- Email: [email protected]
A spatiotemporal study on the distribution of intraperitoneally injected nitroxide radical in the rat head using an in vivo ESR imaging system
Magnetic
ELSEVIER
l
Resonance
Imaging, Vol. 14, No. 5, pp. 559-563, 1996 Copyright 0 1996 Elsevier Science Inc. Printed in the USA. All rights reserved 0730-725X/96 $15.00 + .OO
PII: SO730-725X( 96) 00022-8
Original Contribution A SPATIOTEMPORAL STUDY ON THE DISTRIBUTION OF INTRAPERITONEALLY INJECTED NITROXIDE RADICAL IN THE RAT HEAD USING AN IN VIVO ESR IMAGING SYSTEM HIDEKATSU
YOKOYAMA, MIDORI
*-t TATEAKI HIRAMATSU*
OGATA,$ NOBUAKI AND NORIO MORI?
TSUCHIHASHI,~
*Institute for Life Support Technology, Yamagata Technopolis Foundation, Yamagata 990, Japan tFukushima Medical College, Fukushima 960-12, Japan $Faculty of Engineering, Yamagata University, Yonezawa 992, Japan We have developed a rapid-scan in vlvo electron spin resonance (ESR) imaging system operating at 700 MHz based on an air-cored two-coil Helmholtz designed resistive magnet. Using this system, we performed ESR-CT for the intraperltoneally injected nitroxide radical, 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-loxyl, in the rat head. The imaging data were collected over the time course range from 5 to 47 min after injection at an interval of 3 min and a series of ESR-CT images were reconstructed at the same slice plane (1 cm anterior to interaural line). The series of ESR-CT images thus obtained by rapid scans provided detailed spatiotemporal information on the distribution of the injected nitroxide radical in the rat head. The brain was imaged as a nitroxide-deficient area while the blood vessels and/or extracranium tissues as a nitroxlde-rich area. During periods when high intensities of ESR signals were maintained, spots of nitroxlde-accumulation were imaged at the central part of the brain. The spots were assigned to the middle sized blood vessels in the brain. Keywords:
In vivo ESR-CT; Nitroxide radicals; Rapid scan; Air-cored
coil; Spatiotemporal
study.
scanning system was recorder-control type. Repeating fast field scan by iron-cored magnet was difficult due to magnetic hysteresis.’ Furthermore, maximum scan rate by recorder-control was 15 mT/30 s. Thus, about 40 min was required to obtain one ESR-CT image. Therefore, we were not able to observe temporal changes of the distribution of a nitroxide radical. In this study, we developed a rapid-scan ESR-CT system equipped with an air-cored resistive magnet. Using this system, we examined a spatiotemporal distribution of an exogenously given nitroxide radical in the rat head.
INTRODUCTION In recent years, the field of in vivo electron spin resonance (ESR) imaging has increasingly advanced. In general, the continuous wave method, combined with stationary field gradients has been used for the imaging, because the relaxation times of the resonance system is fast.lW6 The time required for the ESR imaging, when based on the continuous wave method, depends on the rate of the field scan. In the previous study, we demonstrated the ESR-CT imaging of the head of a living rat after intraperitoneal injection of a stable nitroxide radical6 In that study, a commercial-based ESR spectrometer (modified FE-3X, JEOL) was used as a main magnet and a field scanning system. The main magnet was iron-cored resistive electromagnet and the field
MATERIALS
AND METHODS
ESR-CT System The ESR-CT system consisted of a main electromagnet, a pair of field scan coils, a pair of field gradient Foundation, Kurumanomae-683, Numagi, Yamagata 990, Japan.
7131195; ACCEFTED l/29/96. Address correspondence to Dr. Hidekatsu Yokoyama, Institute for Life Support Technology, Yarnagata Technopolis RECEIVED
559
Magnetic ResonanceImaging 0 Volume 14, Number 5, 1996
560
.. .. . . .&
I
I
involved the resonator and the modulation coils. The details of the gradient coil and the ESR unit were described previously.6,8
101mm &......
1
Fig. 1. Schema of the outer view of the main electromagnet (A), the field scan coil (B), and the field gradient coil (C) . The main magnet is air-cored, water-cooled and two-coil Helmboltz designed resistive electromagnet.
coils, power supplies, a personal computer, and a 700 MHz microwave ESR unit. The main electromagnet, which is air-cored, water-cooled, and two-coil Helmholtz designed, was newly developed. It can generate 60 mT of the maximum static magnetic field, by applying a maximum current of 10 A. Its details are as follows. Size of the coil: 407 mm in inner diameter, 633 mm in outer diameter, 62 mm in axial length; 1736 turns (28 lines X 62 layers) ; resistance: 31 R; cooling water: more than 4 Urnin; size of the shield case: 688 mm in outer diameter, 102 mm in axial length. The field scan coil is a supplementary Helmholtz coil. Its details are as follows. Size of the coil: 272 mm in inner diameter, 333 mm in outer diameter, 4 mm in axial length; 128 turns (four lines X 32 layers ) ; resistance: 5 R; cooling water: more than 0.8 Umin (Fig. 1) . The magnetic field was scanned by regulating the current of the field scan coils at a maximum scan rate of 15 mT/s. We may call it as a rapid scan. Because of the absence of magnetic hysteresis, the aircored coil can make repeating rapid scans. Linear magnetic field gradients along the x-, y-, zaxes were produced by the gradient coils (up to 1 mT/cm in the range of 20 mm from the center). The distance between the surfaces of the gradient coils was 101 mm. Current power supplies for the field scan coils and the field gradient coils were controlled by a personal computer (IBM PS/V2450 486DX 33MHz) via four digital-analog converters. The personal computer was also used for collecting data via an analogdigital converter. The 700 MHz microwave ESR unit
I
Spatiotemporal ESR-CT Images of the Rat Head Four male Wistar rats each weighing 200 g were used. A nitroxide radical, 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine- 1-oxyl ( Carbamoyl-PROXYL) was dissolved in saline at 0.2 M. The animals received intraperitoneal administration of 5 ml (1 mmol) of the Carbamoyl-PROXYL solution under pentobarbital anesthesia. The rat was restrained in the 700 MHz microwave ESR unit, with its head placed in the center of the loop-gap resonator (41 mm in inner diameter and 10 mm in axial length) (Fig. 2). The ESR unit was operated at 735 MHz under a static magnetic field of 26 mT. The ESR-CT images were constructed by three-dimensional zeugmatography’*” as described previously.6 For this purpose, the field gradient was applied at 1 mT/cm, changing its directions in 20-degree steps, providing data of nine spectra for each twodimensional projection. For one ESR-CT, data of nine two-dimensional projections were obtained. Thus, data of 81 spectra were obtained for one ESR-CT.6 Also, a scanning with zero gradient was performed, and the line shape of the zero gradient spectrum was obtained for deconvolution of the data of 81 spectra obtained under the field gradients. In this study, 1.4 s was needed to gain the data for one scan: 1.2 s for each field scan, 0.1 s for data storage for the each scan, and 0.1 s for powering of the next field gradient. Eventually, the time for collection of data for one ESR-CT was 2.5 min [ 1.4 s X 82 scans (81 gradient and 1 zero-
Fig. 2. Photograph of the ESR system in which a rat is positioned.
Spatiotemporalstudy of ESR imaging of rat 0 H.
10
30
20 Time
after
injection
40
50
(min)
Fig. 3. Typical plot of signalintensity of the CarbamoylPROXYL in the rat headvs. time after intraperitonealadministration. Signal intensity is the peak-to-peakheight of the low-field component of the Carbamoyl-PROXYL triplet spectrumobtainedunder zero-gradient.ESR conditionsare as follows: microwave power, 20 mW; microwave frequency, 735 MHz; static magnetic field, 26 mT; sweep width, 15 mT; gain, 500; modulation,0.2 mT at 100 kHz.
gradient scans) + 30 s for manual operation]. Thus, the data collection time for imaging was curtailed by a factor of 10 from our previous work.6 The same ESR measurement was repeated every 3 min, from 5 min to 47 min after injection of the Carbamoyl-PROXYL. Data of ESR spectra were deconvoluted by the fast Fourier transform method.” ESR-CT images of the same plane (1 cm anterior to interaural line) were reconstructed from the deconvoluted data by a filtered back projection. The spatial resolution was determined based on the full-width at half-maximum of the deconvoluted spectrum, which was obtained under the zero gradient. The lowest components of the CarbamoylPROXYL triplet spectra obtained under zero gradient were used for evaluation of the sequential pattern of ESR signal intensities. Under a 1 mT/cm field gradient, data for the 150 mm (= 15 mT) line on x, y, z directions were assigned to 300 data points, for instance, 1 point for 0.5 mm. Among the 300 X 300 points of the data, 100 X 100 points (50 x 50 mm) of the central fractions were used for the picture, The region of the linear gradient well fit the resonator and included the rat head sufficiently. The data processing was carried out using an NEC PC9801BA. RESULTS Figure 3 shows an example of time course of the
ESR signal intensities of the Carbamoyl-PROXYL
in
YOKOYAMA
ETAL.
561
the rat head. The signal intensities began to increase about 5 min after injection of the nitroxide radical, reaching the peak at about 15 min and maintaining maximal levels for about 15 min. Then, they began to decrease gradually about 30 min after the injection. Figure 4 shows a typical chronological pattern of ESR-CT images. The thickness of each slice was 1.5 mm and the spatial resolution was 2.3 mm. The nitroxide-deficient area corresponds to the brain, and the n&r-oxide-rich area represents extracranial vessels, or tissues, or both.6 At 5 min, only the extracranial area was vaguely imaged due to the low signal intensity (see Fig. 2). However, the images became clear 11 min after injection. The images taken 11-43 min after the injection showed some accumulation of nitroxide at the central part of the area corresponding to the brain. Figure 5 is the picture obtained by superimposing the brain atlas of rat, ‘* which is the site of measurement to the brain area of the example of the ESR-CT image (Fig. 4, 21 min) to assign the position of nitroxide accumulation. The image taken 47 min after the injection showed disappearance of the nitroxide from the central part of brain. This image roughly corresponded with that obtained in our previous study.6 Similar spatiotemporal patterns were obtained in all four animals tested. DISCUSSION In the present study, the Carbamoyl-PROXYL was intraperitoneally given to rats. Subsequently, ESR signals of the Carbamoyl-PROXYL from the rat head were observed. The sequential pattern of the ESR signals shows that the nitroxide rapidly moves to the head. The nitroxide is reduced to nonparamagnetic species in the biological system.13 Then, the signal intensities gradually decreased via stationary state. The pattern was different from the exponential decay of nitroxide signals after intravenous injection seen in our previous work’ or others’.14-16 However, the sequential pattern of nitroxide signals obtained from mouse head after intraperitoneal injection was similar to the present pattern.” Thus, the change of the signal intensities followed a time course was considered to depend on the route of injection, The brain was imaged as a nitroxide-deficient area, while an extracranial area was imaged as an area rich in the nitroxide at the initial (Fig. 4, 5 min) and the final stage (Fig. 4, 47 min). This is not inconsistent with the fact that the Carbamoyl-PROXYL is a watersoluble compound that does not pass the blood-brain barrier (BBB) .6 The pictures taken during periods when high intensities of ESR signals were maintained
Magnetic ResonanceImaging l Volume 14, Number 5, 1996
562
q
5 min
11 min
17 min
m lcm ~4 mm
~4 mn
Fig. 4. Chronological ESR-CT images of coronal section (1 cm anterior to interaural line) of the rat head. 5 min, 11 min, 14 min, etc. mean the time after injection of the Carbamoyl-PROXYL. Thickness of the slice is 1.5 mm. Spatial resolution is 2.3 mm.
(Fig. 3) showed spots of the nitroxide accumulation at the central part of the brain (Fig. 4, 1l-43 mm). Because of BBB impermeability of CarbamoylPROXYL, the nitroxide in the middle-sized vessels and their branches (arteriolae and/or venulae) in the brain may be imaged, where it will concentrate in the blood. The spot of nitroxide accumulation appears to the correspond to the longitudinal fissure, where the anterior cerebral artery exists (Fig. 5). This may be in contrast to our previous study,6 in which brain vessels were not imaged. In our previous study,6 the time required for collection of data for one ESR-CT image was about 40 min. Also, it was difficult to maintain
the resonant frequency, and the frequency was compensated the frequency by manual operation during collection of data. Furthermore, we dealt the data without deconvolution. These factors blurred the images in our previous study. The rapid scan ESR-CT system overcame these problems. Quaresima et al.i8 showed the time course of the pattern of the distribution of a nitroxide radical in the whole body of the rat, using one-dimensional projection method. It was a pioneering work on spatiotemporal study in vivo. They also demonstrated two-dimensional projection, but they needed 6 min for one image. Here, we have performed a more time-resolved (2.5
urn
ca”&al
putamen
5mm Fig. 5. Picture obtained by superimposing the brain atlas of rat (1 cm anterior to interaural line) to the enlarged image of Fig. 4, 21 min.
Spatiotemporalstudy of
ESR
imaging of rat 0
min for the each image) and three-dimensional ( 1.5 mm in the thickness of the slice of ESR-CT image) imaging in the rat head.
REFERENCES 1. Berliner, L.J.; Fujii, H. Magnetic resonance imaging of biological specimens by electron paramagnetic resonance of nitroxide spin labels. Science 227517-519; 1984. 2. Fujii, H; Berliner, L.J. One- and two-dimensional EPR imaging studies on phantoms and plant speciments. Magn. Reson. Med. 2:275-282; 1985. 3. Berliner, L.J.; Fujii, H.; Wan, X.; Lukiewicz, S.J. Feasibility study of imaging a living murine tumor by electron paramagnetic resonance. Magn. Reson. Med. 4:380384; 1987. 4. Colacicchi,S.; Indovina, P.L.; Mono, F; Sotgiu, A. Lowfrequency three-dimensional ESR imaging of large samples. J. Phys. E: Sci. Instrum. 21:910-913; 1988. 5. Alecci, M.; Colacicchi, S.; Indovina, P.L.; Mono, F.; Pavone, P.; Sotgiu, A. Three-dimensional in vivo ESR imaging in rats. Magn. Reson. Imaging 8:59-63; 1990. 6. 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. Imaging lo:21 -27; 1992. 7. Oikawa, K.; Ogata., T.; Lin, Y.; Sato, T.; Kudo, R.; Kamada H. Rapid field scan L-band electron spin resonance computed tomography system using an air-core electromagnet. Anal. Sci. 11:885-888; 1995. 8. 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. 34:1317-1323; 1989.
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ET-AL.
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9. Lauterbur, PC. Image formation by induced local interaction: Example employing nuclear magnetic resonance. Nature 242: 190- 191; 1973. 10. Lauterbur, P.C.; Lai, C.M. Zeugmatography by reconstruction from projections. IEEE Trans. Nucl. Sci. NS27:1221-1231; 1980. 11. Jansson, P.A. Deconvolution With Application in Spectroscopy. New York: Academic Press; 1984. 12. Paxinos, G. The Rat Brain in Stereotaxic Coordinates. Sydney: Academic Press; 1986. 13. Couet, W.R.; Eriksson, U.G.; Tozer, T.N.; Tuck, L.D.; Wesbey, G.E.; Nitecki, D.; Brasch, R.C. Pharmacokinetits and metabolic fate of two nitroxides potentially useful as contrast agents for magnetic resonance imaging. Pharmaceut. Res. 1:203-209; 1984. 14. Berliner, L.J.; Wan, X. In vivo pharmachokinetics by electron spin resonance spectroscopy. Magn. Reson. Med. 9:430-434; 1989. 15. Bacic, G.; Nilges, M.J.; Magin, R.L.; Walczak, T.; Swartz, H.M. In vivo localized ESR spectroscopy reflecting metabolism. Magn. Reson. Med. 10:266-272; 1989. 16. Ferrari, M.; Quaresima, V.; Ursini, C.L.; Alecci, M.; Sotgiu, A. In vivo electron paramagnetic resonance spectroscopy-imaging in experimental oncology: The hope and the reality. Int. J. Radiat. Oncol. Biol. Phys. 29:42 l-425; 1994. 17. Gomi, F.; Utsumi, H.; Hamada, A.; Matsuo, M. Aging retards spin clearance from mouse brain and food restriction prevents its age-dependent retardation. Life Sci. 5212027-2033; 1993. 18. Quaresima, V.; Alecci, M.; Ferrari, M; Sotgiu, A. Whole rat electron paramagnetic resonance imaging of a nitroxide free radical by a radio frequency (280 MHz) spectrometer. Biochem. Biophys. Res. Commun. 183: 829-835; 1992.
ELSEVIER
l
Resonance
Imaging, Vol. 14, No. 5, pp. 559-563, 1996 Copyright 0 1996 Elsevier Science Inc. Printed in the USA. All rights reserved 0730-725X/96 $15.00 + .OO
PII: SO730-725X( 96) 00022-8
Original Contribution A SPATIOTEMPORAL STUDY ON THE DISTRIBUTION OF INTRAPERITONEALLY INJECTED NITROXIDE RADICAL IN THE RAT HEAD USING AN IN VIVO ESR IMAGING SYSTEM HIDEKATSU
YOKOYAMA, MIDORI
*-t TATEAKI HIRAMATSU*
OGATA,$ NOBUAKI AND NORIO MORI?
TSUCHIHASHI,~
*Institute for Life Support Technology, Yamagata Technopolis Foundation, Yamagata 990, Japan tFukushima Medical College, Fukushima 960-12, Japan $Faculty of Engineering, Yamagata University, Yonezawa 992, Japan We have developed a rapid-scan in vlvo electron spin resonance (ESR) imaging system operating at 700 MHz based on an air-cored two-coil Helmholtz designed resistive magnet. Using this system, we performed ESR-CT for the intraperltoneally injected nitroxide radical, 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-loxyl, in the rat head. The imaging data were collected over the time course range from 5 to 47 min after injection at an interval of 3 min and a series of ESR-CT images were reconstructed at the same slice plane (1 cm anterior to interaural line). The series of ESR-CT images thus obtained by rapid scans provided detailed spatiotemporal information on the distribution of the injected nitroxide radical in the rat head. The brain was imaged as a nitroxide-deficient area while the blood vessels and/or extracranium tissues as a nitroxlde-rich area. During periods when high intensities of ESR signals were maintained, spots of nitroxlde-accumulation were imaged at the central part of the brain. The spots were assigned to the middle sized blood vessels in the brain. Keywords:
In vivo ESR-CT; Nitroxide radicals; Rapid scan; Air-cored
coil; Spatiotemporal
study.
scanning system was recorder-control type. Repeating fast field scan by iron-cored magnet was difficult due to magnetic hysteresis.’ Furthermore, maximum scan rate by recorder-control was 15 mT/30 s. Thus, about 40 min was required to obtain one ESR-CT image. Therefore, we were not able to observe temporal changes of the distribution of a nitroxide radical. In this study, we developed a rapid-scan ESR-CT system equipped with an air-cored resistive magnet. Using this system, we examined a spatiotemporal distribution of an exogenously given nitroxide radical in the rat head.
INTRODUCTION In recent years, the field of in vivo electron spin resonance (ESR) imaging has increasingly advanced. In general, the continuous wave method, combined with stationary field gradients has been used for the imaging, because the relaxation times of the resonance system is fast.lW6 The time required for the ESR imaging, when based on the continuous wave method, depends on the rate of the field scan. In the previous study, we demonstrated the ESR-CT imaging of the head of a living rat after intraperitoneal injection of a stable nitroxide radical6 In that study, a commercial-based ESR spectrometer (modified FE-3X, JEOL) was used as a main magnet and a field scanning system. The main magnet was iron-cored resistive electromagnet and the field
MATERIALS
AND METHODS
ESR-CT System The ESR-CT system consisted of a main electromagnet, a pair of field scan coils, a pair of field gradient Foundation, Kurumanomae-683, Numagi, Yamagata 990, Japan.
7131195; ACCEFTED l/29/96. Address correspondence to Dr. Hidekatsu Yokoyama, Institute for Life Support Technology, Yarnagata Technopolis RECEIVED
559
Magnetic ResonanceImaging 0 Volume 14, Number 5, 1996
560
.. .. . . .&
I
I
involved the resonator and the modulation coils. The details of the gradient coil and the ESR unit were described previously.6,8
101mm &......
1
Fig. 1. Schema of the outer view of the main electromagnet (A), the field scan coil (B), and the field gradient coil (C) . The main magnet is air-cored, water-cooled and two-coil Helmboltz designed resistive electromagnet.
coils, power supplies, a personal computer, and a 700 MHz microwave ESR unit. The main electromagnet, which is air-cored, water-cooled, and two-coil Helmholtz designed, was newly developed. It can generate 60 mT of the maximum static magnetic field, by applying a maximum current of 10 A. Its details are as follows. Size of the coil: 407 mm in inner diameter, 633 mm in outer diameter, 62 mm in axial length; 1736 turns (28 lines X 62 layers) ; resistance: 31 R; cooling water: more than 4 Urnin; size of the shield case: 688 mm in outer diameter, 102 mm in axial length. The field scan coil is a supplementary Helmholtz coil. Its details are as follows. Size of the coil: 272 mm in inner diameter, 333 mm in outer diameter, 4 mm in axial length; 128 turns (four lines X 32 layers ) ; resistance: 5 R; cooling water: more than 0.8 Umin (Fig. 1) . The magnetic field was scanned by regulating the current of the field scan coils at a maximum scan rate of 15 mT/s. We may call it as a rapid scan. Because of the absence of magnetic hysteresis, the aircored coil can make repeating rapid scans. Linear magnetic field gradients along the x-, y-, zaxes were produced by the gradient coils (up to 1 mT/cm in the range of 20 mm from the center). The distance between the surfaces of the gradient coils was 101 mm. Current power supplies for the field scan coils and the field gradient coils were controlled by a personal computer (IBM PS/V2450 486DX 33MHz) via four digital-analog converters. The personal computer was also used for collecting data via an analogdigital converter. The 700 MHz microwave ESR unit
I
Spatiotemporal ESR-CT Images of the Rat Head Four male Wistar rats each weighing 200 g were used. A nitroxide radical, 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine- 1-oxyl ( Carbamoyl-PROXYL) was dissolved in saline at 0.2 M. The animals received intraperitoneal administration of 5 ml (1 mmol) of the Carbamoyl-PROXYL solution under pentobarbital anesthesia. The rat was restrained in the 700 MHz microwave ESR unit, with its head placed in the center of the loop-gap resonator (41 mm in inner diameter and 10 mm in axial length) (Fig. 2). The ESR unit was operated at 735 MHz under a static magnetic field of 26 mT. The ESR-CT images were constructed by three-dimensional zeugmatography’*” as described previously.6 For this purpose, the field gradient was applied at 1 mT/cm, changing its directions in 20-degree steps, providing data of nine spectra for each twodimensional projection. For one ESR-CT, data of nine two-dimensional projections were obtained. Thus, data of 81 spectra were obtained for one ESR-CT.6 Also, a scanning with zero gradient was performed, and the line shape of the zero gradient spectrum was obtained for deconvolution of the data of 81 spectra obtained under the field gradients. In this study, 1.4 s was needed to gain the data for one scan: 1.2 s for each field scan, 0.1 s for data storage for the each scan, and 0.1 s for powering of the next field gradient. Eventually, the time for collection of data for one ESR-CT was 2.5 min [ 1.4 s X 82 scans (81 gradient and 1 zero-
Fig. 2. Photograph of the ESR system in which a rat is positioned.
Spatiotemporalstudy of ESR imaging of rat 0 H.
10
30
20 Time
after
injection
40
50
(min)
Fig. 3. Typical plot of signalintensity of the CarbamoylPROXYL in the rat headvs. time after intraperitonealadministration. Signal intensity is the peak-to-peakheight of the low-field component of the Carbamoyl-PROXYL triplet spectrumobtainedunder zero-gradient.ESR conditionsare as follows: microwave power, 20 mW; microwave frequency, 735 MHz; static magnetic field, 26 mT; sweep width, 15 mT; gain, 500; modulation,0.2 mT at 100 kHz.
gradient scans) + 30 s for manual operation]. Thus, the data collection time for imaging was curtailed by a factor of 10 from our previous work.6 The same ESR measurement was repeated every 3 min, from 5 min to 47 min after injection of the Carbamoyl-PROXYL. Data of ESR spectra were deconvoluted by the fast Fourier transform method.” ESR-CT images of the same plane (1 cm anterior to interaural line) were reconstructed from the deconvoluted data by a filtered back projection. The spatial resolution was determined based on the full-width at half-maximum of the deconvoluted spectrum, which was obtained under the zero gradient. The lowest components of the CarbamoylPROXYL triplet spectra obtained under zero gradient were used for evaluation of the sequential pattern of ESR signal intensities. Under a 1 mT/cm field gradient, data for the 150 mm (= 15 mT) line on x, y, z directions were assigned to 300 data points, for instance, 1 point for 0.5 mm. Among the 300 X 300 points of the data, 100 X 100 points (50 x 50 mm) of the central fractions were used for the picture, The region of the linear gradient well fit the resonator and included the rat head sufficiently. The data processing was carried out using an NEC PC9801BA. RESULTS Figure 3 shows an example of time course of the
ESR signal intensities of the Carbamoyl-PROXYL
in
YOKOYAMA
ETAL.
561
the rat head. The signal intensities began to increase about 5 min after injection of the nitroxide radical, reaching the peak at about 15 min and maintaining maximal levels for about 15 min. Then, they began to decrease gradually about 30 min after the injection. Figure 4 shows a typical chronological pattern of ESR-CT images. The thickness of each slice was 1.5 mm and the spatial resolution was 2.3 mm. The nitroxide-deficient area corresponds to the brain, and the n&r-oxide-rich area represents extracranial vessels, or tissues, or both.6 At 5 min, only the extracranial area was vaguely imaged due to the low signal intensity (see Fig. 2). However, the images became clear 11 min after injection. The images taken 11-43 min after the injection showed some accumulation of nitroxide at the central part of the area corresponding to the brain. Figure 5 is the picture obtained by superimposing the brain atlas of rat, ‘* which is the site of measurement to the brain area of the example of the ESR-CT image (Fig. 4, 21 min) to assign the position of nitroxide accumulation. The image taken 47 min after the injection showed disappearance of the nitroxide from the central part of brain. This image roughly corresponded with that obtained in our previous study.6 Similar spatiotemporal patterns were obtained in all four animals tested. DISCUSSION In the present study, the Carbamoyl-PROXYL was intraperitoneally given to rats. Subsequently, ESR signals of the Carbamoyl-PROXYL from the rat head were observed. The sequential pattern of the ESR signals shows that the nitroxide rapidly moves to the head. The nitroxide is reduced to nonparamagnetic species in the biological system.13 Then, the signal intensities gradually decreased via stationary state. The pattern was different from the exponential decay of nitroxide signals after intravenous injection seen in our previous work’ or others’.14-16 However, the sequential pattern of nitroxide signals obtained from mouse head after intraperitoneal injection was similar to the present pattern.” Thus, the change of the signal intensities followed a time course was considered to depend on the route of injection, The brain was imaged as a nitroxide-deficient area, while an extracranial area was imaged as an area rich in the nitroxide at the initial (Fig. 4, 5 min) and the final stage (Fig. 4, 47 min). This is not inconsistent with the fact that the Carbamoyl-PROXYL is a watersoluble compound that does not pass the blood-brain barrier (BBB) .6 The pictures taken during periods when high intensities of ESR signals were maintained
Magnetic ResonanceImaging l Volume 14, Number 5, 1996
562
q
5 min
11 min
17 min
m lcm ~4 mm
~4 mn
Fig. 4. Chronological ESR-CT images of coronal section (1 cm anterior to interaural line) of the rat head. 5 min, 11 min, 14 min, etc. mean the time after injection of the Carbamoyl-PROXYL. Thickness of the slice is 1.5 mm. Spatial resolution is 2.3 mm.
(Fig. 3) showed spots of the nitroxide accumulation at the central part of the brain (Fig. 4, 1l-43 mm). Because of BBB impermeability of CarbamoylPROXYL, the nitroxide in the middle-sized vessels and their branches (arteriolae and/or venulae) in the brain may be imaged, where it will concentrate in the blood. The spot of nitroxide accumulation appears to the correspond to the longitudinal fissure, where the anterior cerebral artery exists (Fig. 5). This may be in contrast to our previous study,6 in which brain vessels were not imaged. In our previous study,6 the time required for collection of data for one ESR-CT image was about 40 min. Also, it was difficult to maintain
the resonant frequency, and the frequency was compensated the frequency by manual operation during collection of data. Furthermore, we dealt the data without deconvolution. These factors blurred the images in our previous study. The rapid scan ESR-CT system overcame these problems. Quaresima et al.i8 showed the time course of the pattern of the distribution of a nitroxide radical in the whole body of the rat, using one-dimensional projection method. It was a pioneering work on spatiotemporal study in vivo. They also demonstrated two-dimensional projection, but they needed 6 min for one image. Here, we have performed a more time-resolved (2.5
urn
ca”&al
putamen
5mm Fig. 5. Picture obtained by superimposing the brain atlas of rat (1 cm anterior to interaural line) to the enlarged image of Fig. 4, 21 min.
Spatiotemporalstudy of
ESR
imaging of rat 0
min for the each image) and three-dimensional ( 1.5 mm in the thickness of the slice of ESR-CT image) imaging in the rat head.
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