Date sent: Fri, 22 Dec 2000 British Journal of Radiology 73: (875) 1206-1208 November 2000 URL: http://www.bir.org.uk/bjr/bjr_online.shtml (home page) http://www.bir.org.uk/bjronline/bjr/73/875/00105/index.html (text) Figs. 1-3 (cannot be viewed without a password): http://www.bir.org.uk/bjronline/bjr/73/875/00105/F01-00105.gif http://www.bir.org.uk/bjronline/bjr/73/875/00105/F02-00105.gif http://www.bir.org.uk/bjronline/bjr/73/875/00105/F03-00105.gif [Short communication] Proton magnetic resonance spectroscopy and morphometry of the Hippocampus In Chronic Fatigue Synsrome ------------------------------------------------------------------------- (1,2) C W BROOKS, PhD, (1)N ROBERTS, PhD, (1)G WHITEHOUSE, DSc, FRCR and (3)T MAJEED, FRCP (1) Magnetic Resonance and Image Analysis Research Centre, University of Liverpool, Liverpool L69 3BX, (2) Pain Relief Foundation, Clinical Sciences Centre, Lower Lane, Liverpool L9 7AL and (3) Department of Neurology, Royal Preston Hospital, Preston PR2 9HT, UK Received 23 March 2000 and accepted 7 July 2000. Address correspondence to Dr JCW Brooks, Magnetic Resonance and Image Analysis Research Centre, University of Liverpool, Pembroke Place, Liverpool L69 3BX, UK. Abstract Seven patients with chronic fatigue syndrome (CFS) were matched with ten healthy control subjects of similar age. Hippocampal volume, obtained from magnetic resonance images using an unbiased method, showed no difference between the two groups, whereas proton magnetic resonance spectroscopy showed a significantly reduced concentration of N-acetylaspartate in the right hippocampus of CFS patients (p=0.005). -------------------------------------------------------------------------------- The hippocampus has a critical role both in working memory and in long-term memory storage and retrieval. The right hippocampus is 8% smaller compared with controls in patients with combat-related post-traumatic stress disorder (PTSD), a condition in which there is impaired memory function [1]. Patients with chronic fatigue syndrome (CFS) also have reduced ability at verbal and non-verbal memory tasks [2]. Proton magnetic resonance spectroscopy (1H-MRS) is a method of assessing the concentration of cerebral metabolites. The major metabolite peaks visible on 1H spectra include: N-acetylaspartate (NAA) compounds, a putative marker of neuronal density and probably also function; creatine/phosphocreatine (Cr), an indicator of cellular bioenergetics; choline-containing compounds (Cho), a constituent of cell membranes and a precursor of acetylcholine; and myo-inositol (mI), thought to be a glial cell marker. Reduced concentration of NAA has been reported in the hippocampi of patients with PTSD, especially in the right hippocampus [3, 4]. To our knowledge, there have been no reports of hippocampal volume estimation and 1H-MRS in CFS. MATERIALS AND METHODS Seven patients with a clinical diagnosis of CFS, based on the definition of Holmes et al [5], were recruited for this study (five males, two females; age 26-40 years). Ten healthy male subjects (age 21-41 years) underwent neuropsychological screening and formed a control group. Written informed consent was obtained from patients and controls. All MR data were acquired on a 1.5 T Signa whole body MRI system (General Electric, Milwaukee, USA). A high resolution T_1 weighted image volume was acquired in the plane approximately perpendicular to the long axis of the hippocampus using the three-dimensional fast inversion recovery prepared GRASS pulse sequence (124 contiguous slices, 1.6 mm thick, TE=3.5 ms, flip angle=30, TI=450 ms, field of view=20 cm). Morphometric analysis was performed using Analyze software (Mayo Foundation, MN, USA), and unbiased estimates of the volume of both hippocampi were obtained using the Cavalieri method in combination with point counting [6]. Spectra were acquired with the STEAM sequence and the following parameters: TE=30, 72 and 144 ms; TR=3 s; 128 averages; and an 8 step phase cycle. From inspection of the high resolution T_1 images, a 3x1x1 cm^3 voxel was positioned to contain the right hippocampus, with its largest dimension parallel to the long axis. Time constraints limited the ^1H-MRS examination to the right hippocampus. Imaging-based compartmentation [7] was performed, with the cerebrospinal fluid (CSF) fraction of the voxel determined from heavily T_2 weighted images acquired with a fast spin echo sequence, and total water fraction of the voxel (brain water plus CSF) determined from proton density weighted images acquired with a fast spoiled gradient echo sequence. Spectra were analysed using the time-domain VARPRO technique available in MRUI software. Metabolite T_2 relaxation times were calculated by log-linear fitting of peak areas against TEs of 30 ms, 72 ms and 144 ms. Briefly, calibration of the metabolite signal intensities was performed as follows. Signal amplitudes were corrected for T_1 (published values [8]) and T_2 relaxation (calculated) time differences. Compartmentation analysis enabled correction for the presence of CSF in the selected voxel. Subsequently, metabolite signal amplitudes were converted to absolute concentrations, expressed in millimoles per litre of brain tissue, by referencing to the signal recorded from an external standard containing pure water. RESULTS Comparison of hippocampal volume between CFS patients and controls showed no significant difference (Figure 1). The mean volume (p/m SE) of the right hippocampus was 2.97 p/m 0.12 cm^3 in CFS patients and 3.20 p/m 0.10 cm^3 in controls. Typical spectra recorded from CFS patients and controls are presented in Figure 2. The mean (p/m SE) concentrations for NAA, Cr and Cho in CFS patients were 10.8 p/m 0.6, 8.6 p/m 0.5 and 2.5 p/m 0.2 mmol 1^-1 of brain tissue, respectively. The corresponding values for controls were 14.1 p/m 0.7, 10.9 p/m 1.1 and 3.1 p/m 0.2 mmol 1^-1 of brain tissue. The reduction in NAA concentration was statistically significant (p=0.005) (Figure 3). DISCUSSION Hippocampal volumes of CFS patients examined in the present study were not significantly smaller than those measured in controls, the absolute volumes being in the normal range for subjects aged between 20 and 40 years [9]. Whilst we did not standardize recorded hippocampal volumes for differences in intracranial volume, only a weak correlation has been found between these two measurements [9]. The concentrations of NAA, Cr and Cho were reduced in patients compared with controls, although this was significant only for NAA. By using an external standard to calibrate metabolite concentrations, we excluded the possibility that these reductions were due to a change in brain water content. The observed fall in NAA may be interpreted in several ways: either as a reduction in neuronal and/or glial cell density, or as reflecting reduced neuronal and/or glial cell metabolism. The recent finding that mature oligodendrocytes in cell culture contain similar amounts of NAA to that found in neurons [10] precludes further comment about the cellular origin of the fall in NAA in CFS. Given that hippocampal volume is preserved in CFS, it is likely that the NAA decrease reflects reduced neuronal/glial metabolism rather than reduced cell density. The possibility that these changes might be age related [11] was excluded through the use of age-matched controls. However, the patient and control groups were not precisely sex matched, and this may have contributed to the differences observed in this study, although a recent paper [8] did not report sex-dependent modification of hippocampal metabolite concentrations. Future work will be to match these results with clinical details in individual cases, stratifying patients according to their psychopathology, and to compare psychometric testing with NAA levels in CFS patients. ACKNOWLEDGMENTS We wish to thank the ME Association of Great Britain for their support of this project, and Dr Enis Cezayirli and Dr Clare Mackay for their assistance in obtaining the hippocampal volume measurements. The MRUI software package was provided by Aad van den Boogaart ( http://carbon.uab.es/mrui/mruiHomePage.html ). MRUI development is currently funded by the European Union project TMR/Networks ERB-FMRX-CT970160. FIGURE CAPTIONS Fig. 1. Unbiased estimates of the volume of the right (circles) and left (triangles) hippocampus in chronic fatigue syndrome patients (solid symbols) and in controls (open symbols). Also shown are the means and error bars (p/m 2 SE) for estimated volumes. Fig. 2. Spectra recorded from a 3 cm 3 voxel placed over the right hippocampus (a) in a patient with chronic fatigue syndrome and (b) in a healthy control subject. Spectra were acquired with the STEAM sequence, with TE/TM/TR572/13.7/3000 ms and 128 averages. mI, myo-inositol; Cho, choline-containing compounds; Cr, creatine/ phosphocreatine; NAA, N-acetylaspartate. ppm, parts per million. Fig. 3. Right hippocampal N-acetylaspartate (NAA) concentrations plotted for patients and controls. Mean concentration and error bars (p/m 2 standard errors) are also shown. The observed difference is significant (p=0.005). REFERENCES 1. Bremner JD, Randal P, Scott TM, Bronen RA, Seibyl JP, Southwick SM, et al. 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