A CT using longitudinally detected ESR (LODESR-CT) of intraperitoneally injected nitroxide radical in a rat's head

Magnetic

ELSEVIER

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Resonance

Imaging. Vol. 15, No. 6, pp. 701-708, 1997 0 1997 Elsevier Science Inc. All rights reserved Printed in the USA. 0730-725X/97 $17.00 + .OO

97) 0008-8

Original Contribution A CT USING LONGITUDINALLY DETECTED ESR (LODESR-CT) INTRAPERITONEALLY INJECTED NITROXIDE RADICAL IN A RAT’S HEAD HIDEKATSU

YOKOYAMA, * TOSHIYUKI SATO,~ NOBUAKI TSUCHIHASHI,$ HIROAKI OHYA-NISHIGUCHI, * 8 AND HITOSHI KAMADA

TATEAKI *

OF

OGATA,~

*Institute for Life Support Technology, YamagataTechnopolisFoundation,Yamagata990, Japan,TYamagataResearch Institute of Technology, Yamagata990, Japan,$FukushimaMedical College,Fukushima960-12,Japan,$Faculty of Engineering,YamagataUniversity, Yonezawa 992, Japan We have developed an in vivo longitudinally detected ESR (LODESR) imaging system operating at 700 MHz based on a loop-gap resonator and a pair of saddle-type pickup coils. A good linear relationship between the LODESR signal intensity and the applied power in a range up to 15.8 W was obtained. The detection of LODESR signals was barely affected by variations in the resonant properties. The characteristic of LODESR is suitable for in vivo examination. Using this system, we succeeded in obtaining LODESRCT images of the head region of a rat after the intraperitoneal injection of a nitroxide radical. 0 1997 Elsevier Science Inc.

Keywords: In vivo LODESR-CT;

Rat head; Nitroxide

radicals.

INTRODUCTION

one-tenth lower than that of conventional X-band ESR (9 Ghz).’ Because of the lower frequency, however, the apparatus has less than one-tenth the sensitivity of the X-band ESR spectrometers. The sensitivity on an ESR measurement is just proportional to the square root of the irradiation power, even for low levels below saturation. Thus high microwave power can not be efficiently applied. Furthermore, any slight perturbation in resonant properties causes considerable disturbance in measurement. In an in vivo experiment, motion in a live animal due to respiration, heartbeat, and blood flow readily affects the resonant properties. LODESR is expected to overcome these problems of conventional in vivo ESR. Recently, Nicholson et al. developed a LODESR system, which consisted of a birdcage resonator (length, 30 mm; diameter, 38 mm) and a solenoid pickup coil (length, 25 mm; diameter, 33 mm). Implementing the system at a frequency of 300 MHz, they succeeded in obtaining a LODESR

Longitudinally detected ESR (LODESR) spectroscopy is one of the techniques that can be utilized to obtain signals from paramagnetic species. With this technique, the signal is derived from a longitudinal oscillation of the spin magnetization parallel to the z-axis under modulated ESR irradiation.‘,’ LODESR has some potential advantages. Bloch’s equation indicates that the signal intensity of LODESR increases linearly with the applied irradiation power below saturation levels.3 Furthermore, it is anticipated that the detection of LODESR signals will not be affected by relatively small variations in the resonant properties.’ In recent years, the field of in vivo ESR imaging has advanced at an increasing rate.4-6 These ESR imaging systems for conducting samples such as small animals applies lower frequency microwaves (less than 1 GHz), where the dielectric losses are 914196;ACCEPTED l/30/97. Addresscorrespondence to Dr. HidekatsuYokoyama, In-

stitute for Life SupportTechnology,YamagataTechnopolis Foundation,2-2-l Matsuei, Yamagata990, Japan.

RECEIVED

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signal from a sample tube that contained an aqueous solution of a nitroxide radical at a low concentration ( 10 -6 M) . They also demonstrated a one-dimensional (z-axis) projection of two pieces of l,l-diphenyl-%picrylhydrazyl that were placed in a magnetic field gradient. Their more recent work presents two-dimensional projection using LODESR for an intravenously injected nitroxide radical in a rat.8’9 In three-dimensional imaging (i.e., CT), linear magnetic field gradients along the x-, y-, and z-axes must be produced by the gradient coils.lo.” The requisite field gradient for ESR imaging is very large ( l100 mT/cm) compared with that for NMR imaging (0.001-0.1 mT/cm) because the linewidth of an ESR spectrum is usually wider than that of a NMR spectrum. Thus there are many technical difficulties (such as control of large current and cooling) in fabricating gradient coils for ESR imaging. As shown in Fig. la, when the combination of a birdcage resonator and a solenoid pickup coil is used, the distance (d) between the upper and lower parts of the gradient coil for the x- or y-axis must be wider than the outer diameter of the resonator because the sample entrance faces the zaxis ( B0 direction). In this case, the production of large and liner gradients is consider to be very difficult.” We have been developing an in vivo 700 MHz microwave ESR-CT system equipped with a loop-gap resonator, where the sample entrance does not face the z-axis.6,‘3,‘4 In this case, the direction of B,, is perpendicular to that of sample insertion. As shown in Fig. lb, in our system d can equal the most suitable value that is derived from Anderson’s equation.” In this study, we built an in vivo LODESR-CT system based on the combination of a loop-gap resonator and a pair of saddle-type pickup coils. Using this system, we investigated the influences of applied microwave power and perturbation of resonant properties upon the signal intensities obtained from a nitroxide radical solution. We also performed LODESR-CT for an intraperitoneally injected nitroxide radical in a rat’s head.

METHODS LODES?-CT System A block diagram of our LODESR-CT system is shown in Fig. 2. The main magnet is a commercially available electromagnet (modified RE3X, JEOL) . The field scan coil is a supplementary Helmholtz coil. The magnetic field was scanned by controlling the current in the field scan coils at a maximum scan rate of 7.5 mT/s. The gradient coils, which have already been described in detaiL6 provide a linear magnetic field gradient to 4 mT/cm in a range from the center to 20

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Gradient

coils

Fig. 1. Schematic representation of positions of a resonator and gradient coils for x-axis. “d” is the distance between the upper and lower parts of a gradient coil. When a combination of a birdcage resonator and a solenoid pickup coil is used, d must be wider than the outer diameter of the resonator because the entrance for the sample faces the z-axis (a). When a loop-gap resonator is used that has an entrance for the sample that does not face the z-axis, d can be set to the most suitable value (derived from Anderson’s equation) (b) .

mm along the x-, y-, and z-axes. The distance between the surfaces of the gradient coils was 79 mm. The currents supplied for the field scan coils and the field gradient coils were controlled by a personal computer (PC9801BA, NEC) via 4 digital-analog converters. The loop-gap resonator, a two-gap type, the details of which were previously described,13,r5 is 10 mm in axial length and 41 mm in inner diameter. The resonator was driven at a frequency of approximately 700 MHz, using an oscillator (POS-767, Mini circuit) and a power amplifier ( AlOOO- 1050, R&K). The on/off modulation of the microwaves was operated by using a pin switch (ZYSWA, Mini circuit) at a modulation frequency of 600 kHz. The LODESR signal is detected by a pair of pickup coils placed inside the loop-gap

LODESR-CT of nitroxide in rat head0 H. YOKOYAMA

600 A-ekHz

I

ETAL.

Power-

ND .iTJ 1

]Personal

1

Fig. 2. Block diagramof our LODESR-CT system.

resonator. The pickup coil is a saddle-type coil (30 mm in outer diameter), which is constructed from 15 turns of copper wire (0.3 mm in diameter). The distance between the pickup coil and inner surface of the loop-gap resonator is 4 mm (Fig. 3). The signal induced in the pickup coils by the longitudinal oscillation of the spin magnetization was amplified by a preamplifier (SA-430F5, NF Electronic Instruments), followed by detection, using a lock-in amplifier (5302, PARC ) at the modulation frequency. The internal oscillator of the lock-in amplifier is used as the modulation/reference signal source. The LODESR spectral data are collected via an analog-digital converter, using the personal computer. LODESR Signal Intensity and Microwave Power Characteristics A nitroxide radical, 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine- 1-oxyl (carbamoyl PROXYL ) was used. The sample tube, which contained 15 ml of a 1 mM solution of carbamoyl-PROXYL dissolved in saline, was placed in the center of the loop-gap resonator; then LODESR measurements were performed at an operating frequency of approximately 710 MHz. An average power of 7.92, 9.97, 12.6, or 15.8 W was applied. The signal intensity of carbamoyl-PROXYL was derived from the peak-height of the lowest component (M, + 1) of the triplet spectrum.

Influence of Perturbation of Resonant Properties The sampletube, which contained 10 ml of a 1 mM solution of carbamoyl-PROXYL dissolvedin water, was placed in the center of the loop-gap resonator; then the first measurementwas made at a resonant frequency of approximately 7 10 MHz and an average power of 15.8 W. Subsequently,a smallsampletube containing0.5 ml of salinewas insertednext to the sampletube of carbamoylPROXYL to perturb the resonantproperties.The second measurementwas made without retuning. Becausein an in vivo measurement,the perturbation of resonantproperties due to motion of a live animal occurs continuously, it is difficult to retune the spectrometerwhenever the resonant propertiesare perturbe4I.So the spectrometerwas not retuned in this experiment after perturbations.To compare with a conventional in vivo ESR, the measurementswere madeat a power level of 40 mW using 700 MHz microwave ESR spectrometer,employing an otherwiseidentical procedure.The detail of the spectrometerhave been describedelsewhere.‘” LODESR-CT Imaging of Rat’s Head Male Wistar rats (each weighing 200 g) were used. Carbamoyl-PROXYL was dissolved in saline at 0.2 M. The animalsreceived intraperitoneal administration of 5 ml ( 1 mmol) of the carbamoyl-PROXYL solution. Fifteen min after the injection and under pentobarbital anesthesia, the rat was restrained in the imaging sys-

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Fig. 3. Schemaof the outer view of the loop-gapresonatorand the saddle-typepickup coils. Dimensionsare in mm.

tern, with its head placed at the center of, and its interaural line aligned 15 mm posterior to the edge of the loop-gap resonator. In this condition, the unloaded and loaded Q-factor of the resonator was about 150 and 50, respectively. LODESR measurements were made at an operating frequency of approximately 720 MHz and an average power of 7.92 W. For threedimensional zeugmatography, the field gradient was set at 1 mT/cm, changing direction in 20-degree steps. Thus data on 81 spectra were obtained under the field gradients for one set of CT images.14 Spectral data obtained under the field gradients were deconvoluted, using the line-shape of the zero gradient spectrum, by a fast Fourier transform method under low pass filtering.16,17 CT images were reconstructed from the deconvoluted data by a filtered back projection. The spatial resolution was determined on the basis of the full-width at half-maximum (FWHM) of the deconvoluted spectrum that was obtained under the

zero-gradient. Signals lower than 25% of the maximum intensity in all slices were regarded as noise. The image was reproduced in 256 colors. To compare with a previous ESR, measurements were made by using the aforementioned ESR spectrometer and a power level of 40 mW; and procedures similar to those already described were employed. RESULTS

AND DISCUSSION

LODESR Signal Intensity and Microwave Power Characteristics As shown in Fig. 4a, 3 hyperfine lines were observed from the sample tube containing 15 ml of a saline of 1 mM carbamoyl-PROXYL using our LODESR system. The spectra were obtained from an average of 16 accumulations of 2 s scans. Figure 5 shows signal intensities plotted against the average power level of irradiation (Values are means +SE from

LODESR-CT of nitroxide in rat head 0 H. YOKOYAMA MI=+1

M,=O

M,=-1

s$ .a E 2 .G

6000

4000

0

2

Fig. 4. Typical LODESR signal of carbamoyl-PROXYL observed from a sample tube containing 15 ml of a 1 mM solution of the carbamoyl-PROXYL dissolved in saline (a), and from the rat’s head after the intraperitoneal injection of 5 ml of 0.2 M carbamoyl-PROXYL (b). The instrument settings are: microwave average power, 15.8 W (a) or 7.92 W (b); microwave frequency, 710 MHz (a) or 720 MHz (b); scan rate, 5 mT/s (a) or 7.5 mT/s (b); accumulation number, 16 (a) or 4 (b); modulation frequency, 600 kHz; time constant, 1 ms.

MI=0

4

6

8

Microwave

1 mT

MI=+1

705

ET AL.

10

12

14

16

power/W

Fig. 5. Signal intensities of LODESR spectra obtained from a 1 mM solution of carbamoyl-PROXYL dissolved in saline ( 15 ml) plotted against the average power level of irradiation. Values are means ? SE; 4 separate measurements.

MI=-1 MI=+1

M,=O

M,=-1

a Fig. 6. Examples of LODESR (a) and ESR (b) spectra before and after perturbation of resonant properties. The sample tube, which contained 10 ml of a 1 mM solution of carbamoyl-PROXYL dissolved in water, was placed at the center of the loopgap resonator; then the first measurement was made (upper). A small sample tube containing 0.5 ml of saline was inserted next to the sample tube of carbamoyl-PROXYL to perturb the resonant properties; then the second measurement was made without retuning (lower). The signal intensity derived from the second measurement was normalized by that of the first measurement, which was defined as the % post-perturbation intensity ( 100 X 1,/I, ).

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4 independent determinations). We obtained a good linear relationship between the LODESR signal intensity and the applied power, with high reproducibility (correlation coefficient = 0.9990 + 0.0004). The finding indicates that applied power levels (in the range up to 15.8 W) were below the saturation level.

InJuence of Perturbation

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Figure 6 shows examples of LODESR (a) and ESR (b) spectra with and without the sampletube of saline. The signal intensity is the peak-height for LODESR, and the peak-to-peak height for ESR of the lowest component (M, + 1) of the triplet spectrum. The signal intensity derived from the measurement with saline was normalized by that when measurementwas made without saline (defined as the % post-perturbation intensity, 100 X 12/11). As shown in Fig. 7, the % postperturbation intensity in LODESR was 92.3 ? 3.6, and in the former ESR, 22.3 + 1.1 (4 separate measurements). This indicates that the detection of LODESR signals is barely affected by the variations in resonant properties as opposed to the detection of ESR signals. Thus we believe that where the resonant properties are perturbed by the motion of living animals, the sensitivity of in vivo experiments when the LODESR system is used will not be reduced.

LODESR-CT

Imaging

of Rat’s Head

After the injection of carbamoyl-PROXYL, threeline LODESR signals, which closely resembled those obtained from the sampletube of carbamoyl-PROXYL (Fig. 3a), were observed from the rat’s head (Fig. 4b). The spectra were obtained from an average of 4 accumulations of 2 s scans. The S/N ratio of zero gradient spectrum for LODESR and ESR was about 20 and 60, respectively. CT images were successfully reconstructed. Figure 8 shows a typical set of LODESR-CT images (upper) and former ESR-CT images (lower) of the z-x plane in the rat’s head after the injection of nitroxide. The number at the bottom of each slice image indicates the distance from the posterior edge of the loop-gap resonator in the direction of the nasal side. The thickness of each slice was 0.5 mm. One spectral set of data for CT was obtained from one scan and the total collection time was 220 s. No change in the body temperature (rectal temperature) of the rats was observed before and after microwave irradiation. The spatial resolution of LODESRCT and ESR-CT was 3.22 mm FWHM and 2.30 mm FWHM, respectively. Both LODESR-CT and ESR-CT images show similar pattern: i.e., the brain is imaged as a nitroxide-deficient area (arrows in Fig. 8)) while the extracranial area is imaged as an area rich in ni-

Fig. 7. The % post-perturbationintensity in LODESR and ESR. Values are means*SE; 4 separatemeasurements.

troxide. This is not inconsistent with the fact that carbamoyl-PROXYL is a water-soluble compound does not pass through the blood-brain barrier.6,‘4 In this study, we succeededin obtaining LODESRCT images of a rat’s head after the intraperitoneal injection of nitroxide in vivo. Although the in vivo measurementswere performed at power level of about 8 W, no elevation in the rat’s body temperature was observed. However some energy is dissipated as heat in the rat’s head. The energy dissipation, which is estimated to be about 5 W on the basisof the change in the resonator Q after loading, is invasive to the biological system. In the future improvements in the sensitivity of the apparatuswill be necessaryto reduce the applied power. The LODESR-CT imageshave a slightly lower spatial resolution when compared to the ESR-CT im-

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and preamplifier were not matched in this study. Overcoming this problem will also contribute to greater sensitivity of our LODESR-CT system. REFERENCES 1. Nicholson, I.; Robb, F.J.L.; Lurie. D.J. Imaging paramagnetic species using radiofrequency longitudinally detected ESR (LODESR). J. Magn. Reson. B 104:284-

288; 1994. 2. Colligiani, A.; Leporini, D.; Lucchesi, M.; Martnelli,

3. 4.

lcm

5.

6.

ESR-CT Fig. 8. A typical set of LODESR-CT images (upper) and ESR-CT images (lower) of the z-x plane in the rat’s head 15 min after carbamoyl-PROXYL was injected. The thickness of each slice was 0.5 mm; the spatial resolution was 3.22 (upper) or 2.30 mm FWHM (lower). The number at the bottom of each slice image indicates the distance from the posterior edge of the loop-gap resonator in the direction of the nasal side. Both LODESR-CT and ESR-CT images show similar pattern: i.e., the brain is imaged as a nitroxidedeficient area (arrows ) , while the extracranial area is imaged as an area rich in nitroxide.

ages. This seemsto be due to the relatively low S/N ratio of LODESR because the spatial resolution depends on this ratio.16,” However, signal intensities of LODESR are unaffected by the perturbation of resonant properties. The characteristic of LODESR is important advantage for in vivo experiments. The pickup coils used here were placed near the loop-gap resonator (distance, 4 mm). Therefore eddy currents

occurring

in the loop-gap

resonator

will coun-

7. 8.

9.

10.

II.

12. 13.

terbalance signal currents induced in the pickup coils by the longitudinal oscillations. When the eddy currents are reduced,

more

efficacious

detection

will

be

achieved. Finally, the impedances of the pickup coils

14.

M. Longitudinal detection of ESR: Comparison between continuous wave and low power pulse techniquies. In: N.D. Yordanov (Ed). Electron Magnetic Resonance of Disorder Systems. Singapore: World Scientific; 1991: pp. 16-37. Bloch, F. Nuclear induction. Phys. Rev. 70:460-474; 1946. Berliner, L.J.; Fujii, H. Magnetic resonance imaging of biological specimens by electron paramagnetic resonance of nitroxide spin labels. Science 227517-5 19; 1984. Alecci, M.; Colacicchi, S.; Indovina, P.L.; Mono, F.; Pavone, P.; Sotgiu, A. Three-dimensional in vivo ESR imaging in rats. Magn. Reson. Imag. 8:59-63; 1990. Ishida, S.; Matsumoto, S.; Yokoyama, H.; Mori, N.; Kumashiro, H.; Tsuchihashi, N.; Ogata, T.; Yamada, M.; Ono, M.; Kitajima, T.; Kamada, H.; Yoshida, E. An ESR-CT imaging of the rat head of a living rat receiving an administration of a nitroxide radical. Magn. Reson. Imag. 10:21-27; 1992. Ray, R.S. Broadband complex refractive indices of ice and water. Applied Optics 11:1836-1844; 1972. Nicholson, I.; Foster, M.A.; Robb, F.J.L.; Hutchison, J.M.S.; Lurie, D.J. Two-dimensional in-vivo imaging in detected ESR the rat using radiofrequency longitudinally (LODESR IMAGING). In: 2nd Int. Workshop on In Vivo ESR and ESR Imaging, L’Aquila, 1995: pp. 76. Nicholson, I.; Foster, M.A.; Robb, F.J.L.; Hutchison, J.M.S.; Lurie, D.J. Two-dimensional in-vivo imaging using radiofrequency longitudinally detected ESR (LODESR IMAGING). In: 19th Int. EPR Symposium, Denver, 1996: pp. 133. Lauterbur, P.C. Image formation by induced local interaction: Example employing nuclear magnetic resonance. Nature 242:190- 191; 1973. Lauterbur, P.C.; Lai, C.M. Zeugmatography by reconstruction from projections. IEEE Trans. Nucl. Sci. NS27:1221-1231; 1980. Anderson, W.A. Electrical current shims for correcting magnetic fields. Rev. Sci. Instrum. 32:241-250; 1961. Ishida, S.; Kumashiro, H.; Tsuchihashi, N.; Ogata, T.; Ono, M.: Kamada, H.; Yoshida, E. In vivo analysis of nitroxide radicals injected into small animals by L-band ESR technique. Phys. Med. Biol. 34: 13 17- 1323; 1989. Yokoyama, H.; Ogata, T.; Tsuchihashi, N.; Hiramatsu, M.; Mori, N. A spatiotemporal study on the distribution

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of intraperitoneally injected nitroxide radical in the rat head using an in vivo ESR imaging system. Magn. Reson. Imag. 14559-563; 1996. 15. Hirata, H.; Ono, M. Resonance frequency estimated of a bridged loop-gap resonator used for magnetic resonance measurements. Rev. Sci. Instrum. 67: l-5; 1996.

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16. Jansson, P.A. Deconvolution with Application in Spectroscopy. New York: Academic Press; 1984. 17. Yokoyama, H.; Fujii, S.; Yoshimura, T.; Ohya-Nishiguchi, H.; Kamada, H. In vivo ESR-CT imaging of the liver in mice receiving subcutaneous injection of nitric oxide-bound iron complex. Magn. Reson. Imag. 15:249 -253; 1997.