Biomedical Imagingand Bioengineering (NIH / NIBIB) National BiomedicalTechnology Resource Center. The authors thankDr. Thomas C. O’Haver (University of Maryland, CollegePark, MD) for providing code useful for the deconvolu-tion of spectral peaks, Sally Zimney for carefully readingthe manuscript, and Dr. Laurence Hedlund for valuablediscussions regarding the development of thisexperiment.

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