In vivo pO2 imaging in the porcine model with perfluorocarbon F-19 NMR at low field

ELSEVIER

l Original Contribution

Magnetic Resonance Imaging, Vol. 14, No. 1, pp. 103-l 14, 1996 Copyright 0 1996 Elsevier Science Inc. Printed in the USA. All rights reserved

0730-725X/96 $15.00 + .OO

0730-725X(95)02046-2

IN VIVO PO2 IMAGING IN THE PORCINE MODEL WITH PERFLUOROCARBON F-19 NMR AT LOW FIELD

STEPHEN R. THOMAS,* RONALD G. PRATT,* RONALD W. MILLARD,~ RANASINGHAGE C . SAMARATUNGA, * YOSEPH SHIFERAW, * $

ANTHONY J. MCGORON,* AND KIM KIAT TAN*§

University of Cincinnati Medical Center, Departments of *Radiology and ?Pharmacology & Cell Biophysics, Cincinnati, OH 45267-0579, USA

Quantitative pOz imaging in vivo has been evaluated utilizing F-19 NMR in the porcine model at 0.14 T for the lungs, liver, and spleen following IP administration of the commercial perfluorotributylamine (FC-43)-based perfluorocarbon (PFC) emulsion, Oxypherol-ET. Calculated Tt maps obtained from a two spin-echo saturation recovery/inversion recovery (SR/IR) pulse protocol are converted into quantitative pO1 images through a temperature-dependent calibration curve relating longitudinal relaxation rate (l/T,) to ~0~. The uncertainty in pOZ for a T1 measurement error of +5% as encountered in establishing the calibration curves ranges from +lO torr (*40%) at 25 torr to k16 torr (kll%) at 150 torr for FC-43 (37°C). However, additional uncertainties in Zfr dependent upon the signal-to-noise ratio may be introduced through the SR/IR calculated Z’r pulse protocol, which might severely degrade the pO1 accuracy. Correlation of the organ image calculated pOZ with directly measured pOz in airway or blood pools in six pigs indicate that the PFC resident in lung is in near equilibrium with arterialized blood and not with airway pOZ, suggesting a location distal to the alveolar epithelium. For the liver, the strongest correlation implying equilibrium was evident for venous blood (hepatic vein). For the spleen, arterial blood pO1 (aorta) was an unreliable predictor of pO* for PFC resident in splenic tissue. The results have demonstrated the utility and defined the limiting aspects of quantitative pOZ imaging in vivo using F-19 MRI of sequestered PFC materials.

Keywords: Oxygen imaging; pOZ imaging; F-19 NMR, Fluorine-19 magnetic resonance imaging (MRI); Perfluo- rocarbon F-19 NMR; Liver; Spleen; Lung.

INTRODUCTION

Blood substitute materials based on perfluorocarbon (PFC) compounds may be expected to assume enhanced importance as a strategy for blood replacement within the transfusion sciences. The indications for this in- clude: (a) the imperative biomedical need in response to the increasing prevalence of viral and other blood- borne diseases, and (b) the reactive symbiotic commercial development of PFC-based blood substitute emulsions.1-3 Significant resources are being committed to the investigation of biomedical applications and per- formance of PFC blood substitutes.4-6 These highly

fluorinated compounds have the unique capacity for transport of substantial volumes of dissolved gases (up to 60 ~01% of oxygen and 120 ~01% of carbon diox- ide’) and are biocompatible when in emulsified form.4-6 The modalities of F-19 nuclear magnetic resonance (NMR) imaging and spectroscopy have opened significant new avenues for high-sensitivity studies of the in vitro and in vivo properties and diagnostic capabilities of fluorine-based compounds in general, and PFC compounds in specific.8 One opportunity, which serves as the basis of this report, is the potential for in vivo oxygen imaging and quantitative determination of partial pressure of oxygen (PO,) in tissues concentrating PFC

RECEIVED 3/9/95; ACCEPTED 7/12/95. E560 MSB, PO Box 670579, Cincinnati, OH 45267-0579. Address correspondence to Stephen R. Thomas, Ph.D., $Current affiliation: National Institutes of Health, In Vivo

University of Cincinnati Medical Center, Department of Ra- NMR Research Center. diology, Division of Medical Physics, 23 1 Bethesda Avenue, §Current affiliation: Philips Laboratories.

103

104 Magnetic Resonance Imaging 0 Volume 14, Number 1, 1996

(e.g., lung, reticuloendothelial system, marrow, and sites of macrophage accumulation) following IV or IP administration of PFC emulsions.

In vivo quantitative assessment of tissue oxygenation may be achieved through utilization of NMR techniques sensitive to the paramagnetic effect of dissolved molecular oxygen (O,), which reduces the F-19 spin- lattice relaxation parameter T, .*-16 The well-established linear relationship between the longitudinal relaxation rate T;’ and p028J7J8 is used as a calibration curve to derive pOZ values following in vivo measurement of F-19 T, . The fluorocarbon phase and biological aqueous phase are immiscible; thus, as required for fidelity of pOZ determination, changes in the F-19 T, correlate with local oxygen concentration (dissolved, in equilibrium) and are independent of various bioconstituent components residing in the surrounding aqueous environment .I9

The ability to monitor pOz in vivo noninvasively presents a significant diagnostic opportunity. Even mini- mal reductions in the normal oxygen supply maintaining cellular homeostasis in tissue may compromise physiological function. Quantitative pOZ imaging has potential application in evaluation of oxygenation status, for example, as related to differentiation of normal vs. pathologic conditions associated with ischemia. A com- prehensive capability exists for tracking oxygen distribution with F-19 NMR from the lungs through the blood to tissues sequestering PFCs.*’ The objective of the research reported here is to document the utility and accuracy of quantitative p02 imaging utilizing F-19 NMR in the porcine model at low field for the lungs, liver, and spleen following IP administration of the commercial PFC emulsion, Oxypherol-ET.’

METHODS AND MATERIALS

PFC Emulsion Oxypherol-ET’ (Alpha Therapeutic Corporation,

Los Angeles, CA) is composed of the perfluorocarbon perfluorotributylamine [(CF$F, CF2 CF2)s N; commercial name FC-431 emulsified with the surfactant copolymer polyoxyprolene-polyoxyethylene (Pluronic F-68). The Pluronic F-68 surfactant, which forms a single molecule monolayer on the surface of the spheri- cal FC-43 droplet, maintains the emulsion stability.*l The emulsion contains 20 wt/vol 70 FC-43 (i.e., 0.2 g FC-43/ml emulsion). The emulsion particle size distribution exhibits a weighted average diameter of approximately 0.09 pm with no particles larger than 0.6 pm diameter (65% < 0.10 pm).** In general, PFC NMR spectra exhibit multiple lines of varying intensity as a result of nonequivalent chemical environments of the individual fluorine atoms within the molecule. The

chemical shift spectrum for FC-43 is shown in Fig. 1 for various magnetic field strengths. Although not the subject of this article, our experimental protocol at low magnetic field involved acquisition of the entire spectrum followed by application of deconvolution al- gorithms, which provides improved signal-to-noise characteristics (relative to that from utilization of a single line over equivalent imaging times) and allows image reconstruction free of interfering chemical shift artifacts.23,24

Ty’/pO, Calibration Curves Calibration curves relating the longitudinal relax-

ation rate T;’ to p02 are shown in Fig. 2 as a function of temperature in the range 25 to 42°C. These curves were determined through equilibration of neat liquid PFC samples with calibrated gas standards con- taining 0%) lo%, 20%) 30%) and 60% oxygen (Wright Brothers, Inc., Cincinnati, OH). The experimental conditions involved bubbling and sealing the temperature- controlled sample standards under glove bag conditions, which ensured no contamination by ambient atmosphere. All magnetic resonance imaging and relaxation time measurements were performed at 0.14 T using the low field MRI research system designed and con- structed within the Division of Medical Physics at the University of Cincinnati. 25 Radiofrequency (RF) coils used were designed for proton (5.96 MHz) and/or fluorine (5.63 MHz) imaging at this field.26*27 The T, values were measured using the inversion recovery (IR) method with the sealed standards maintained at temperature in a water jacket assembly designed to fit within the RF coil. For each T, determination, the IR

Aj 7.06T 1 i

L

0.66T

B

Fig. 1. Chemical shift spectra for FC-43: (A) 7.06 T, (B) 0.66 T, (C) 0.14 T. The spectral width displayed is 62 ppm. The separation between the CF3 and (Y-CF~ lines is approximately 45 ppm.13

RECTOp02 imaging in porcine model 0 S.R. THOMAS ET AL. 105

6

I 200 400 600 800

PO* (torr)

Fig. 2. Calibration curves for FC-43 relating the observed longitudinal relaxation rate, l/T,, to the partial pressure of oxygen, pOZ, at 0.14 T as a function of temperature. The linear regression fit parameters (slope, a and intercept, b) are given in Table 1. The error bars for the relaxation rate represent a +5% uncertainty in r, (see text). The error bars shown for pOZ were calculated using Eq. (3) setting the error term 6T,/T, to 0.05 (i.e., 5%). They indicate the uncertainty in p02 that would result from utilization of the calibration curve for a k5% measurement error in Tr (also see Table 2).

pulse sequence was run using 16 different delay inter- vals. The range of delays used for a given T, measurement was chosen as appropriate for the T, value of the sample under study with the time between repetitions set to three to five times the expected Tl value to en- sure adequate magnetization recovery. Tl values were calculated by fitting the data set using a three-parameter nonlinear least squares fitting routine. The magnetization at the end of each delay interval was measured by integrating the free induction decay (FID). Thus, because the magnetization is sampled in the time domain, the Tl values reported represent a weighted average T, for all the spectral lines in the PFC emulsion. This approach is appropriate in particular for the relatively low field strength used (0.14 T) as the individual spectral lines are not fully resolved (see Fig. 1). In addition, the deconvolution techniques employed during image formation effectively combine the signal intensity from all lines.23,24

Magnetic Resonance Imaging The pulse protocol utilized to obtain calculated T,

images for conversion into quantitative regional tissue

p02 images through application of the calibration curves was the combined saturation recovery/inversion recovery (SR/IR) sequence shown in Fig. 3.28*29 Two spin-echo data sets Sl and S2 are acquired, each of which is used to form an image. By taking the ratio ‘% of the two signal intensities, T2-dependent terms can- cel out:

= 1 - 2e-“Tl + 2e-‘r+‘,-TE/2’/T, _ e-(7+&,)/T,

1 - 2e-(‘b-TE/2VT~ + e-tb/T,

(1)

The timing parameters t,, 7, and tb are defined in Fig. 3 with the pulse sequence repetition time TR = t, + 7 + tb . These parameters should be selected such that the first echo (Sl) acquired during the saturation recovery portion of the pulse sequence will provide a spin density-weighted image whereas the second echo (S2) acquired during the inversion recovery segment will be T, weighted. In addition, the formalism assumes that the signal decays to zero between successive RF pulses, precluding refocusing of any residual transverse magnetism. In practice, this may be ensured through application of appropriate gradients within the imaging sequence.28 ‘% is dependent only on T, , as well as the various timing parameters, and may be used to determine Tl via look-up tables. This protocol offers the advantage of providing calculated Tl data within a single imaging sequence, thus minimizing artifacts associated with subject motion or other experimental in- stabilities that might occur during a repeated sequence. To optimize the timing parameters for maximum accuracy in the calculated T, image, graphical simula- tion of !B was performed.

The criteria for optimization center on two aspects: (1) the change in 8 per change in Tl (= AWAT,) should be maximized to provide adequate sensitivity for the

90” I ad

RF II; *

18d sd’ IS&

. .

Signal

Fig. 3. The saturation recovery/inversion recovery (SRAR) pulse protocol utilized to obtain calculated Tr images.** The ratio of the two echoes acquired (‘8 = S2/Sl) is a function of Tr and the timing parameters t,, r, tb [see Eq. (l)].


relevant range of Tl ‘s involved, and (2) the magnitude of the second echo S2 should not be reduced to an extent that would introduce significant uncertainty through degraded signal-to-noise ratio (SNR). For the fluorine images presented in this article, a two-dimensional projection protocol (slice-select gradient off) was utilized to compensate for the relatively low SNR encountered in the in vivo models at 0.14 T. Therefore, the calculated pOZ values represent an average across the projection volume. Imaging times were approximately 25 min, which provided second echo SNRs greater than 20: 1 in the liver and spleen under room air breathing conditions. pOz maps are presented qualitatively using grey-scale and/or pseudocolor images whereas quantitative results are shown via labeled isobar contour plots.

Porcine Model Preparation Six young domestic female swine (7-l 1 kg) under ke-

tamine sedation (10 mg/kg, IM) were administered an IP injection of 500 ml warmed (37°C) fluorocarbon emulsion (Oxypherol-ET, - 100 g FC-43) 4 to 7 days prior to the MR imaging. On the day of the study, animals were anesthetized with sodium thiamylal(l0 mg/ kg IV initially, then supplemented as needed) and in- dwelling catheters were placed in the aorta, pulmonary artery, and hepatic vein via peripheral access with the aid of fluoroscopy for blood pool sampling (systemic arterial, mixed venous, and hepatic venous, respectively) in correlation with the MR imaging. The anesthetized, intubated animals were supported with mechanical ventilation during MR imaging with the fraction inspired oxygen (FiOz) controlled from 0.21 (ambient air) to near 0.95 (carbogen). A ventilatory period of 15 to 30 min was allowed to establish equilibrium before imaging for each FiOz step. Airway (trachea) pOZ was monitored on expiration through direct gas sampling. Blood and air samples were analyzed for oxygen partial pressure on a BMS Mark 3 Radiometer Clinical Gas Analyzer (Radiometer, Copenhagen, Denmark). The animal core temperature (rectal) was monitored before and after each imaging session, allowing selection of the appropriate calibration curve for the calculated pOz images. The animals were euthanatized following completion of the MR imaging protocols with tissues taken for quantitation of FC-43 concentration.

RESULTS

T,-‘/p02 Calibration Curves The calibration curves for FC-43 at 0.14 T as a func-

tion of temperature encompassing the physiologically relevant range are shown in Fig. 2. In accord with the protocol described above, T, values reported corre-

spond to an average response of the unresolved spectral lines at 0.14 T.

The linear relationship between the observed longitudinal relaxation rate l/T, and p02 of the form

l/T, = a(p0,) + b (2)

is well established.i7,” The intercept b represents the intrinsic relaxation rate due to all mechanisms not involving relaxation via dipolar coupling to the unpaired electron spin of the oxygen molecule (02) in solution. The relaxation rate proportionality with pOZ arises from the reasonably ideal Henry’s law solubility characteristics of O2 gas in PFCs. The slope, a, will depend on the particular PFC, the resonance line(s) measured, temperature, and magnetic field strength.

One objective of this research was to define the absolute uncertainty in the estimated pOz as a consequence of measurement errors in Tl. Under the experimen- tally observed conditions in which the fitting errors for the calibration curve slope, a, and intercept, b, are neg- ligible, the fractional uncertainty (relative error) in pOz is given by the expression: 3o

~PWPOZ = (6T,/T,) [(I + l/(rl~O,)l (3)

where 6pOz and AT, represent the respective uncertainties and 7 = a/b is defined as the PFC response index. Equation (3) indicates that for a given fractional uncertainty in Tl (i.e., 6Tl /T,), it is desirable that q be as large as possible to minimize the fractional uncertainty in ~0~. Qualitatively this means that the change in relaxation rate with respect to pOZ (slope, a) should be large whereas the intrinsic relaxation rate (intercept, b) should be small. Table 1 presents the parameters a, b, and r] as a function of temperature for FC-43.

In general, for our experimental system using the IR method, the coefficient of variation for repeated (con- secutive) T, measurements on the same reference sample standard was I 1%. However, day-to-day Tl reproduc- ibility was observed to exhibit a coefficient of variation

Table 1. FC-43 calibration curve parameters at 0.14 T: Slope (a), intercept (b) , and response index (r])

FC-43

Temperature Slope, a Intercept, b 7 (“C) (torr-’ s-l) W1) (torr-‘)

25.5 7.09 x 10-3 1.55 4.6 x 1O-3 31.0 6.90 x 1O-3 1.27 5.4 x 10-J 37.0 6.23 x 1O-3 1.09 5.7 x 10-3 42.6 5.98 x 1O-3 0.94 6.4 x 1O-3

RECTOpOz imaging in porcine model 0 S.R. THOMAS ET AL. 107

15%. As shown by Eq. (3), the fractional uncertainty in pOZ also depends upon the absolute value of pOZ as well as 6T/T, and 7. Table 2 provides an estimate of the uncertainty in pOZ to be expected for FC-43 at 37°C as a function of T, measurement error. For a +5% uncertainty, the variation in p02 is from *9 torr (+ 180%) to +24 torr (+8%) over the range 5 to 300 torr. As will be discussed below, determination of T, through the MR imaging protocol involving use of 8 look-up tables will introduce additional uncertainties dependent upon the SNR encountered.

Pulse Sequence Optimization The accuracy of the calculated T, images derived

from the SR/IR two-point pulse sequence shown in Fig. 3 and the 3 look-up table protocol based on Eq. (1) depends upon the interrelationships between the timing parameters (t, , 7, tb), the T, regime of interest, and the in vivo signal-to-noise leve1.29 Thus, the opti- mized pulse sequence must be tailored to the specific pOZ range under investigation. To satisfy the require- ments stated previously that the first echo (Sl) will provide a spin density-weighted image while the second echo (S2) will be Tl weighted, the timing parameters must be chosen in relationship to the maximum Tl expected (T,,,,) in the region of interest. To assure full relaxation within the pulse sequence, t, and tb would ideally be -5 T,,,,; however, this extreme would ex- tend the image acquisition time to an unacceptable de- gree. Thus, traditional compromises are required. Some graphical solutions of the ratio !B as a function of Tl [Eq. (l)] for various pulse sequences are shown in Fig. 4. For adequate look-up table sensitivity as related to T, discrimination accuracy, the timing parameters should be chosen such that the T, range of interest occurs in the rapidly falling portion of the curve

l-

0.75-

0.5 -

5 0.25-

B s o-

-0.25-

-0.5-

1504/r/1504 TE=lms

0 200 400 600

Tl (ms)

1200

Fig. 4. The graphical solution of % as a function of T, from Eq. (1) for the pulse sequence 1504/7/1504 (notation: f,/r/& units in ms) with TE = 8 ms. For optimal look-up table sensitivity, the operational parameters should be chosen such that the T, range of interest is in the falling portion of the % curve (steepest slope) but before the zero crossing.

where the slope A!I?/AT, is relatively large. However, adequate signal level S2 must be maintained to provide sufficient signal-to-noise in the second echo image. Otherwise, noise propagation/amplification introduced through the image ratio process would significantly degrade the calculated Tl image resulting in unacceptable uncertainty levels. With the pulse sequence notation &/r/t, (units in ms), typical values used for the in vivo porcine imaging studies with FC-43

Table 2. FC-43 fractional uncertainty in pOZ for a f 5%, lo%, and 20% error in rr at 0.14 T and 37°C

FC-43

PO2 (torr)

Tl

(ms) 5%*

6 Tl (ms)

lo%* 20%* 5%

6 PO2/PO, 6 p02 (torr)

10% 20% 5% 10% 20%

5 892 45 89 178 1.80 3.60 7.20 9 18 36 10 868 43 87 174 0.93 1.85 3.70 9 18 37 25 803 40 80 161 0.40 0.80 1.60 10 20 40 50 713 36 71 143 0.23 0.45 0.90 11 22 45

100 584 29 58 117 0.14 0.28 0.55 14 27 55 150 494 25 49 99 0.11 0.22 0.43 16 32 65 200 428 21 43 86 0.094 0.19 0.38 19 37 75 250 378 19 38 76 0.085 0.17 0.34 21 42 85 300 338 17 34 68 0.079 0.16 0.32 24 47 95

*Refers to the +% error in TI .


were 1504/800/1504 with TE = 8 ms. Generally, TE was in the range 7 to 11 ms. [The t,, tb values of 1504 ms arise because the pulse program coding utilized a format involving (t, - TE/2) and (tb - TE/2) (see Fig. 3). Thus, with the round number of 1500 ms (= to - TE/2) chosen for input convenience and TE = 8 ms, t, = 1504 ms. Similarly, tb = 1504 ms.]

The effect of image noise on the precision of the calculated Ti values may be evaluated under a model in which the noise is considered to be the uncertainty in the spin-echo magnitudes of the first and second echo data sets, namely, 6Sl and 6S2, respectively. Using standard propagation of error methods, the fractional uncertainty in !B is given as:

&X2/% = [(SSl/Sl)2 + (SS2/S2)2] 1’2 (4)

The resulting fractional uncertainty in 7’i for the pulse sequence 1504/r/1504 with TE = 8 ms is shown in Fig. 5 normalized to a constant noise representative of a SNR of 10 and 20 for the second echo at Ti = 600 ms (corresponding to a p02 -100 torr at 37°C for FC-43). In regions where ‘% does not vary significantly as a function of Ti (i.e., for small values of T, as shown in Fig. 4 where As/AT, - 0), small errors in determining !X translate into relatively large errors in T,. As $I ap- proaches zero (i.e., the magnitude of S2 is decreasing), the uncertainty in T, begins to increase again. Thus, for the 1504/800/1504 sequence with a SNR of 10 or 20 as defined, the uncertainty in the image measured Tl over the range 900 to 300 ms is approximately 12- 20% and 7-10070, respectively. As presented in Table 2, a &20% error in Tl would contribute a relatively large uncertainty in p02 of k40 and *65 torr at 25 and 150 torr, respectively.

Tissue FC-43 Concentration Following the MR imaging sessions (4 to 7 days post-

administration), the pigs were euthanatized with anes- thesia excess and organ tissue harvested for PFC concentration analysis. The quantitative technique involved NMR signal intensity analysis of the tissue samples in comparison with a FC-43 reference standard.31 The tissue analysis results for the six pigs are shown in Table 3. FC-43 concentration in samples taken from different lobes of the liver and from various locations within the spleen indicated that the PFC distribution in these organs (for individual animals) was relatively uniform. The lung data exhibited a slight trend toward higher concentrations in the base region relative to the apex; however, the differences were not statistically significant. The average concentrations of FC-43 in the right and left lungs were equivalent. In addition, there was no observable systematic difference in FC-43 concentration in tissues harvested from pigs euthanatized at different times within the 4- to 7-day period after IP administration. This is consistent with the known long biological residence time of FC-43 in vivo ( Tl,2 - 500 days32).

Calculated p02 Images Figure 6 shows an example of the image data sets

including proton, F-19 first spin-echo, calculated p02 images, and quantitative p02 isobar contour plots for one pig in both the coronal and sagittal views. The proton images (5 mm slice thickness) serve as anatomic localizers to define organ morphology. The lung field and liver are clearly identified. The spleen, which wraps around the left side of the abdomen from anterior to posterior, is not visualized in the proton image as a consequence of the tomographic (thin slice) presentation.

SNR = lOA 0.5

dms)

01 100 300 500 700 900 110013001500

Tl(ms)

V.”

0.5 -

0.4-

g '; 0.3- t;,

0.2-

B SNR=20:1

2 4 dms)

wo*

"ii0 3b0 5k-1 7im 960 nw&oi5bo Tl (ms)

Fig. 5. The fractional uncertainty 6T, /T, as a function of T, for the SR/IR pulse sequences 1504/600/1504, 1504/800/1504, and 1504/1000/1504 (units in ms) with TE = 8 ms. The noise has been taken as constant and equal to (A) 10% (SNR = 10) or (B) 5% (SNR = 20) of the second echo amplitude at T, = 600 ms (corresponding to -100 torr at 37°C for FC-43).

RECTOp02 imaging in porcine model 0 S.R. THOMAS ET AL. 109

Table 3. Tissue FC-43 concentration in six pigs

FC-43 concentration (mg FC-43/g tissue)

Organ Mean +I SD

Liver 93 23 Spleen 353 128 Right lung 90 31 Left lung 89 32

Oxypherol(500 ml) had been administered IP 4 to 7 days prior to sacrifice.

The F-19 first echo projection images, which demonstrate the PFC biodistribution, exhibit signal intensity proportional to the total PFC content (spin density weighted) whereas the second spin-echo image (not shown) is Tl weighted. PFC localization in the liver, spleen, and lungs is evident. PFC uptake in the thymus is noted also. The S2/S 1 ratio obtained on a pixel-by- pixel basis is used as described previously in combina- tion with the T, look-up table and calibration curve to provide the calculated pOZ projection image and contour plots. Pseudocolor calculated p02 images as a function of Fi02 for another pig are presented in Fig. 7.

The inspired oxygen ranged from approximately 150 torr (ambient) to -500 torr. However, as the image data constitutes a projection through whole organs, the effect of pOZ volume averaging must be considered when interpreting the calculated pOZ values.

Correlation of Image Calculated p02 With Airway or Blood ~0,

Regional pOZ values calculated from the mean S2/Sl ratios in the F-19 images have been correlated with the directly sampled pOZ measurements (Table 4). In general, image calculated pOZs for lung, liver, and spleen track positively with FiOz (although to varying degrees), which is reflective of the overall driving force of the inspired oxygen. For the lung, three different potential oxygen sources for equilibration with fluorocarbon resident in tissue are considered: airway, capillary or postcapillary arterialized blood in the pulmonary vein (sampled as arterialized blood from the aorta), and precapillary mixed venous blood (sampled as blood from the pulmonary artery). The calculated pOZ values, taken from regions-of-interest established within the caudal portions (base) of each lung such as not to overIap with liver, trachea, or major bronchi, represent an average p02 for these areas. Composite data from the six pigs for calculated pOZ vs. measured pOZ

Fig. 6. Coronal (A-D) and sagittal (E-H) image data sets for one pig. (A, E) Proton images (5 mm slice thickness) serving as anatomic localizers. The heart (H) is visualized within the thoracic cavity. (B, F) F-19 first echo projection images. Signal intensity is proportional to the total fluorine content across the projection. PFC accumulation is observed in the right and left lungs (RL, LL), liver (Liv), spleen (Sp), and thymus (T). (C, G) Calculated pOZ images for the high FiOl condition. The calculated pOZ signal intensity is dependent on the oxygen level and independent of the PFC concentration. High-intensity artifacts resulting from the processing algorithm present at the edge of the organs are attributed to anomalies associated with the rapidly changing signal-to-noise characteristics in this signal transition region. Regions-of-interest (ROIs) for p02 analysis were chosen to exclude these edge transition zones. (D, H) Isobar contour plots for the high FiOz providing a quantitative readout of pOZ values.

Magnetic Resonance Imaging 0 Volume 14, Number 1, 1996

Fig. 7. Pseudocolor calculated pOz images for another pig in the coronal (A-C) and sagittal (D-F) projections. Three different levels of inspired oxygen are presented: (A, D) ambient air breathing; (B, E) -3 l/min supplemental oxygen, and (C, F) -6 I/min supplemental oxygen. Regions of high pOz are coded red with the lower pOz areas shown in blue. In addition to

the lungs (right, RL; left, LL), liver (Liv), and spleen (Sp), significant concentrations of PFC are observed in the thymus (T) and in marrow of the long bones (humerus, Hu) and vertebrae (V).

in expired air, aortic blood, and pulmonary artery are shown in Fig. 8. The correlation with a slope most nearly approximating unity and a relatively small intercept with respect to the pOZ range was observed for arterialized aortic blood. This suggests that the PFC in lung may be near equilibrium with this oxygen source.

For expired air, the slope was significantly less than 1, indicating reduced oxygenation of the resident pulmonary PFC relative to airway ~0~. Correlation of the calculated lung pOZ with the precapillary mixed venous blood of the pulmonary artery demonstrated a large positive slope with negative intercept straightforwardly

Table 4. Linear regression analysis for six pigs

Organ Site of measured

PO2

No. points

Slope Intercept (torr) Correlation

m SE b SE coefficient

Right lung

Left lung

Liver

Expired air Aorta Pulmonary artery

Expired air Aorta Pulmonary artery

Hepatic vein Aorta

Spleen Aorta

21 21 21 21 21 21 19 19 21

0.61 0.09 -3.7 25.2 1.12 0.11 -36.9 21.3 8.52 1.94 -246.6 92.4 0.64 0.06 -13.9 16.3 1.11 0.08 -38.0 15.5 9.56 1.45 -297.8 69.3 0.95 0.21 -11.4 8.6 0.11 0.02 -8.6 4.6 0.37 0.09 -8.7 16.4

0.87 0.91 0.71 0.93 0.95 0.70 0.73 0.71 0.70

Image calculated p02 in lung, liver, and spleen in correlation with directly measured pOz in airway (expired air), aorta (arterialized blood), pulmonary artery (mixed venous blood), and hepatic vein (hepatic venous blood). (PO&~ = rn(~O~),,,~~~ + b.

RECTOpOz imaging in porcine model 0 S.R. THOMAS ET AL.

0 200 400 600 0 loo 200 300 400 0 10203040506070 Expired Air pO2 (torr) Arterial p02 (torr) Mixed Venous p02 (torr)

300

200

loo

0

111

0200400600 0 100200300400 0 20 40 60 80 Expired Air p02 (~orr) Altcrirl p02 (torr) Mixed Venous pO2 (tom)

Fig. 8. Image calculated pOZ for the right and left lung for all six pigs as a function of directly measured pO1 in the airway (A, D), arterialized blood (aorta) (B, E), and mixed venous blood (pulmonary artery) (C, F). Each individual pig is represented by a different symbol. The linear regression parameters are presented in Table 4. The representative error bars shown (reflect- ing the uncertainty analysis discussed in the text) are taken as +lO% for the high ~0, regime (high SNR) and -+40% for low PO,.

consistent with the fact that the oxygenation level of precapillary venous blood is markedly below that of the airway.

For the liver, linear regression correlation with the venous blood of the hepatic vein (Fig. 9A) showed a slope close to unity consistent with the interpretation that the PFC resident in liver has a p02 in equilibrium with that of venous sinuses. The fact that hepatic oxygen levels under normal physiological conditions are low is reflected in the significantly reduced slope observed in the correlation of the liver calculated pOz with the aortic blood pOa (Fig. 9B). As shown in Fig. 9C, the spleen also exhibits a reduced slope when correlated

with arterial blood. These observations suggest that arterial blood is an unreliable predictor of p02 for PFC resident in splenic tissue. In general, a variable response in calculated pOz was observed for the spleen with some animals exhibiting little or negative enhancement at the highest FiOz and arterial p02 compared to others.

DISCUSSION AND CONCLUSIONS

Systemic oxygen distribution demonstrates a contin- uous gradient from inspired atmosphere air (20.9% oxygen at sea level) to the electron transport chain (near 0%) within cell mitochondria. Oxygen partial pressure

; (6) Liver

i

40

30

v

8 NA

20 fOo* A

10 A 0 .

I50 S leen

(Cl 0 x 0

loo 0 D 0

50

P “.

l 0

0 0 IO 20 30 40 50 60 0 loo 200 300 400 0 loo 200 300 400

Hepatic Vein pO2 (tom) Arterial pO2 (torr) Aaerial pO2 (torr)

Fig. 9. Image calculated pOZ for all six pigs as a function of directly measured p02 in the hepatic vein (A) and aorta (B, C) for the liver and the spleen. Each individual pig is represented by a different symbol. The linear regression parameters are presented in Table 4. The representative error bars shown for the liver are *20% (high ~0~) and +40% (low ~0~). For the spleen, the representative error bars shown are k 10% and *20% at high and low pOZ, respectively.


(~0~) of water vapor equilibrated ambient air at 37°C found in the trachea approximates 150 torr. Pulmonary ventilation at sea level results in an alveolar pOZ between 120 and 100 torr in the normal lung whereas pulmonary diffusion elevates mixed venous blood pOZ from 40 torr to an arterial blood pOz level of approximately 90 to 100 torr. 33 For most organs, it is this arterial blood with fully oxygen saturated hemoglobin that is the sole source of tissue oxygen. In some sinu- soid organs like liver and spleen, significant blood sta- sis and hemoglobin desaturation may occur as the arterial blood transits the organ. In addition, the spleen has the capacity to alter its blood volume and thereby its internal pOZ environment while the liver receives not only oxygenated arterial blood but significantly deoxygenated blood from the gut via the portal vein. The inherent pOZ in each tissue where PFC resides will thus depend upon the nature and composition of the blood supply, the internal architecture of the vascular spaces in the organ, and, understandably, the balance between oxygen supply and local oxygen consumption, which depends on local metabolic rate.

This research has outlined methodology for in vivo monitoring of pOZ in tissues sequestering PFC blood substitute materials using F-19 MRI at low field in the porcine model as an example. Under the experimental configuration involving optimization of the parameters t,/r/tb utilized with the two-point SR/IR pulse sequence described for calculating Tl, it is estimated that the uncertainty in Tl determination for FC-43 may vary from approximately 5% to 20% dependent upon the SNR present over the T, range from 300 to 900 ms corresponding to pot values of -360 torr to -0 torr at body temperature (37°C). Based on the calibration curves, for a f 5% error in T, , the percentage error in the estimated pOZ would be approximately +lO torr (+40%) at 25 torr and k16 torr (kll%) at 150 torr. For a T, error of *20%, the uncertainty in pOZ would increase to +40 torr (k 160%) and +65torr (+43%) at 25 and 150 torr, respectively.

The calculated pOZ projection images of the lung demonstrate a heterogeneous oxygen distribution with maximum pOZ tracking the inspired oxygen. This ob- servation is consistent with the fact that pulmonary blood flow has been reported to be regionally hetero- geneous34 and that local areas of the lung may vary in ventilation efficiency. 33 The strong correlation of image calculated pOZ in the lung with arterialized blood pOZ suggests that the PFC is resident below the alveolar epithelial surface potentially at an interstitial location that permits near equilibrium with this oxygen pool rather than with that of the external airway. Light and electron microscopy studies of lung tissue following infusion of FC-43 (Oxypherol) in rabbits by Narmey

et al.35 demonstrated that alveolar interstitial phagocytes accumulate PFC extensively without any concentration observed in alveolar type 1 and 2 cells. In addition, lesser concentrations were noted in endothelial cells of the pulmonary capillaries within alveolar walls and muscular arteries.

The calculated liver PFC pOZ values (16 + 7 torr) obtained at room air (for arterial and venous blood pOZ values of 75 + 21 torr and 37 f 6 torr, respectively) were consistent with those obtained by Kessler et a1.36 using a lo-15 pm platinum wire electrode in mammalian liver in situ (pOZ range 1 to 56 torr with a mean of 33 torr). We obtained only modest elevations in liver pOZ values for significant increases in arterial ~0~. A strong correlation is evident between the pOZ of PFC in liver and that of venous blood (hepatic vein). This response is reflective of the fact that the liver receives deoxygenated blood (via portal vein) as well as systemic arterial blood (via hepatic artery). PFC uptake is observed to be primarily within the Kupffer cells to an extent where the cytoplasm may be engorged with PFC vacuoles allowing significant opportunity for pOZ equilibration with the blood in the sinusoids.35 PFC particles were also observed in the endothelial cells but only rarely detected within hepatocytes. The calculated pOa values for PFC in spleen (25 f 4 torr) were also consistent with those previously reported by Vaupel et al. ,37 who used gold microelectrodes (l-5 pm tip diameter) to make measurements in spleens of animals breathing room air. They observed that the splenic red pulp pOZ distribution ranged from 15 to 95 torr with a mean of 53 torr when arterial and venous blood pOZ values were 95 and 55 torr, respectively. In our studies, we found the spleen to exhibit variable results with dramatic elevation in pOZ at the highest FiOz in some animals and little or negative enhancement in others. In the rabbit studies of Nanney et a1.p5 the PFC has been found to reside in macrophages that focally in- vade the red pulp of the spleen, often with distention of the venous sinusoids. The white pulp was consider- ably less involved and then usually around arterioles. Although no splenic venous blood samples were taken, our data are consistent with the possibility that PFC residing in splenic tissue is in equilibrium with the venous blood pool, in agreement with measurements in other mammalian model systems.37

The current work has demonstrated the feasibility of quantitative pOZ imaging in vivo using F- 19 MRI of sequestered PFC materials and has defined points of relevance associated with evaluation of the accuracy obtainable. Projection images were utilized to improve the SNR encountered at low magnetic field. However, interpretation of such projection data in the context of physiological functions has recognized limitations. Fu-

RECTOpOl imaging in porcine model 0 S.R. THOMAS ET AL. 113

ture investigations at higher fields will allow implementation of slice selection protocols as required to determine the true three-dimensional distribution of oxygen within organs. Integration of three-dimensional magnetic resonance imaging of pOZ with other mark- ers of physiological function including, for example, perfusion and metabolism (also obtainable from MR protocols), hold the promise of yielding important new physiological correlations of relevance for diagnosis of regional oxygen distribution and utilization.

Acknowledgments-The authors thank Scott K. Holland, Ph.D., for helpful comments upon review of the manuscript. Supported in part by NIH Grants ROl HL45243, PO1 HL22619 (Core I), and HL07382.

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In vivo pO2 imaging in the porcine model with perfluorocarbon F-19 NMR at low field

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