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Glutamate measurement in Parkinson's disease using MRS at 3 T field strength
KICKLER, Nils, et al.
KICKLER, Nils, et al . Glutamate measurement in Parkinson's disease using MRS at 3 T field strength. NMR in Biomedicine , 2007, vol. 20, no. 8, p. 757-762
DOI : 10.1002/nbm.1141 PMID : 17334978
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Glutamate measurement in Parkinson’s disease using MRS at 3 T field strength
yNils Kickler,1,2* Paul Krack,3,4Vale´rie Fraix,3,4Jean-Franc¸ois Lebas,6Laurent Lamalle,1,2Franck Durif,5 Alexandre Krainik,1,2Chantal Re´my,1,2Christoph Segebarth1,2and Pierre Pollak3,4
1INSERM, U594, Neuroimagerie fonctionnelle et me´tabolique, Grenoble, F-38043 France
2Universite´ Joseph Fourier, Grenoble, F-38043 France
3INSERM, U318, Neurosciences Pre´cliniques, Grenoble, F-38043 France
4De´partement de Neurologie, Grenoble University Hospital, Grenoble, F-38043 France
5INSERM, unite´ EMI 9904, Faculte´ de Me´decine et de Pharmacie, Clermont-Ferrand, F-63001 France
6INSERM, IFR 1, RMN biome´dicale unite´ IRM 3T, Grenoble University Hospital, Grenoble, F-38043, France
Received: 13 June 2006; Revised 30 November 2006; Accepted 1 December 2006
ABSTRACT: Loss of nigral dopamine neurons in Parkinson’s disease induces abnormal activation of glutamate systems in the basal ganglia. The purpose of this study was to assess these changes in the lentiform nucleus using MRS with optimized glutamate sensitivity (TE-averaged method). Ten patients with Parkinson’s disease and 10 healthy controls were examined.
Compared with healthy controls, no significant differences in glutamate were measured in patients, but a trend to lower total creatine was observed. Copyright#2007 John Wiley & Sons, Ltd.
KEYWORDS: glutamate; Parkinson’s disease; MRS; TE averaged Point-Resolved Spectroscopy (PRESS)
INTRODUCTION
Parkinson’s disease is characterized by degeneration of the dopaminergic melanized neurons of the substantia nigra pars compacta. Dopamine denervation induces an increase in corticostriatal glutamate transmission (1) that is central to the pathophysiology of Parkinson’s disease (2). Several studies have suggested that dopamine lesion may also increase glutamate transmission in the basal ganglia output structures, especially the pallidum, presumably as a result of the abnormal activation of the subthalamic nucleus. Moreover, glutamate-mediated mechanisms are thought to play a role in the development of dyskinesias induced by L-Dopa (levodopa, L-3,4- dihydroxyphenylalanine) (3). An increase in extracellular glutamate has been observed in the striatum of dopamine-lesioned rats after either acute (4) or repeated (3)L-Dopa injections.
Proton MRS allows non-invasive measurement of brain metabolites. Only a few MRS investigations have been conducted so far to assess glutamate in patients with Parkinson’s disease (5–7) and only one included administration of an anti-Parkinsonian treatment (apo- morphine) (5). None of these studies showed alterations in the glutamate region of the spectrum at 2.3 ppm.
However, the sequences used were not optimized for glutamate detection, i.e. the 2.3 ppm peak of the proton spectrum results from the overlap of resonance lines due to protons from glutamate but also from glutamine, N-acetyl-aspartate (NAA) and g-aminobutyric acid (GABA). Any change in glutamate concentration might be masked by concomitant changes in other metabolite contents, especially glutamine.
In this study, we used an MRS sequence optimized for glutamate detection,TE-averaged PRESS (8), at 3 T field strength. This sequence minimizes the contribution of overlapping resonance lines of glutamine, NAA and GABA, and thus the 2.3 ppm peak is mainly due to glutamate. We aimed to assess changes in glutamate concentration in the lentiform nucleus of patients with Parkinson’s disease in relation to administration of levodopa and compared with healthy controls.
METHODS
Ten patients with Parkinson’s disease, according to the UK Parkinson’s Disease Society Brain Bank criteria, and
NMR Biomed.2007;20: 757–762
Published online 2 March 2007 in Wiley InterScience (www.interscience.wiley.com) DOI:10.1002/nbm.1141
*Correspondence to:N. Kickler, INSERM U 594, CHU, Pavillon B, BP 217, F-38043 Grenoble cedex 09, France.
E-mail: Nils.Kickler@ujf-grenoble.fr
yA preliminary account of this work was published as an abstract at the 22nd annual meeting (Basel, Switzerland) of the European Society for Magnetic Resonance in Medicine and Biology (ESMRMB).
Abbreviations used: L-Dopa, levodopa, L-3,4-dihydroxyphenylala- nine; FWHM, frequency width at half maximum; GABA, g-aminobutyric acid; glx, glutamate with eventual contributions from glutamine, NAA, GABA; NAA,N-acetylaspartate; tCho, total choline (cholineþphosphorylcholineþglycerophosphorylcholine); tCr, total creatine (creatineþphosphocreatine); PRESS, Point-resolved spec- troscopy.
10 healthy subjects participated in this study and underwent two MRS examinations. All participants gave written consent; the study was approved by the local ethics committee (Comite´ Consultatif pour la Protection des Personnes se preˆtant a` la Recherche Biome´dicale, Grenoble University Hospital). The meanSD age of the patients was 605 years with disease duration of 113.5 years (range 5–16). All were being treated with levodopa at the time of the study and had motor fluctuations. For further details, see Table 1. At the time of the first MRS examination, patients had not received levodopa for at least 12 h (off-drug condition). The second examination was performed after administration of a supra-threshold levodopa dose, defined as the usual levodopa morning dose plus 20%, plus the levodopa dose equivalent to dopamine agonist drugs usually taken by the patient (9). This dose corresponded to the dose necessary to achieve the best on-drug conditions during the examination. The on-drug condition was confirmed by a decrease in motor symptoms to a state typical for the individual patient. The control group (meanSD age 564 years) was matched for age with the patient group.
Examinations were performed with a Medspec 3 T whole-body imager (Bruker, Ettlingen, Germany; 63 cm bore diameter) using a standard-quadrature head coil for emission and signal reception. For spectroscopy, a rectangular volume measuring 20 mm20 mm in the coronal plane and 30 mm in the anterior–posterior direction was centered on the lentiform nucleus. To ensure reliable placement in repeated examinations, its position was chosen relative to the anterior and posterior commissure line, compared with an anatomical atlas (10).
Volumes excited for the different metabolites differed slightly from the chosen position, as the pulse carrier frequencies were centered on the water resonance. For glutamate at 2.3 ppm, this displacement accounts for 2.2–3.5 mm in the coronal plane and 5.2 mm in the anterior–posterior direction (bandwidths of 2 ms hermite pulse: excitation/refocusing in coronal plane, 2.7 kHz and 1.71 kHz; refocusing in the anterior–posterior direction, 1.71 kHz). Shimming was performed with FASTMAP (11) and resulted in a frequency width at half maximum
(FWHM) of about 11 Hz for NAA. This value is the average of the frequency widths at half maximum obtained for the line fit of NAA in the spectra acquired throughout the study. MRS usedTE-averaged PRESS (8), which is the sum of PRESS spectra acquired at different echo times. Spectra with echo times of 35–195 ms, at intervals of 10 ms, were acquired and stored separately.
For each PRESS spectrum, the total echo time was divided symmetrically between the first and second echo time. A spectral width of 5000 Hz was acquired on 4096 points, accumulating eight scans and using an eight-step phase-cycling scheme (12). With a repetition time of 3 s, the total acquisition duration was 6.8 min. The unsup- pressed water signal from the same voxel was also acquired for later normalization of the metabolite amplitudes, using an echo time of 136 ms.
To estimate signal contributions in the 2.3 ppm region, spectra using the same parameters as in vivo were acquired from solutions containing 50 mM glutamate, glutamine, NAA or GABA. Samples were prepared in phosphate buffered saline (Sigma, St. Louis, USA), pH 7.3, containing, in addition to the metabolite, about 0.25 mM chelated gadolinium complex (Dotarem, Guer- bet, France) as relaxant and 3-(trimethylsilyl)propionic acid as phase and frequency reference. On the in vitro spectrum of NAA, line broadening was adjusted to obtain a FWHM of about 9 Hz on the singlet peak at 2.0 ppm.
Afterwards, amplitudes of this peak in the NAA spectra acquired at increasing echo times for laterTEaveraging were adjusted to simulate a decay corresponding to aT2of 199 ms (13). The adjustments applied to NAA were then applied to the spectra of glutamate, glutamine and GABA.
Data processing of thein vivospectra used jMRUI (14).
Each individual spectrum of the TE-averaged PRESS acquisition was corrected for frequency shift and phase using the NAA signal as reference. The glutamate signal (glx) at 2.3 ppm (mainly glutamate, minimized contri- butions of glutamine, NAA, GABA), total choline (tCho;
sum of choline, phosphorylcholine, glycerophosphoryl- choline, contributions of taurine and myo-inositol) at 3.2 ppm, total creatine (tCr; creatine, phosphocreatine) at 3.0 ppm and NAA at 2.0 ppm were quantified using Table 1. Patient data
Patient Age (years)
Disease duration (years)
LEDD (mg/day)
UPDRS III motor score on-drug condition
UPDRS III motor score off-drug condition
Levodopa dose administered for exam in on-drug condition (mg)
1 56 9 700 6 27 275
2 67 12 600 22 45 200
3 64 16 1450 9 46 250
4 61 6 900 28 48 300
5 58 5 1000 17 33 400
6 56 10 1650 2 15 300
7 61 12 1400 15 45 250
8 50 12 1500 14 34 200
9 61 14 1650 15 42 200
10 64 14 1500 10 43 250
LEDD, levodopa equivalent daily dose; UPDRS, Unified Parkinson Disease Rating Scale.
Copyright#2007 John Wiley & Sons, Ltd. NMR Biomed.2007;20: 757–762
DOI: 10.1002/nbm
758 N. KICKLERET AL.
AMARES (15) as implemented in jMRUI. The glx signal, as well as NAA, tCr and tCho, was fitted by a single Lorentzian line. Signals above 3.2 ppm were not fitted as they were corrupted by water suppression in some spectra. Data were further processed to obtain an estimation of metabolite T2 relaxation times for NAA, tCr and tCho. Only spectra acquired at echo times longer than 55 ms, where a flat baseline was observed, were used.
The flat baseline allowed the assumption that macro- molecule contributions had decayed sufficiently. Statisti- cal evaluation used Wilcoxon (paired) or Mann–Whitney (unpaired) non-parametric tests without correction for multiple comparisons.
RESULTS
Figure 1 (bottom) shows TE-averaged PRESS spectra obtained from solutions of glutamate, glutamine, NAA and GABA. The spectra were weighted with the indicated factors to simulate relative concentrations of 9.25 mmol/kg for glutamate, 4.4 mmol/kg for glutamine, 12.25 mmol/kg for NAA and 1.6 mmol/kg for GABA (16). Even though a small contribution of glutamine, NAA and GABA resonances to the signal observedin vivo cannot be excluded, the region near 2.3 ppm is dominated
by a signal stemming from glutamate. The sum of the individual metabolite spectra is shown in the top panel of Fig. 1 (dotted line) together with an AMARES fit (solid line) of the signal near 2.3 ppm and of the signal at 2 ppm.
Figure 2 shows a spectrum obtained from a patient with Parkinson’s disease in the off-drug condition (top) together with the fit function (middle) and the residual (bottom). Even though the glx signal is only visible with difficulty in the spectrum, it is well fitted by AMARES.
The placement of the volume used for spectroscopy, centered on the lentiform nucleus, is visualized in Fig. 3.
The quality of metabolite quantification can be assessed via Cramer-Rao lower bounds. These provide a minimal standard deviation for peak amplitude determination. For glutamate, the Cramer-Rao lower boundaries ranged from 10% to 37% (mean 18%) of the
Figure 1. TE-averaged PRESS spectra obtained from solutions of glutamate, glutamine, NAA and GABA (bottom).
The spectra were broadened to obtain a FWHM of 9 Hz, as measured on the NAA singlet, and weighted as indicated, to simulate relative concentrations of 9.25 mmol/kg for gluta- mate, 4.4 mmol/kg for glutamine, 12.25 mmol/kg for NAA and 1.6 mmol/kg for GABA (16). Near 2.3 ppm, the pre- eminent signal stems from glutamate. Top, Sum of the metabolite spectra (dotted line) and an AMARES fit (solid line) of the signal near 2.3 ppm and of the signal at 2 ppm
Figure 2. Top,TE-averaged PRESS spectrum from the lenti- form nucleus of a patient with Parkinson’s disease in the off-drug condition. The signal at 2.3 ppm is dominated by glutamate. NAA is visible at 2.0 ppm, total creatine at 3.0 ppm, total choline at 3.2 ppm. Signals at 3.7 ppm (glu- tamateþglutamine) and 3.9 ppm (total creatine) were not evaluated as they were attenuated by water suppression in some spectra. No exponential broadening was applied;
FWHM of the NAA signal, as determined by AMARES, is about 9 Hz in this spectrum. Middle, AMARES fit of NAA, glx, tCr and tCho. Bottom, Residual, after subtraction of the AMARES fit from thein vivospectrum
peak amplitude. For a stronger signal such as NAA, the Cramer-Rao lower boundaries ranged only from 1% to 2%.
Figure 4 summarizes the metabolite signal measure- ments. Theyvalues do not represent concentration ratios, as the metabolite and the water reference spectra were not acquired using the same receiver gain and number of accumulations.
No significant change associated with levodopa administration was detected in glx/water, NAA/water, tCr/water or tCho/water ratios in the patients. Results from the first and second examinations of the controls were not significantly different and were averaged. No significant differences in glx/water, NAA/water and tCho/
water were seen between patients (on- or off-drug condition) and controls, whereas a slight (6%) reduction in tCr/water was found in the patient group [tCr/water signal ratio (meanSEM) 0.3170.005 for controls, 0.2980.007 for patients in the off-drug condition and
0.2940.009 for patients in the on-drug condition;
P<0.052 and P¼0.05, respectively, Mann–Whitney].
A power analysis (StatMate; GraphPad Software) choosing a¼0.05 and b¼0.2 (corresponding to a 5%
probability of Type I error and 20% probability of Type II error) permits determination of the size of a glx/water change that could have been identified in this study. We assumed that 58% of the acquisition volume is the lentiform nucleus (17) and the rest is white matter. For a gray matter/white matter glutamate ratio of 9.4 mM to 4.5 mM (18), we estimated that a 30% difference in the glx signal from the lentiform nucleus between the on-drug and off-drug conditions would have led to a statistically significant result on analysis by pairedttest. Comparing patients with controls, a change of 41% would have led to a significant result using an unpaired ttest.
Transverse relaxation times (T2) of NAA, tCr and tCho showed no significant differences between the on- drug and off-drug conditions and between patients and controls. Values for the control group are (meanSEM) 2297 ms for NAA, 1273 ms for tCr and 198 8 ms for tCho and are comparable to values in the literature (13).
DISCUSSION
The aim of this study was to measure glutamate in patients with Parkinson’s disease in the off-drug and on-drug condition and to compare these values with those from healthy controls.
Figure 1 shows that, assuming relative metabolite concentrations of the healthy brain, the signal observed near 2.3 ppmin vivois dominated by glutamate and can be reasonably fitted by a single Lorentzian lineshape.
Performance of ourTE-averaged PRESS implementation is comparable to results in the literature (8). As glutamate and glutamine are not resolved from each other in vivo, small changes in the glutamate concentration may become masked by concomitant changes in glutamine in the opposite direction. Measurement of glutamate may also be hampered by the presence of macromolecule resonances in the spectrum (resonance M6 (19)).
Transverse relaxation times of macromolecules are, however, relatively short (24–40 ms (19,20)). Therefore, in the individual spectra of a TE-averaged PRESS acquisition, which have been acquired with echo times of 65 ms and longer, macromolecule signals are not observed, and the baseline is essentially flat. In the TE-averaged spectrum, which is the sum of spectra with echo times of 35–195 ms, macromolecule contributions should therefore not be important.
Even with this MRS sequence optimized for glutamate detection, we found no significant changes in glutamate (glx/water) in a voxel centered on the lentiform nucleus despite clear changes in the patients’ motor performances after levodopa administration. Comparing patients with Figure 4. Ratios of metabolite/water signal for patients and
controls. On- and off-drug measurements on the patients as well as 1st and 2nd measurements on the controls are connected by a line to show evolution between examinations Figure 3. Voxel of 202030 mm3size centered on the lentiform nucleus, as used for acquisitions
Copyright#2007 John Wiley & Sons, Ltd. NMR Biomed.2007;20: 757–762
DOI: 10.1002/nbm
760 N. KICKLERET AL.
controls, no differences in glutamate concentration could be found either.
Some microdialysis studies (1,4) have reported large increases (þ45% to 146%) in extracellular striatal glutamate after lesion of the nigrostriatal pathway in rats. Acute (4) or repeated (3)L-Dopa administration was also shown to increase extracellular glutamate in the striatum of this model, the reported increase ranging from 224% to about 350% of the basal value. However, there remains controversy, as other studies (3,21,22) report unchanged glutamate concentrations after dopamine lesion.
Signals measured with MRS represent the pool of extracellular and intracellular glutamate, which amounts to about 10 mM (16). Extracellular glutamate, with a concentration of 2–3mM (23), as measured in the microdialysis studies cited above, is only a tiny fraction of the total glutamate pool. Variations in extracellular glutamate, if present, are not necessarily accompanied by large changes in the total glutamate pool, which was measured by MRS in our study. Furthermore, our study was not performed on newly diagnosed patients but on patients with disease duration and treatment ranging from 5 to 16 years. The long-term treatment might interfere with glutamate measurements even after 12 h ofL-Dopa deprivation.
Our data indicate a trend to reduced tCr/water ratios in patients with Parkinson’s disease which is not caused by a decrease in tCr transverse relaxation time, as indicated by ourT2measurements. No clear evidence of altered water concentrations in Parkinson’s disease has been reported.
Relaxation times for water were not measured in this study; in the literature, increased (24) as well as reduced (25,26) water T2 values have been reported for the putamen and globus pallidum of patients with Parkinson’s disease and have been related to altered iron deposition.
Although we cannot exclude the possibility that changes in the water signal amplitude have occurred due to T2
alterations, the reduced tCr/water found in this study may also be interpreted as a decrease in the combined cerebral creatineþphosphocreatine concentration, which may signify changes in energetic metabolism in response to Parkinson’s disease. Further research with a larger group size and including measurement of water relaxation time are necessary to substantiate this finding. Our measure- ments do not confirm a significantly increased tCho/water in Parkinson’s disease as measured by some authors (6), but, consistently, we did not detect changes in NAA/
water.
CONCLUSION
Glutamate concentrations in patients with Parkinson’s disease measured with an MRS sequence optimized for glutamate detection at 3 T field strength do not show any differences from control values. If present, any changes in
the total glutamate pool remain below the level of detection of the acquisition protocol applied.
Acknowledgement
We thank Dr Marc Savasta for helpful discussions.
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