1H magnetic resonance spectroscopic imaging of permanent focal cerebral ischemia in rat: longitudinal metabolic changes in ischemic core and rim

Brain Research 907 (2001) 208–221 www.bres-interactive.com

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H magnetic resonance spectroscopic imaging of permanent focal cerebral ischemia in rat: longitudinal metabolic changes in ischemic core and rim q Hironaka Igarashi a,b,c , *, Ingrid L. Kwee a,b , Tsutomu Nakada a,b,d , Yasuo Katayama c , Akiro Terashi c a

Neurochemistry and Magnetic Resonance Research Laboratories, VANCHCS, Martinez, CA 94553, USA b Department of Neurology, University of California, Davis, CA 95616, USA c Second Department of Internal Medicine, Nippon Medical School, 1 -1 -5 Sendagi, Bunkyo-ku, Tokyo 113 -8602, Japan d Department of Integrated Neuroscience, Brain Research Institute, University of Niigata, 1 Asahimachidori, Niigata City, Niigata, Japan Accepted 9 May 2001

Abstract The purpose of this study was to determine whether regional differences in metabolites can be seen chronologically in permanent focal cerebral ischemia using 1 H magnetic resonance spectroscopic imaging (MRSI), and whether these changes reflect pathological outcome. Regional variation in metabolites after permanent focal ischemia were investigated longitudinally in rats using 1 H MRSI for a total of 7 days and then compared to histopathological findings. Four hours after the induction of ischemia, N-acetyl-L-aspartate (NAA) levels in the lateral caudo-putamen and the somatosensory cortex, core ischemic regions, decreased 22 and 40%, respectively. This reduction in NAA was coupled with a marked rise in lactate. In the medial caudo-putamen, the ischemic rim, however, NAA was preserved in spite of a marked increase in lactate. By 24 h post ischemia, the levels of NAA in medial caudo-putamen (ischemic rim in caudate) also decreased significantly. However NAA in cingulated cortex (ischemic rim in cortex) decreased more gradually between 24 and 48 h. This regional difference can reflect the severity of metabolic derangement in the acute stage. After 96 h following ischemia, the levels of all metabolites detected by 1 H MRSI had decreased and the levels of NAA decline reflected the severity of histopathological damage. In conclusion, the regional metabolic differences could be assessed by 1 H MRSI chronologically, and the depth of NAA decline reflected histopathological changes in the chronic stage.  2001 Elsevier Science B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Ischemia Keywords: Cerebral ischemia; Magnetic resonance; Chemical shift imaging; Stroke; Rat

1. Introduction 1

H-magnetic resonance spectroscopy (MRS) is now widely used to investigate neuronal pathological state in cerebral ischemia [11,63], epilepsy [28,42], multiple sclerosis [15,39], Parkinson disease [31], amyotropic lateral sclerosis [48] and others. In cerebral ischemia, drastic spectral changes are seen in the acute stage, within 24 h q

Published on the World Wide Web on 4 June 2001. *Corresponding author. Tel.: 181-3-5814-6272; fax: 181-3-38224865. E-mail address: [email protected] (H. Igarashi).

after onset, and some of these changes are believed to have prognostic value for clinical outcome [19,46]. However, few reports have correlated longitudinal change of 1 HMRS with pathological outcome in several brain regions simultaneously. It is generally held that there are two relatively spatially distinct populations of neurons in the acute stages of a cerebral ischemia, a core of irreversibly injured neurons surrounded by potentially viable ischemic cells. Identification of this surrounding region, the socalled ischemic ‘penumbra’, is of particular importance due to obvious clinical implications [1,29,32]. The molecular processes leading to cell damage and death following an ischemic insult are complex multi-factorial events and it

0006-8993 / 01 / $ – see front matter  2001 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 01 )02579-3

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is difficult, if not impossible, to define the penumbra as a single rigid term applicable to every situation [17,36,40]. Nevertheless, the fundamental concept appears to reside in the common denominator of a ‘time limited reversibility of metabolic derangements associated with the ischemic insult’ [32]. In practice, however, this time-dependent appearance and disappearance of the penumbra has rendered researching it extremely technically challenging. Therefore, it is highly desirable to utilize a method that is capable of simultaneous data collection from multiple brain regions at multiple time points. In this study magnetic resonance spectroscopic imaging (MRSI) was used. MRSI is an imaging version of MRS that allows for analysis of MRS data in both pictorial and spectral format [16,21,47]. The pictorial presentation provides information regarding the spatial distribution of a single chosen metabolite detected by MRS, whereas the spectral presentation provides a means of quantifying multiple metabolites within a target volume of interest, identical to conventional single voxel MRS. The metabolites detectable by proton MRS, namely, N-acetyl-aspartate (NAA), glutamate, glutamine, g-aminobutyric acid (GABA), creatine, and lactate, represent key index cerebral metabolites which are especially useful in the investigation of brain ischemia or anoxia [11,33,49]. Particularly, NAA, a cytosolic amino acid primarily limited to neurons, which has a distinct peak in 1 H-MRS [61,62]. Additionally, alterations in NAA levels have been thought to reflect loss of neuronal density [11,28,43,63], although recent studies have revealed perpetual metabolism of NAA in normal brain [5,7]. The question then arises whether the loss of NAA during the acute stage of cerebral ischemia reflects an alteration of NAA metabolism as opposed to a simple decrease in neuronal density. Also, although lactate is a sensitive indicator of anaerobic glycolysis during ischemia, there are few reports investigating chronological changes in lactate in several regions, e.g. ischemic core and rim, simultaneously [30]. In this study, we used 1 H-MRSI to investigate biochemical changes in various regions of rat brain after induction of focal ischemia. To overcome limitations encountered with MRSI, we applied adiabatic RF pulses and a surface coil for RF tranceiving. Animals were studied longitudinally, from 4 h through 1-week after induction of ischemia, in an effort to delineate a biochemical profile of the ischemic penumbra. Later the metabolic data and the histopathological data were compared.

2. Materials and methods

2.1. Animals Adult male Sprague–Dawley rats weighing 290–320 g were used. Ten animals served as controls for baseline MRSI studies and regional cerebral NAA assay. A total of

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37 experimental animals underwent permanent right middle cerebral artery and MRSI studies were performed chronologically for 1 week. At the conclusion of the MRSI studies, brains of experimental animals were harvested and fixed for histopathological studies. All experiments were performed in accordance with the animal welfare guidelines and laws of the United States of America, and were approved by the local ethical committee for animal experiments.

2.2. Proton magnetic resonance imaging ( MRI) and spectroscopic imaging ( MRSI) All MRI and MRSI studies were performed on a General Electric (GE) Omega 7T system equipped with a horizontal super-conductive magnet operating at 7 T (120 mm usable bore). Animals were anesthetized using 1–1.5% halothane with a 70 / 30% mixture of N 2 O / O 2 while breathing spontaneously. The head was fixed in a custom designed head holder, and the animal was placed into the MR magnet in the supine position. Rectal temperature was maintained at 37.060.58C using a custom made heating system. For MR studies, a one-turn surface coil (round, 26 mm diameter) was used as the radio frequency (RF) coil. To compensate for B 1 field in-homogeneity inherent to surface coils, adiabatic pulses were adopted [21,24,50]. T 2 and diffusion-weighted images were used to confirm the ischemic region and for co-registration of SI images. T 2 weighted images were obtained using the following pulse sequence. An adiabatic pulse (B 1 insensitive refocusing pulse: BIR4 [58]) was used for RF excitation and refocusing, and a hyperbolic secant pulse [13] (HBS) was used for a one-dimensional image selected in vivo spectroscopy (1D-ISIS) for slice selection. The parameters were: TR of 2000 ms; TE of 120 ms; FOV of 30 mm; image matrix of 2563256; and slice thickness of 2 mm. Diffusion-weighted images were taken with the identical parameters to T 2 -weighted images except for a b-value of 600 s 2 / mm, diffusion gradient time of 48 ms, and gradient spacing time of 78 ms. The MRSI pulse sequence was essentially the three dimensional Fourier transform (3 DFT) version of the sequence introduced by Garwood and Ke [24,50] based on solvent suppressive adiabatic pulses (SSAP) [58]. In brief, slice selection was accomplished with 1D-ISIS with hyperbolic secant adiabatic inversion pulse (slice thickness 4 mm). The sweep width was 3 kHz. The excitation and refocusing pulses for the 3D-FT sequence were jump and return type SSAP pulses, respectively. A field of view (FOV) of 30330 mm was observed. The data matrix size was 16316. Echo signals were stored into 512 memory blocks, and eight transients were averaged for each k-space data point. Recycle time was 1.3 s and echo time was 272 ms. The total time to complete a single SI session was |45 min. Data were processed using conventional 3D-FT. For

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chemical shift dimension, 7 Hz of exponential apodization was applied as a noise filter to both sides of the echo signals. For spatial dimensions, sine bell apodization was applied to avoid intervoxel signal contamination and the matrix was zero filled to a 32332 matrix. Spatial resolution was then enhanced to a 64364 matrix by interpolation. Metabolites were assessed by utilizing spectral presentation of the volume of interest (1.9431.9434 mm) indexed by SI images and co-registered to DWI or T2weighted images (Fig. 1). Quantification of each peak was made by fitting peaks using a Lorentzian curve (Fig. 2). A fitted spectra is shown in Fig. 2. Seven peaks representing (1) choline compounds, (2) creatine1phosphocreatine, (3) NAA1aspartate, (4) glutamine1NAA, (5) glutamate1 GABA, (6) NAA, and (7) lactate1fat were identified in spectra of all 10 brains studied. At the TE selected in this study (272 ms), the fat signal was efficiently suppressed making it unnecessary to utilize fat saturation or outer volume suppression. Although J-modulation could affect peak height of multiplet spectra (NAA1aspartate, glutamine1NAA, glutamate1GABA, lactate), changes in these peaks could be assessed chronologically and used to calculate ratios of these metabolites with respect to stable metabolite levels in the contralateral non-ischemic side. Cortical NAA was selected as an internal standard, the levels of which are accepted as stable in the steady state [7,23,30,51].

(cross-linkage)) column (200–400 mesh, hydrogen form, Sigma, St. Louis, MO), 5 cm resin height. The column was washed with 2.5 ml of HPLC grade water for a total sample of 3.0 ml. Samples (15 ml) were run on an Alltech Partisil SAX 10 m, 250 mm34.6 mm anion exchange column. A 2.0 mm in-line column prefilter was used with 0.1 M KPO 4 (monobasic) and 0.025 M KCI, pH 4.5, as the mobile phase, at a rate of 1.5 ml / min. The recovery rate was 96%.

2.4. Experimental group

Ten normal rats were evaluated by MRSI to establish whether there were regional differences in NMR metabolite visibility and to assess the accuracy of quantification of spectra in each voxel. After MRSI studies, brains were frozen in situ using liquid nitrogen, and 33334 mm pieces from the caudo-putamen and the parietal cortex were taken. These specimens were used for NAA assays.

2.4.1. Induction of focal cerebral ischemia Under 1.5% of halothane anesthesia with 70 / 30% mixture of N 2 O / O 2 , permanent focal cerebral ischemia was induced using the intraluminal filament occlusion model developed by Koizumi et al. [37] with the modification suggested by Memezawa et al. [41]. This model provides an area of infarct with a high degree of reproducibility. In brief, following a midline skin incision in the anterior aspect of the neck, the right internal carotid artery was exposed from the level of the carotid bifurcation to the base of the cranium at the site of the tympanic bulla. The external carotid artery was then ligated with 9-0 silk suture, 1 mm distal from the carotid bifurcation. The pterygopalatine artery was encircled with suture and retracted to the left side of the experimental animal to prevent incorrect insertion of the occluder. Subsequently, unilateral circle of Willis occlusion was achieved by inserting a 0.25-mm-diameter nylon monofilament thread (Berkeley Outdoor Technologies Group, Iowa) at |3 mm proximal from carotid bifurcation and advancing it into the internal carotid artery to a point 19 mm distal from the bifurcation. Catheters were inserted into the tail artery and vein for blood sampling and blood pressure monitoring. Rectal temperature was kept at 37.060.58C throughout the procedures using a heating pad.

2.3.1. NAA assay Absolute NAA contents in cortex and caudo-putamen in normal rats were determined using high performance liquid chromatography (HPLC). Each sample was homogenized in four volumes of 0.5 N perchloric acid (PCA) and centrifuged at 16,000 rev. / min for 30 min. The pellet was re-suspended in two volumes of 0.5 N PCA and centrifuged as before. The first and second supernatants were combined and brought to a pH of 9.0–9.5 with KHCO 3 using one drop of methyl orange as an indicator. After KClO 4 was precipitated in ice for 15 min the sample was centrifuged at 16,000 rev. / min for 20 min at 48C. The supernatant was saved. A Hewlett-Packard Series 1050 HPLC equipped with autosampler, quarternary pump and UV/ VIS detector was used for all HPLC measurements. NAA separation was performed as described by Koller et al. [38]. Extraction samples, 0.5 ml, were filtered through an AG 50W38 (%

2.4.2. Inclusion criteria and experimental design In order to obtain the highest ischemic uniformity in the regions defined below, only animals that met the following parameters were used. (1) Consistent clinical findings (Bederson’s grade 2 to 4 [9]) 1 h after the induction of ischemia, (2) consistent area of parietal cortex infarction defined by diffusion-weighted MRI image 10 h after the induction of ischemia, and (3) consistent area of infarction on pathological specimens obtained 7 days after induction of infarction. Thirty-two of 37 animals in which infarction was induced met these criteria. Twenty-three of the 32 animals survived throughout the 1 week experimental duration. Data from these 23 animals were used for spectral analysis, and data from 12 to 17 measurements from 23 animals were collected at each time point. MRSI data in pictorial format obtained 10 h after induction of infarction were utilized to co-register NAA images to the corresponding T 2 -weighted images. Sub-

2.3. Control group

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Fig. 1. Representative diffusion weighted image (DWI) at 24 h after the induction of ischemia, utilized for definition of the region of interest (ROI). Square boxes ‘1’, ‘2’, ‘3’ and ‘4’ correspond to the lateral caudo-putamen, medial caudo-putamen, somatosensory cortex in ischemic hemisphere, and medial caudo-putamen in contra-lateral hemisphere, respectively. The square box ‘r’ is the area of normal cortex utilized for reference. Five regions of interest in temporal cortex correspond to ‘a’ through ‘e’ (upper image). The lower three images show the ROI in the ischemic rim and the growth of the MRI positive area. DWI positive lesions and the ROI in the ischemic rim (arrow and arrowhead) do not overlap at 4 h after the induction of ischemia, however at 10 and 24 h they overlap due to the growth of MRI positive lesion.

sequently, MRSI spectra from voxels (1.9431.9434 mm) which corresponded to the following areas: (1) lateral caudo-putamen (Fig. 1 – 1), (2) medial caudo-putamen

(Fig. 1 – 2), and (3) somatosensory cortex (Fig. 1 – 3), (4) contralateral (non-ischemic) lateral caudo-putamen (Fig. 1 – 4) were assessed chronologically. In this study,

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2.4.3. Histopathologic study Following the completion of MRSI studies, animals were sacrificed by pentobarbital overdose. Each brain was perfused with phosphate buffer (pH 7.2), removed, and fixed in 5% formaldehyde. Paraffin embedded sections (sliced (12 mm) and stained with hematoxylin-eosin) identical to the MRSI images were selected for pathological evaluation. Each specimen was inspected by two researchers in a blinded fashion, and divided into two groups according to the width of the band of selective neuronal loss at the medial caudo-putamen. The zone was measured in 1 mm increments (top to bottom) in the medial caudo-putamen. Rats showing broader transient zones than 500 mm in average width were considered to have a consistent transient zone. Fig. 2. Representative proton spectra (spectral presentation) obtained from somato-sensory cortex of normal rat (a), fitted peaks (b) and residual (c). Each numbered peak corresponds to the following metabolites: 1, choline compounds; 2, creatine1phosphocreatine; 3, NAA1aspartate; 4, glutamine1NAA; 5, glutamate1GABA; 6, N-acetyl-aspartate (NAA); 7, lactate1residual fat.

area (1), lateral caudo-putamen is the ischemic core and area (2), medial caudo-putamen, is the ischemic rim. These areas were determined for the following reasons: (1) there are regional differences in 1 H-MRS visible metabolites in normal rat brain, including differences in NAA concentration between caudate and cortex. Thus comparison of NAA concentration changes between these areas can be difficult in a 1 H-MRSI study. (2) Post ischemic depolarization, which can occur in ischemic cortex but not in ischemic caudate in the suture model, may modify changes of metabolites in cortex [13]. (3) Areas (1) and (3) are areas where cerebral blood flow (CBF) drops to less than 10 ml / 100 g / min at 30 min after induction of infarction, while the medial caudo-putamen is the area where CBF is maintained between 15 and 30 ml / 100 g / min [40] which is within the level of CBF in penumbra determined by Astrup et al. [1]. On the other hand, cortical ischemic rim or ‘penumbra’, as mentioned in the Results section, exhibited a gradient of NAA loss as seen in other metabolites [57]. Thus we set the 0.9230.9234 mm five contagious VOI in parietal to cingulate cortex, and chronological changes of distribution of NAA concentration was analyzed (Fig. 1a–e). Variability in brain slices between each scanning session was made virtually negligible by fixing the head of the animals to an identical location with respect to the MR system. This was accomplished through placement of the animals in a custom designed non-magnetic head fixation device based on conventional stereotaxic head fixation. MRSI was performed sequentially at eight time points (before ischemia and 4, 10, 24, 48, 72, 96 and 168 h after the induction of ischemia). At each time point, animals were anesthetized as described previously.

2.5. Statistics Data are expressed as mean6S.D. Student t-test was used for comparing differences in each peak between cortex and caudo-putamen in the control study. Repeated measures ANOVA with Bonferroni / Dun multiple comparison post-hoc test was used to compare differences at different time points. Data at each time point was compared to the control (pre-ischemic) value of the identical region. Comparison of NAA level depended on pathological changes. Repeated measures ANOVA with post-hoc Scheffe’s F test was used to compare the difference between the two groups at each time point. A value of P,0.05 was regarded as statistically significant.

3. Results

3.1. Regional differences in 1 H-MRS visible metabolites in normal rat brain In normal rats, NAA content was higher in cortex than caudo-putamen. The absolute quantity of NAA calculated from HPLC analysis was 10.960.9 mg / g in cortex and 8.360.8 mg / g in caudo-putamen. The ratio of NAA content in cortex to caudo-putamen was 0.76160.082 (P,0.01), which matched the MRSI findings (0.74960.118). The glutamate / GABA overlapping peak at 2.3 ppm was also larger in cortex than in caudo-putamen (P,0.05). The concentrations of the remaining metabolites were almost identical between the two regions. The concentrations of 1 H-MRS visible metabolites in these regions are summarized in Fig. 3.

3.2. Spectral changes in permanent focal ischemia Physiological variables before and after the induction of ischemia remained constant as summarized in Table 1. Representative metabolic images at each time point are shown in Fig. 4, and typical proton spectra from the lateral

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Fig. 3. Differences in metabolite concentrations in normal brain between cortex (closed bar) and caudo-putamen (open bar) obtained from MRSI peak area, *P,0.01, **P,0.05.

caudo-putamen and medial caudo-putamen are presented in Fig. 5. Fig. 6 presents chronological changes of each metabolite in four different regions, namely lateral caudoputamen and somato-sensory cortex (the ischemic core), medial caudo-putamen (the ischemic rim) and contralateral (non-ischemic) lateral caudo-putamen. In the acute stage of ischemia, a marked elevation in lactate was seen in the ischemic areas, though less drastically in the medial caudoputamen. After 24 h of ischemia, lactate levels had decreased and were not significantly different from preischemic levels at 96 h after induction. NAA began to decrease in the lateral caudo-putamen and somato-sensory cortex 4 h after induction of ischemia. In the medial caudo-putamen, however, although NAA levels had dropped, they were not significantly different from preischemic levels at 4 h. The creatine compounds peak, consisting mainly of creatine and phosphocreatine, began to decrease 24 h after induction of ischemia and continued

Table 1 Physiological variables before and 15 min after MCA occlusion

Values are Mean6SD; MCAO, middle cerebral artery occlusion; MABP, mean arterial blood pressure.

to decrease until 96 h after ischemia. The peak at 2.3 ppm, thought to contain glutamate and GABA, increased during the acute ischemic stage, then decreased gradually. The glutamine1NAA peak at 2.5 ppm decreased gradually during the acute stage in the lateral caudo-putamen and somato-sensory cortex, but at 10 h after ischemia there was a transient increase in the medial caudo-putamen. In contagious areas in parietal to cingulate cortex, as shown in changes of DWI in Fig. 2 and MRSI in Fig. 4, spreading of the lesion and decline of NAA concentration in this occurred later than that in medial caudo-putamen. The area with NAA loss spread gradually from the parietal cortex to cingulate cortex even in 48 h after the induction of ischemia (Fig. 7).

3.3. Histopathologic correlation to spectroscopic findings One week after the induction of ischemia, the ischemic core (lateral caudo-putamen and somato-sensory cortex) showed pan-necrosis in all animals. The ischemic rim (medial caudo-putamen), however, showed one of two patterns. In 10 of the 23 experimental animals there was a consistent transition zone consisting of selective neuronal loss and patchy necrosis (Fig. 8). In the remaining 13, there was a sharp demarcation between normal and necrotic tissue. The animals demonstrating a transition zone had significantly higher NAA (P,0.05) at 96 h and 168 h after induction of ischemia (0.3860.027 vs. 0.2760.025 (relative to peak area at contra-lateral somato-sensory cortex) at 96 h, and 0.4260.14 vs. 0.1660.012 at 168 h, Fig. 9). No correlation was observed between the other metabolites and histopathological findings.

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Fig. 4. Representative images (pictorial presentations) for each metabolite peak from MRSI data. The faint round structure observed outside normal brain in the lactate1fat image represents subcutaneous fat tissue. Although pictorial representation is not suitable for quantitative analysis, it can be quite useful for qualitative assessment, especially for definition of the area to be sampled for spectral analysis.

4. Discussion In this study, the chronological changes of metabolites detected by 1 H-MRS in both topological and spectral format are demonstrated. Using adiabatic pulses and a surface coil conjugated with relatively long echo-time, each metabolite could be depicted clearly without additional fat suppression or outer volume suppression. This advantage is clearly demonstrated in the lactate images. Without outer volume suppression, we could obtain lactate images of a complete head slice, with presumably minimal fat contamination. On the surface of the cortex, the lactate peak is usually masked by large fat peaks originating in cranial bone marrow and subcutaneous fat when using conventional pulses with surface coil [10]. Although the disadvantages of using a surface coil, such as B 1 field inhomogeneity, high-flux signals from sub-cutaneal fat tissue [10], are almost eliminated, the advantages of

utilizing a surface coil, such as high signal to noise ratio and low RF power are preserved with our method. With complete brain slice images, NAA concentrations between cortex and caudate can be compared with MRS. The NAA concentration was higher in cortex with both HPLC methods and 1 H-MRS, and the ratio between cortex and caudate (0.76160.082 vs. 0.74960.118) was almost identical in both methods. Although there is no other article showing the difference of NAA concentration between cortex and caudate in rat, other mammals show the same trend. Miyake et al. measured NAA in rabbit brain, finding NAA concentrations of 10.8 mmol / kg in cortex and 5.39 mmol / kg in caudate [43]. This difference may be due to the neuronal density of each area, although differences in activity of asparto-acylase, which is responsible for NAA breakdown, may also contribute. The enzyme activity is three times higher in white matter than in gray matter [14], and caudate is nerve-fiber-rich com-

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Fig. 5. Representative proton spectra from somato-sensory cortex (region 3), lateral caudo-putamen (region 1) and medial caudo-putamen (region 2). Note that the NAA in the core 4 h after the induction of the focal ischemia has decreased drastically, while that in the rim is preserved.

pared to cortex. Thus turnover of NAA may be higher in caudate than in cortex. In this model, the areas constituting the ischemic core [40], namely the lateral caudo-putamen and somatosensory cortex, exhibited a marked decrease in NAA 4 h after induction of ischemia. The metabolic findings in our study correspond to the NAA reduction, as measured by HPLC, in ischemic lesions 4 h after MCA occlusion [51]. Higuchi et al. [30] have found significant decreases in NAA in the ischemic core as early as 80 min after MCA occlusion using MRSI, although these findings have not been corroborated by HPLC studies [51]. The decline of NAA concentration in the ischemic region is drastic during the first 10 h after the induction of ischemia. In our MRI data, assuming pre-ischemic NAA concentration in caudate is 8.3 mmol / kg from HPLC measurements, and the relaxation times of NAA do not change within 24 h after the induction of ischemia [59], the rate of decline of NAA concentrations up to 10 h after induction of ischemia, calculated with linear regression, were 0.44 mmol / kg / h in lateral caudo-putamen (ischemic core) and 0.29 mmol / kg / h in medial caudo-putamen (ischemic rim). The former value is comparable with the data obtained with HPLC [51]. There are some possible explanations for the drastic decline of NAA in acute stage.

Firstly, NAA synthesis can be severely impaired in the ischemic condition. Because NAA is synthesized from acetyl-CoA and aspartate by the enzyme L-aspartate Nacetyl transferase which exists mainly in the mitochondria of the neuron, NAA production by this enzyme is inhibited on impairment of mitochondrial energy production [8]. Another possible explanation is the accelerated hydrolysis of NAA. NAA and its derivative N-acetyl-L-aspartylglutamate (NAAG) is perpetually metabolized [60] in a metabolic compartment system among neurons, oligodendrocytes and astrocytes in normal brain [7]. NAA is delivered from neurons, and catabolized to aspartate and acetate by aspartoacylase which is thought to be present in oligodendrocytes [6]. Under ischemia, NAA is delivered into the extracellular space [52,53]. This extracellular NAA can be catalyzed by aspartoacylase presumably on the cell membrane of the oligodendrocyte or in the extracellular space [5]. Sager et al. suggested aspartoacylase resides inside glia and NAA enters the glia with a specific transport mechanism in the normal condition [54]. However this transport mechanism might be impaired in the ischemic region which suffered from energy failure, since this transport mechanism is inhibited by ouabain or the decline in the transmembrane sodium gradient. The escape of NAA into the blood is less likely. In the

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Fig. 6. Chronological changes in concentration of key metabolites. LCP, lateral caudo-putamen; MCP, medial caudo-putamen; SSC, somato-sensory cortex; contra-LCP, lateral caudo-putamen on non-ischemic side. In the ischemic hemisphere concentration of metabolites changed drastically, while the LCP in the non-ischemic hemisphere did not show any significant changes during the experimental period, showing inter-measurement stability of magnetic resonance spectroscopy. Numbers on top of glutamate1NAA peaks show the respective number of animals imaged at each time point.

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Fig. 7. Chronological changes of the distribution of NAA in parietal to cingulate cortex are shown. Note the area showing loss of NAA is spreading gradually from parietal cortex to cingulated cortex.

ischemic core where cerebral blood flow is strictly compromised in this model [40,41], NAA declines more rapidly than in the ischemic rim where cerebral blood flow is higher [41]. As Baslow claims [7], NAA is metabolized perpetually in brain. Drastic NAA decline in the acute stage of ischemia is thought to reflect the consequences of dynamic metabolic changes rather than the density of the surviving neurons. After 24 h of focal ischemia, NAA levels declined more slowly than in acute stage, and in the ischemic core it fell to about 10% of pre-ischemic levels although not to zero, despite histology showing pan-necrosis and almost no surviving neurons. Sager et al. showed that NAA in caudate declined to 10 to 20% of pre-ischemic levels at 7 days after the focal ischemia in mice and that NAA was trapped in cell debris with no surviving neurons [55]. This may explain the residual NAA in the ischemic core. In parietal to cingulate cortex, NAA decline occurred between 24 and 48 h after the induction of ischemia, later than that in medial caudo-putamen. Nowiki et al. [45] reported ATP depression in parietal cortex occurred between 24 and 48 h after ischemia, although in striatum ATP depressed within 2 h. The reduction in rCBF is milder in the temporal cortex compared with medial caudoputamen (0.69 ml / g / min in temporal cortex and 0.31 ml / g / min in medial caudo-putamen at 180 h after the induction of ischemia [40]), because of co-lateral perfusion from the anterior cerebral artery. Therefore metabolic derangement in this area can be mild during the early phase of ischemia [45] and may be enough to maintain NAA metabolism. Or the NAA decline can be caused by the secondary decrease in CBF induced by cerebral edema which reaches maximum at 72 h following focal ischemia [27]. Also, post ischemic depolarization, which occurs only in cortex [12], may contribute to the delayed NAA decline in cingulated cortex, since post ischemic depolar-

ization contributes to infarct spread and maturation in cortex [2]. Histopathologically the neuronal population in the ischemic rim was distinguishable by the presence or absence of a transition zone consisting of selective neuronal loss and patchy necrosis. The presence of a transition zone marked a more benign outcome histopathologically and correlated with higher NAA levels in the sub-acute stage of ischemia, 96 to 168 h after occlusion. NAA levels 72 h or earlier did not differ between the two groups. At 96 h after occlusion pathological changes have already developed, and metabolite levels are relatively stable. As Sager et al. claimed [55], although NAA concentration does not reflect precise neuronal density even in sub-acute stage, it still can be used as a marker of neuronal loss. Clinical studies during the sub-acute stage of ischemia have found marked NAA reductions in affected areas [11,20,22,25,26,34,46], and there is some evidence of a correlation between neuronal outcome and NAA level [63]. Our data suggested a continuous rise of lactate in the ischemic area up to 10 h after the induction of focal ischemia. Higuch et al. showed identical changes in the permanently MCA occluded rat [30]. This indicates that, although oxidative metabolism is severely compromised, glycolysis continues in some cells. In this model, cerebral blood flow in the core severely declines but does not cease and is about 10 ml / 100 g / min [40]. This residual blood flow may provide glucose for metabolism by still surviving but dying cells. Persisting lactate production in cerebral infarction was shown by a 1 H-MRS study with [1- 13 C]glucose administration [18]. Also as Barker [3] and Monsein [44] mentioned, microscopic (cell by cell) heterogeneity of metabolic impairment in the volume of tissue examined should be taken into account. In pictorial images of lactate, the area showing gradual increasing lactate spread up to 10

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Fig. 8. Representative macroscopic (a) and light microscopic (b–d) histology of experimental brain shows an area of selective neuronal loss 1 week after the induction of focal ischemia. Light microscopic images (b), (c) and (d) correspond to contralateral (non-ischemic) lateral caudo-putamen (region 4), lateral caudo-putamen (region 1) and medial caudo-putamen (region 2). Closed squares in (a) show the corresponding areas in light microscopic image (b) and (c), and open squares are the corresponding ROI in MRSI. Pan-necrosis is seen in the lateral caudo-putamen, whereas selective neuronal loss and patchy necrosis is seen in the medial caudo-putamen. The contralateral lateral caudo-putamen (non-ischemic) shows normal histology, bar5200 mm.

h after ischemia, is larger than the area showing ischemic morphological change in pathological specimen. In other words, some areas in the peripheral ischemic zone have mild impairment of oxidative metabolism in the acute stage, but this impairment is not followed by cell death. Though diffusion of lactate from the ischemic core should also be taken into consideration. A maximum increase in the GABA1glutamate peak was observed primarily in the ischemic core 10 h after carotid occlusion. At this time tissue glutamate content is thought to have been exhausted [51] while GABA accumulates during ischemia via the GABA shunt. Since GABA increase has previously been demonstrated in our nonvolume selective in vivo 2D super cosy [4] study using a small surface coil (f 55 mm) [35], GABA is likely to be the main component of the peak with possible contribu-

tions from fat compounds seen at about 2.3 ppm. In contrast to the findings in the ischemic core, glutamine and NAA peaks were observed to rise in the ischemic rim 10 h after ischemia. While the reason for this observation has not been determined, it may be caused by glutamine synthesis up-regulation, or by its conversion from glutamate [56]. In conclusion, serial MRSI measurements in focal brain ischemia provide longitudinal, regional and quantitative information about cerebral metabolites. In the acute stage, changes in metabolite levels reflect dynamic metabolic changes, and as these stabilize, they reflect mainly pathological changes. A decrease in NAA levels during the acute stage of cerebral ischemia identifies the formation of the ischemic core and may be used as a marker for irreversibility in cellular injury. Relative preservation of

H. Igarashi et al. / Brain Research 907 (2001) 208 – 221

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Fig. 9. NAA contents in the medial caudo-putamen (region 2) obtained from the two different groups recognized on histopathological examination 1 week after the induction of focal ischemia. Open bars denote areas in the transition area consisting of selective neuronal loss and patchy necrosis, whereas closed bars represent NAA concentration from the medial caudo-putamen which showed no transition zone. Data are presented as mean6S.D., *P,0.05.

NAA levels in the face of increased lactate appears to be a reliable marker for identifying potentially reversible and treatable areas of neuronal injury during the acute stage of focal cerebral ischemia.

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Acknowledgements We would like to thank Mr. Brian Curran and Mrs. Ann Muramatsu for their thoughtful critique of this manuscript. This work was supported by Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan.

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