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Magnetic resonance imaging of intracellular sodium
JOURNAL
OF MAGNETIC
RESONANCE
83,197-204
( 1989)
Magnetic Resonance Imaging of Intracellular Sodium
*Department of Radiology, Beth Israel Hospital and Harvard Medical School, 330 Brookline Avenue, Boston, Massachusetts 02215; and $ Bruker Instruments, Inc., I9 Fortune Drive, Manning Park, Billerica, Massachusetts 01821 Received December 27, 1988
Sodium nuclear magnetic resonance studies have the potential of contributing physiologic and clinical information which is unavailable from either proton or phosphorus NMR studies. Applications include the study of normal cardiac or renal physiology, electrophysiologic events such as the alteration of intracellular sodium levels due to either pharmacologic intervention or pathologic events, and tumor processes. Recently, sodium NMR spectroscopy, combined with the use of shift reagents, has enabled monitoring of changes in the bulk intracellular sodium levels in excised or perfused organs or cells (Z-9). However, these studies, based on a composite signal from the whole sample, are unable to detect local changes in intracellular sodium. One-dimensional information regarding localized intra- versus extracellular sodium has recently been obtained with a rotating-frame experiment with shift reagents and a perfused rabbit heart ( IO). In vitro or in vivo two-dimensional sodium images have demonstrated local variations in the combined intra- and extracellular sodium in a perfused heart ( 1 I ), excised hearts ( Z2), and the normal brain and have demonstrated high contrast in regions of pathology ( 13, 14). In vivo intra- plus extracellular sodium images of other organs are also feasible ( 15-Z 7). While these images show promise for detecting earlier pathologic changes or for yielding improved contrast relative to the corresponding proton images, the image interpretation would be greatly enhanced if the spatial variation of intra- or extracellular sodium could be directly observed. In addition, this differentiation would permit the local effects of electrophysiologic events to be studied in perfused organs or in vivo. Until now, several difficulties have precluded magnetic resonance imaging of intracellular sodium. One difficulty is that of separating the signals from the intra- and extracellular sodium. Once these signals can be differentiated, a technique is needed which can image the intracellular sodium while avoiding interference from the extracellular sodium signal. This is made more difficult due to the normally high extracellular sodium signal, such that even a small degree of interference from the extracellular signal can significantly mask the intracellular signal. In addition, the very short NMR relaxation times of sodium lead to hardware constraints of fast gradient switching in order to yield short echo time images. Finally, the inherent sodium NMR signal t To whom correspondence should be addressed. 197
0022-2364189 $3.00 Copyri@t 0 1989 by Academic Res, Inc. All rights of reproduction in any form reserved.
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is relatively weak, resulting in a relatively low signal-to-noise ratio in an image of intracellular sodium unless significant signal averaging is utilized. In this study, two-dimensional images of intracellular sodium in a perfused heart were obtained. This was accomplished through the addition of a shift reagent to the perfusate in combination with the use of a chemical-shift imaging pulse sequence. The shift reagent alters the resonant frequency of the cations with which it has contact. Since the cell membrane is impermeable to the shift reagent, the resonant frequency of only the extracellular cations is altered while the resonant frequency of the intracellular cations is not affected. Therefore, the intra- and extracellular sodium signals each have distinct resonant frequencies. The chemical-shift imaging pulse sequence permits an image of the sodium to be obtained at a particular resonant frequency. As described below, the pulse sequence utilizes two different pulses to obtain full suppression of the extracellular sodium signal. This pulse sequence has the added advantage of being easily altered to a localized spectroscopy sequence. In order to accomplish this, the read and phase-encoding gradients are turned off, and a spectrum is obtained from the slice which is to be imaged. With this localized spectroscopy sequence, the level of suppression of the extracellular resonance can be verified. In addition, microimaging hardware was utilized in order to eliminate some of the technical problems associated with sodium magnetic resonance imaging. Microimaging techniques have several advantages: High pixel resolution allows high resolution images of perfused organs to be obtained, fast gradient switching without the generation of significant eddy currents allows for short echo time acquisitions, and insignificant gradient heating problems allow for high repetition rates for signal averaging. All studies were performed on a Bruker 9.4 T wide-bore spectrometer operating at 105.8 MHz at Bruker Instruments, Inc., Billerica, Massachusetts. The spectrometer was equipped with a microimaging accessory with 50 mm diameter gradient coils. In all cases, the images were obtained with a field of view of 5.7 cm (corresponding to 10.3 G/cm) across a 128 by 128 matrix to yield a resolution of 0.44 mm and a slice thickness of 5 mm (7.5 G/cm). The radiofrequency and gradient pulse sequences used for the sodium imaging are shown in Fig. 1. Sodium imaging experiments were performed on a perfused heart preparation. The heart was excised from a Southern grassfrog (Ram pipiens), and perfused in a 10 mm o.d. NMR tube as previously described (9)) and shown in Fig. 2a. In order to increase intracellular sodium, the perfusate contained 20 PM ouabain and no potassium. The perlusate in this case also contained 6 mM dysprosium tripolyphosphate shift reagent (18). Cross-sectional images were obtained through the ventricular region (Fig. 2b). A 12 mm RF coil was used. The spectra obtained from the perfused heart preparation are shown in Figs. 2c and 2d. The standard spectrum demonstrates a large shifted (extracellular) resonance and a smaller unshifted (intracellular) resonance. The spectrum from the chemicalshift-selective sequence demonstrates complete suppression of the shifted extracellular resonance. The slight (approximately 25%) decrease in the intracellular resonance is probably due to the slightly longer echo time of the chemical-shift sequence (see Fig. 1 legend) and possibly also due to the less efficient refocusing of the relatively long (500 j,~s)180” soft pulse.
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(a) RF
Slice
Phase
Read
Receiver
(b)
SUPP
cs~
Slice
Obs css
FIG. 1. (a) RF and gradient sequence utilized for standard sodium imaging. The slice-selective 90” pulse is a Gaussian of 250 FLSduration and the 180’ pulse is a hard pulse of 150 ps duration. The echo time was 2.6 ms, and the repetition time was 70 ms. In order to obtain spectra from the slices which would be imaged (slice-selective spectra), the read and phase-encoding gradients were turned off. (The slight timing correction to transform the data from the peak of the echo was performed postacquisition.) (b) RF pulse sequence utilized for chemical-shift imaging. A chemical-shift-selective pulse (at the frequency to be suppressed) of 2500 ps duration preceded the slice-selective 90” pulse (250 ps), which was followed by a chemical-shift-selective 180” pulse (at the frequency to be observed) of 500 ps duration. All pulses were Gaussian shaped. The echo time was 2.9 ms. Chemical-shift slice-selective spectra were obtained by running the same sequence without the read and phase-encoding gradients. The combined use of the two chemical-shift-selective pulses was found to yield much better chemical-shift selectivity than either alone. Similar results have recently been demonstrated with proton chemical-shift imaging (29).
A standard sodium image of a perfused heart is shown in Fig. 2e. As expected, the brightest signal arises from the saline in the ventricular cavity. The chemical-shift image, with suppression of the extracellular signal, is shown in Fig. 2f. As indicated from the chemical-shift-selective spectrum in Fig. 2d, the signal arises totally from the intracellular ions and therefore represents an image of intracellular sodium in the myocardial wall. The drop out of one side of the myocardial wall from the image is most likely due to loss of signal due to motion effects, since the free wall (which was not positioned against the wall of the tube), exhibited a significant amount of motion due to the action of the peristaltic perfusion pump. The suppression of the extracellular signal in the spectrum was verified both before and after the imaging sequence. The future application of these techniques to the in vivo situation holds great promise. The shift reagent dysprosium triethylenetetramine hexaacetic acid (19) has been demonstrated to be nontoxic in vivo (20)) and may therefore be useful for in vivo studies in organs such as the heart. However, its full distribution in vivo is still under question, especially in the brain where it may not cross the blood-brain barrier (21,
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22). Alternatively, two other techniques have been suggested as a means of differentiating between intra- and extracellular sodium in NMR studies. The first is based on the observation that intracellular sodium ions may have a component of the NMR spin-lattice relaxation time on the order of several milliseconds (23). Therefore, if this short T2 component was imaged (by subtracting late echo images from early echo images after suitable correction for T2 decay) it was suggested that the image would represent a primarily intracellular image. This preferential imaging of cellular regions has been demonstrated in clinical sodium imaging ( 13). However, interstitial T2 relaxation times have not yet been quantified, and therefore it is difficult to determine the degree to which the short T2 or “intracellular” images contain signal from the interstitial ions. In addition, the degree of interference from the interstitial signal may vary under pathologic conditions where various degrees of edema may alter interstitial relaxation times. Another proposal for the separation of intra- and extracellular signals has been for the use of double quantum filter pulse sequences (24). These sequences generate signal only from nuclei which exhibit biexponential T, NMR relaxation (25)) as in the case of the sodium nucleus with spin-z and significant quadrupolar interactions. The basis of the double quantum filter technique for the detection of intracellular sodium is that several studies have shown the intracellular sodium T2 to be biexponential(23,26), and therefore if the extracellular sodium did not display this behavior, the double quantum filter could be used to preferentially detect intracellular sodium. However, it is again unknown if the interstitial ions in general exhibit biexponential relaxation and hence if they contribute to the double quantum filter output. The perfused organ preparation and methods described in this paper are ideal for comparing the results of the shift reagent, relaxation time, and double quantum filter techniques for imaging intracellular sodium, with either perfused organ or in vivo applications. The high resolution in vivo sodium images obtainable with the microimaging apparatus were demonstrated by obtaining in vivo sodium images of tumorbearing nude mice. The mice were injected subcutaneously with COLO 205 (colon
FIG. 2. (a) Schematic illustration of the perfused heart apparatus. Briefly, the atria1 input was cannulated such that the perfusate washed through the atria, into the ventricle, and out the arterial branches. (Frog hearts consist of two atria and one ventricle; there are no coronary arteries.) The efflux was allowed to drip down to the bottom of the tube where it was suctioned, such that the heart was suspended in air although there was a significant perfusate pool in the ventricular cavity. (b) Cross-sectional schematic view of the perfused heart through the ventricular region, corresponding to the imaging plane shown in ( e) and ( f ) . (c) The standard sodium spectrum demonstrates both an intracellular resonance (at approximately 0 ppm ) and an extracellular resonance ( 15 ppm). (d) The chemical-shift-selective spectrum shows that this sequence totally eliminated the signal from the extracellular sodium while retaining the signal from the intracellular sodium. Both spectra were obtained with 5 12 averages. (e) Sodium image of the perfused heart obtained with the standard pulse sequence, corresponding to the spectrum in (c). The brightest signal arises from the saline in the ventricular cavity and along the edge of the tube. The signal from the myocardial wall is due to both intra- and extracellular sodium. The image was obtained with 100 averages. (f) The chemical-shift-selective (intracellular) image, corresponding to the spectrum in (d), displays only the intracellular sodium in the ventricular wall. (The image is displayed on a scale different than that of the standard image.) The image was obtained with 500 averages, although even 200 averages yielded a reasonable signal-to-noise ratio.
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adenocarcinoma) cells (American Type Culture Collection, Rockville, Maryland), and the tumors were allowed to grow for approximately 2 weeks before imaging. A 28 mm RF coil was used for these studies. The images, shown in Fig. 3, clearly delineate two tumors in one case (Fig. 3a), with some structure within the tumors. Figure 3b illustrates an image ofa cyst, with much higher contrast relative to the surrounding tissue sodium. These high resolution, short echo time images will enable changes in total sodium content during tumor growth to be monitored. Although shift reagent may not penetrate fully into tumors or cysts, the well-defined system of perfused organs described above will hopefully enable other techniques to be developed, tested, and compared for imaging intracellular sodium. Changes in intracellular sodium content in the tumors may then possibly be differentiable from changes in extracellular edema. The technique demonstrated in this report has a wide variety of potential applications. One important application is the ability to monitor local variations or local changes in bulk sodium content or intracellular sodium in perfused or in vivo organs (such as the kidney or heart) under normal or pathologic conditions. It should be noted that all measurement techniques have some inherent bias. Microelectrode studies reflect sodium activity; the NMR studies reflect the sodium concentration weighted mainly by the sodium T2 value. This includes the possibility that there may be some fraction of sodium which is not observed through these studies due to a very short T2, such that the signal decays before the echo time. However, a large fraction of the intracellular sodium in hearts has been shown to be NMR observable under a
b
FIG. 3. In vivo sodium images of tumor-bearing nude mice, obtained with the standard pulse sequence. (a) Two tumors (of approximately 1 cm diameter) are seen (arrows). In addition, some structure can be seen within the tumor. The image was acquired with 100 averages. (b) Very high contrast is obtained from a cyst (arrow) of approximately 1 cm diameter. The cyst was located high on the mouse’s back, and the bright area toward the center line is probably due to cerebral spinal fluid. The image was obtained with 80 averages.
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conditions similar to these studies ( 9,27). In addition, in at least one case of pharmacologic intervention (ouabain) in the heart, the Tz of the NMR observable intracellular sodium was found to be invariant with the rise of intracellular sodium levels (23). The bulk sodium relaxation times of excised tissues were also found to be the same in the tumor and the surrounding normal tissue (28). Therefore, in these casesNMR may be used in a straightforward manner to follow relative changes in the local NMR observable sodium content. In general, one must keep in mind that the signal level in a sodium image displays information regarding both the sodium content and the NMR relaxation times, T, and T, . In summary, the first two-dimensional magnetic resonance images of intracellular sodium have been demonstrated with a perfused frog heart. In addition, high resolution in vivo sodium images were obtained from tumor-bearing nude mice. These developed techniques should greatly enhance the recent studies of sodium NMR spectroscopy in which bulk changes in intracellular sodium are monitored, and the recent sodium imaging studies in which information is obtained regarding local variations in the combined intra- and extracellular sodium. ACKNOWLEDGMENTS We thank Dr. Eric Fossel for his continued support and helpful suggestions. This part by Grant HL-38906 (D. Burstein, PI.) from the National Institutes of Health.
work
was funded
in
REFERENCES I. M. J. AVISON, S. R. GULLANS, T. OGINO, G. GIEBISCH, AND R. G. SHULMAN, Am. J. Physiol. 253, C126( 1987). 2. Y. B~ULANGER, P. VINAY, AND M. BOULANGER, Am. J. Physiol. 253, F904 ( 1987). 3. M. M. PIKE, J. C. FRAZER, D. F. DEDRICK, J. S. INGWALL, P. D. ALLEN, C. S. SPRINGER, JR., AND T. W. SMITH, Biophys. J. 48,159 ( 1985). 4. Y. SEO, M. MURAKAMI, T. MATSUMOTO, H. NISHIKAWA, AND H. WATARI, J. Magn. Reson. 72,341
(1987). B. M. RAYSON AND R. K. GUPTA, J. Biol. Chem. 260,7276 ( 1985). M. M. CIVAN, H. DEGANI, Y. MARGALIT, AND M. SHPORER, Am. Physiol. Sot. 245, C213 ( 1983). A. KUMAR, A. SPITZER, AND R. K. GUPTA, Kidney Int. 29,747 ( 1986). C. S. SPRINGER, JR., Annu. Rev. Biophys. 16,375 (1987). 9. D. BURSTEIN AND E. T. FOSSEL, Am. J. Physiol. 252, H 1138 ( 1987). IO. C. T. W. MOONEN, S. E. ANDERSON, AND S. UNGER, Magn. Reson. Med. 5,296 ( 1987). Il. J. L. DELAYRE, J. S. INGWALL, C. MALLOY, AND E. T. FOSSEL, .Science212,935 ( 1982). 12. P. J. CANNON, A. A. MAUDSLEY, S. K. HILAL, H. E. SIMON, AND F. CASSIDY, J. Am. CON.Cardiol. 5. 6. 7. 8.
7,573 (1986).
13. S. K.
HILAL, J. B. RA, C. H. OH, I. K. MUN, S. G. EINSTEIN, AND P. ROSCHMANN, in “Magnetic Resonance Imaging” (D. D. Stark and W. G. Bradley, Jr., Eds.), p. 7 15, Mosby, St. Louis, 1988. II. P. A. TURSKI, L. W. HOUSTON, W. H. PERMAN, J. K. HALD, D. TURSKI, C. M. STROTHER, AND J. F. SACKETT, Radiology 163,245 ( 1987). IS. J. B. RA, S. K. HILAL, C. H. OH, AND I. K. MUN, Magn. Reson. Med. 7,ll ( 1988). 16. J. GRANOT, Radiology 167,547 ( 1988). 17. H. L. KUNDEL, A. SHETTY, P. M. JOSEPH, R. M. SUMMERS, E. A. KASSAB, AND B. MOORE, Magn. Reson. Med. 6,38 1 ( 1988).
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18. R. K. GU~TA AND P. GUPTA, J. Magn. Resort. 47,344 ( 1982). 19. S. C. CHU, M. M. PIKE, E. T. FOSSEL, T. W. SMITH, J. A. BALSCHI, AND C. S. SPRINGER, JR., J. Magn. Reson. 56,33 ( 1984). 20. H. NARITOMI, M. KANASHIRO, M. SASAIU, Y. KURIBAYASHI, AND T. SAWADA, Biophys. J. 52,6 11 (1987). 21. D. BURSTEIN, Biophys. J. 54,191( 1988). 22. H. NARITOMI, M. SASAKI, Y. KURIBAYASHI, AND M. KANASHIRO, Biophys. J. 54,193 ( 1988). 23. D. BURSTEIN AND E. T. FOSSEL,Magn. Reson. Med. 4,26 I ( 1987). 24. J. PEKAR, P. F. RENSHAW, AND J. S. LEIGH, JR., J. Magn. Reson. 72,159 ( 1987). 25. J. PEKAR AND J. S. LEIGH, JR., J. Magn. Reson. 69,582 ( 1986). 26. H. SHINAR AND G. NAVON, Biophys. Chem. 20,275 ( 1984). 27. E. T. FOSSEL AND H. HOEFELER, Magn. Reson. Med. 3,534 ( 1986). 28. L. P. CHEUNG, M.D. thesis, Harvard University-Massachusetts Institute of Technology Division of Health Sciences and Technology, 1988. 29. P. M. JOSEPH AND A. SHETTY, Magn. Reson. Imaging 6,42 I( 1988).
OF MAGNETIC
RESONANCE
83,197-204
( 1989)
Magnetic Resonance Imaging of Intracellular Sodium
*Department of Radiology, Beth Israel Hospital and Harvard Medical School, 330 Brookline Avenue, Boston, Massachusetts 02215; and $ Bruker Instruments, Inc., I9 Fortune Drive, Manning Park, Billerica, Massachusetts 01821 Received December 27, 1988
Sodium nuclear magnetic resonance studies have the potential of contributing physiologic and clinical information which is unavailable from either proton or phosphorus NMR studies. Applications include the study of normal cardiac or renal physiology, electrophysiologic events such as the alteration of intracellular sodium levels due to either pharmacologic intervention or pathologic events, and tumor processes. Recently, sodium NMR spectroscopy, combined with the use of shift reagents, has enabled monitoring of changes in the bulk intracellular sodium levels in excised or perfused organs or cells (Z-9). However, these studies, based on a composite signal from the whole sample, are unable to detect local changes in intracellular sodium. One-dimensional information regarding localized intra- versus extracellular sodium has recently been obtained with a rotating-frame experiment with shift reagents and a perfused rabbit heart ( IO). In vitro or in vivo two-dimensional sodium images have demonstrated local variations in the combined intra- and extracellular sodium in a perfused heart ( 1 I ), excised hearts ( Z2), and the normal brain and have demonstrated high contrast in regions of pathology ( 13, 14). In vivo intra- plus extracellular sodium images of other organs are also feasible ( 15-Z 7). While these images show promise for detecting earlier pathologic changes or for yielding improved contrast relative to the corresponding proton images, the image interpretation would be greatly enhanced if the spatial variation of intra- or extracellular sodium could be directly observed. In addition, this differentiation would permit the local effects of electrophysiologic events to be studied in perfused organs or in vivo. Until now, several difficulties have precluded magnetic resonance imaging of intracellular sodium. One difficulty is that of separating the signals from the intra- and extracellular sodium. Once these signals can be differentiated, a technique is needed which can image the intracellular sodium while avoiding interference from the extracellular sodium signal. This is made more difficult due to the normally high extracellular sodium signal, such that even a small degree of interference from the extracellular signal can significantly mask the intracellular signal. In addition, the very short NMR relaxation times of sodium lead to hardware constraints of fast gradient switching in order to yield short echo time images. Finally, the inherent sodium NMR signal t To whom correspondence should be addressed. 197
0022-2364189 $3.00 Copyri@t 0 1989 by Academic Res, Inc. All rights of reproduction in any form reserved.
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is relatively weak, resulting in a relatively low signal-to-noise ratio in an image of intracellular sodium unless significant signal averaging is utilized. In this study, two-dimensional images of intracellular sodium in a perfused heart were obtained. This was accomplished through the addition of a shift reagent to the perfusate in combination with the use of a chemical-shift imaging pulse sequence. The shift reagent alters the resonant frequency of the cations with which it has contact. Since the cell membrane is impermeable to the shift reagent, the resonant frequency of only the extracellular cations is altered while the resonant frequency of the intracellular cations is not affected. Therefore, the intra- and extracellular sodium signals each have distinct resonant frequencies. The chemical-shift imaging pulse sequence permits an image of the sodium to be obtained at a particular resonant frequency. As described below, the pulse sequence utilizes two different pulses to obtain full suppression of the extracellular sodium signal. This pulse sequence has the added advantage of being easily altered to a localized spectroscopy sequence. In order to accomplish this, the read and phase-encoding gradients are turned off, and a spectrum is obtained from the slice which is to be imaged. With this localized spectroscopy sequence, the level of suppression of the extracellular resonance can be verified. In addition, microimaging hardware was utilized in order to eliminate some of the technical problems associated with sodium magnetic resonance imaging. Microimaging techniques have several advantages: High pixel resolution allows high resolution images of perfused organs to be obtained, fast gradient switching without the generation of significant eddy currents allows for short echo time acquisitions, and insignificant gradient heating problems allow for high repetition rates for signal averaging. All studies were performed on a Bruker 9.4 T wide-bore spectrometer operating at 105.8 MHz at Bruker Instruments, Inc., Billerica, Massachusetts. The spectrometer was equipped with a microimaging accessory with 50 mm diameter gradient coils. In all cases, the images were obtained with a field of view of 5.7 cm (corresponding to 10.3 G/cm) across a 128 by 128 matrix to yield a resolution of 0.44 mm and a slice thickness of 5 mm (7.5 G/cm). The radiofrequency and gradient pulse sequences used for the sodium imaging are shown in Fig. 1. Sodium imaging experiments were performed on a perfused heart preparation. The heart was excised from a Southern grassfrog (Ram pipiens), and perfused in a 10 mm o.d. NMR tube as previously described (9)) and shown in Fig. 2a. In order to increase intracellular sodium, the perfusate contained 20 PM ouabain and no potassium. The perlusate in this case also contained 6 mM dysprosium tripolyphosphate shift reagent (18). Cross-sectional images were obtained through the ventricular region (Fig. 2b). A 12 mm RF coil was used. The spectra obtained from the perfused heart preparation are shown in Figs. 2c and 2d. The standard spectrum demonstrates a large shifted (extracellular) resonance and a smaller unshifted (intracellular) resonance. The spectrum from the chemicalshift-selective sequence demonstrates complete suppression of the shifted extracellular resonance. The slight (approximately 25%) decrease in the intracellular resonance is probably due to the slightly longer echo time of the chemical-shift sequence (see Fig. 1 legend) and possibly also due to the less efficient refocusing of the relatively long (500 j,~s)180” soft pulse.
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(a) RF
Slice
Phase
Read
Receiver
(b)
SUPP
cs~
Slice
Obs css
FIG. 1. (a) RF and gradient sequence utilized for standard sodium imaging. The slice-selective 90” pulse is a Gaussian of 250 FLSduration and the 180’ pulse is a hard pulse of 150 ps duration. The echo time was 2.6 ms, and the repetition time was 70 ms. In order to obtain spectra from the slices which would be imaged (slice-selective spectra), the read and phase-encoding gradients were turned off. (The slight timing correction to transform the data from the peak of the echo was performed postacquisition.) (b) RF pulse sequence utilized for chemical-shift imaging. A chemical-shift-selective pulse (at the frequency to be suppressed) of 2500 ps duration preceded the slice-selective 90” pulse (250 ps), which was followed by a chemical-shift-selective 180” pulse (at the frequency to be observed) of 500 ps duration. All pulses were Gaussian shaped. The echo time was 2.9 ms. Chemical-shift slice-selective spectra were obtained by running the same sequence without the read and phase-encoding gradients. The combined use of the two chemical-shift-selective pulses was found to yield much better chemical-shift selectivity than either alone. Similar results have recently been demonstrated with proton chemical-shift imaging (29).
A standard sodium image of a perfused heart is shown in Fig. 2e. As expected, the brightest signal arises from the saline in the ventricular cavity. The chemical-shift image, with suppression of the extracellular signal, is shown in Fig. 2f. As indicated from the chemical-shift-selective spectrum in Fig. 2d, the signal arises totally from the intracellular ions and therefore represents an image of intracellular sodium in the myocardial wall. The drop out of one side of the myocardial wall from the image is most likely due to loss of signal due to motion effects, since the free wall (which was not positioned against the wall of the tube), exhibited a significant amount of motion due to the action of the peristaltic perfusion pump. The suppression of the extracellular signal in the spectrum was verified both before and after the imaging sequence. The future application of these techniques to the in vivo situation holds great promise. The shift reagent dysprosium triethylenetetramine hexaacetic acid (19) has been demonstrated to be nontoxic in vivo (20)) and may therefore be useful for in vivo studies in organs such as the heart. However, its full distribution in vivo is still under question, especially in the brain where it may not cross the blood-brain barrier (21,
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22). Alternatively, two other techniques have been suggested as a means of differentiating between intra- and extracellular sodium in NMR studies. The first is based on the observation that intracellular sodium ions may have a component of the NMR spin-lattice relaxation time on the order of several milliseconds (23). Therefore, if this short T2 component was imaged (by subtracting late echo images from early echo images after suitable correction for T2 decay) it was suggested that the image would represent a primarily intracellular image. This preferential imaging of cellular regions has been demonstrated in clinical sodium imaging ( 13). However, interstitial T2 relaxation times have not yet been quantified, and therefore it is difficult to determine the degree to which the short T2 or “intracellular” images contain signal from the interstitial ions. In addition, the degree of interference from the interstitial signal may vary under pathologic conditions where various degrees of edema may alter interstitial relaxation times. Another proposal for the separation of intra- and extracellular signals has been for the use of double quantum filter pulse sequences (24). These sequences generate signal only from nuclei which exhibit biexponential T, NMR relaxation (25)) as in the case of the sodium nucleus with spin-z and significant quadrupolar interactions. The basis of the double quantum filter technique for the detection of intracellular sodium is that several studies have shown the intracellular sodium T2 to be biexponential(23,26), and therefore if the extracellular sodium did not display this behavior, the double quantum filter could be used to preferentially detect intracellular sodium. However, it is again unknown if the interstitial ions in general exhibit biexponential relaxation and hence if they contribute to the double quantum filter output. The perfused organ preparation and methods described in this paper are ideal for comparing the results of the shift reagent, relaxation time, and double quantum filter techniques for imaging intracellular sodium, with either perfused organ or in vivo applications. The high resolution in vivo sodium images obtainable with the microimaging apparatus were demonstrated by obtaining in vivo sodium images of tumorbearing nude mice. The mice were injected subcutaneously with COLO 205 (colon
FIG. 2. (a) Schematic illustration of the perfused heart apparatus. Briefly, the atria1 input was cannulated such that the perfusate washed through the atria, into the ventricle, and out the arterial branches. (Frog hearts consist of two atria and one ventricle; there are no coronary arteries.) The efflux was allowed to drip down to the bottom of the tube where it was suctioned, such that the heart was suspended in air although there was a significant perfusate pool in the ventricular cavity. (b) Cross-sectional schematic view of the perfused heart through the ventricular region, corresponding to the imaging plane shown in ( e) and ( f ) . (c) The standard sodium spectrum demonstrates both an intracellular resonance (at approximately 0 ppm ) and an extracellular resonance ( 15 ppm). (d) The chemical-shift-selective spectrum shows that this sequence totally eliminated the signal from the extracellular sodium while retaining the signal from the intracellular sodium. Both spectra were obtained with 5 12 averages. (e) Sodium image of the perfused heart obtained with the standard pulse sequence, corresponding to the spectrum in (c). The brightest signal arises from the saline in the ventricular cavity and along the edge of the tube. The signal from the myocardial wall is due to both intra- and extracellular sodium. The image was obtained with 100 averages. (f) The chemical-shift-selective (intracellular) image, corresponding to the spectrum in (d), displays only the intracellular sodium in the ventricular wall. (The image is displayed on a scale different than that of the standard image.) The image was obtained with 500 averages, although even 200 averages yielded a reasonable signal-to-noise ratio.
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adenocarcinoma) cells (American Type Culture Collection, Rockville, Maryland), and the tumors were allowed to grow for approximately 2 weeks before imaging. A 28 mm RF coil was used for these studies. The images, shown in Fig. 3, clearly delineate two tumors in one case (Fig. 3a), with some structure within the tumors. Figure 3b illustrates an image ofa cyst, with much higher contrast relative to the surrounding tissue sodium. These high resolution, short echo time images will enable changes in total sodium content during tumor growth to be monitored. Although shift reagent may not penetrate fully into tumors or cysts, the well-defined system of perfused organs described above will hopefully enable other techniques to be developed, tested, and compared for imaging intracellular sodium. Changes in intracellular sodium content in the tumors may then possibly be differentiable from changes in extracellular edema. The technique demonstrated in this report has a wide variety of potential applications. One important application is the ability to monitor local variations or local changes in bulk sodium content or intracellular sodium in perfused or in vivo organs (such as the kidney or heart) under normal or pathologic conditions. It should be noted that all measurement techniques have some inherent bias. Microelectrode studies reflect sodium activity; the NMR studies reflect the sodium concentration weighted mainly by the sodium T2 value. This includes the possibility that there may be some fraction of sodium which is not observed through these studies due to a very short T2, such that the signal decays before the echo time. However, a large fraction of the intracellular sodium in hearts has been shown to be NMR observable under a
b
FIG. 3. In vivo sodium images of tumor-bearing nude mice, obtained with the standard pulse sequence. (a) Two tumors (of approximately 1 cm diameter) are seen (arrows). In addition, some structure can be seen within the tumor. The image was acquired with 100 averages. (b) Very high contrast is obtained from a cyst (arrow) of approximately 1 cm diameter. The cyst was located high on the mouse’s back, and the bright area toward the center line is probably due to cerebral spinal fluid. The image was obtained with 80 averages.
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conditions similar to these studies ( 9,27). In addition, in at least one case of pharmacologic intervention (ouabain) in the heart, the Tz of the NMR observable intracellular sodium was found to be invariant with the rise of intracellular sodium levels (23). The bulk sodium relaxation times of excised tissues were also found to be the same in the tumor and the surrounding normal tissue (28). Therefore, in these casesNMR may be used in a straightforward manner to follow relative changes in the local NMR observable sodium content. In general, one must keep in mind that the signal level in a sodium image displays information regarding both the sodium content and the NMR relaxation times, T, and T, . In summary, the first two-dimensional magnetic resonance images of intracellular sodium have been demonstrated with a perfused frog heart. In addition, high resolution in vivo sodium images were obtained from tumor-bearing nude mice. These developed techniques should greatly enhance the recent studies of sodium NMR spectroscopy in which bulk changes in intracellular sodium are monitored, and the recent sodium imaging studies in which information is obtained regarding local variations in the combined intra- and extracellular sodium. ACKNOWLEDGMENTS We thank Dr. Eric Fossel for his continued support and helpful suggestions. This part by Grant HL-38906 (D. Burstein, PI.) from the National Institutes of Health.
work
was funded
in
REFERENCES I. M. J. AVISON, S. R. GULLANS, T. OGINO, G. GIEBISCH, AND R. G. SHULMAN, Am. J. Physiol. 253, C126( 1987). 2. Y. B~ULANGER, P. VINAY, AND M. BOULANGER, Am. J. Physiol. 253, F904 ( 1987). 3. M. M. PIKE, J. C. FRAZER, D. F. DEDRICK, J. S. INGWALL, P. D. ALLEN, C. S. SPRINGER, JR., AND T. W. SMITH, Biophys. J. 48,159 ( 1985). 4. Y. SEO, M. MURAKAMI, T. MATSUMOTO, H. NISHIKAWA, AND H. WATARI, J. Magn. Reson. 72,341
(1987). B. M. RAYSON AND R. K. GUPTA, J. Biol. Chem. 260,7276 ( 1985). M. M. CIVAN, H. DEGANI, Y. MARGALIT, AND M. SHPORER, Am. Physiol. Sot. 245, C213 ( 1983). A. KUMAR, A. SPITZER, AND R. K. GUPTA, Kidney Int. 29,747 ( 1986). C. S. SPRINGER, JR., Annu. Rev. Biophys. 16,375 (1987). 9. D. BURSTEIN AND E. T. FOSSEL, Am. J. Physiol. 252, H 1138 ( 1987). IO. C. T. W. MOONEN, S. E. ANDERSON, AND S. UNGER, Magn. Reson. Med. 5,296 ( 1987). Il. J. L. DELAYRE, J. S. INGWALL, C. MALLOY, AND E. T. FOSSEL, .Science212,935 ( 1982). 12. P. J. CANNON, A. A. MAUDSLEY, S. K. HILAL, H. E. SIMON, AND F. CASSIDY, J. Am. CON.Cardiol. 5. 6. 7. 8.
7,573 (1986).
13. S. K.
HILAL, J. B. RA, C. H. OH, I. K. MUN, S. G. EINSTEIN, AND P. ROSCHMANN, in “Magnetic Resonance Imaging” (D. D. Stark and W. G. Bradley, Jr., Eds.), p. 7 15, Mosby, St. Louis, 1988. II. P. A. TURSKI, L. W. HOUSTON, W. H. PERMAN, J. K. HALD, D. TURSKI, C. M. STROTHER, AND J. F. SACKETT, Radiology 163,245 ( 1987). IS. J. B. RA, S. K. HILAL, C. H. OH, AND I. K. MUN, Magn. Reson. Med. 7,ll ( 1988). 16. J. GRANOT, Radiology 167,547 ( 1988). 17. H. L. KUNDEL, A. SHETTY, P. M. JOSEPH, R. M. SUMMERS, E. A. KASSAB, AND B. MOORE, Magn. Reson. Med. 6,38 1 ( 1988).
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18. R. K. GU~TA AND P. GUPTA, J. Magn. Resort. 47,344 ( 1982). 19. S. C. CHU, M. M. PIKE, E. T. FOSSEL, T. W. SMITH, J. A. BALSCHI, AND C. S. SPRINGER, JR., J. Magn. Reson. 56,33 ( 1984). 20. H. NARITOMI, M. KANASHIRO, M. SASAIU, Y. KURIBAYASHI, AND T. SAWADA, Biophys. J. 52,6 11 (1987). 21. D. BURSTEIN, Biophys. J. 54,191( 1988). 22. H. NARITOMI, M. SASAKI, Y. KURIBAYASHI, AND M. KANASHIRO, Biophys. J. 54,193 ( 1988). 23. D. BURSTEIN AND E. T. FOSSEL,Magn. Reson. Med. 4,26 I ( 1987). 24. J. PEKAR, P. F. RENSHAW, AND J. S. LEIGH, JR., J. Magn. Reson. 72,159 ( 1987). 25. J. PEKAR AND J. S. LEIGH, JR., J. Magn. Reson. 69,582 ( 1986). 26. H. SHINAR AND G. NAVON, Biophys. Chem. 20,275 ( 1984). 27. E. T. FOSSEL AND H. HOEFELER, Magn. Reson. Med. 3,534 ( 1986). 28. L. P. CHEUNG, M.D. thesis, Harvard University-Massachusetts Institute of Technology Division of Health Sciences and Technology, 1988. 29. P. M. JOSEPH AND A. SHETTY, Magn. Reson. Imaging 6,42 I( 1988).