- Email: [email protected]
Differentiating hippocampal subregions by means of quantitative magnetization transfer and relaxometry: preliminary results
www.elsevier.com/locate/ynimg NeuroImage 23 (2004) 1093 – 1099
Differentiating hippocampal subregions by means of quantitative magnetization transfer and relaxometry: preliminary results Claus Kiefer,a,* Johannes Slotboom,a Caroline Buri,b Jan Gralla,a Luca Remonda,a Thomas Dierks,b Werner K. Strik,b Gerhard Schroth,a and Peter Kalusa,b a
Institute for Diagnostic and Interventional Neuroradiology, University Hospital Bern, Switzerland University Hospital of Clinical Psychiatry, Bern, Switzerland
b
Received 27 February 2004; revised 5 June 2004; accepted 29 July 2004 Available online 14 October 2004 The hippocampal formation (HF) of healthy control subjects and schizophrenic patients was examined using an MRI experiment that implements sequences for relaxometry and magnetization transfer (MT) quantification. In addition to the semi-quantitative magnetization transfer ratio (MTR), all of the observable properties of the binary spin bath model were included. The study demonstrates that, in contrast to the MTR, quantitative MT parameters (especially the T2 relaxation time of restricted protons, T2b) are capable to differentiate functionally significant subregions within the HF. The MT methodology appears to be a promising new tool for the differential microstructural evaluation of the HF in neuropsychiatric disorders accompanied by memory disturbances. D 2004 Elsevier Inc. All rights reserved. Keywords: Magnetization transfer; Relaxation; Hippocampus; Schizophrenia
Introduction Magnetization transfer imaging (MTI) (Henkelman et al., 1993) is a technique that is very sensitive to subtle neuropathological changes. As reported by Natt et al. (2003), MTI provides morphologic information on brain tissue that is not accessible by conventional T1 and T2 relaxometry. Therefore, MTI has been used for the evaluation of several brain diseases associated with slight structural tissue alterations. Kabani et al. (2002) reported a significant reduction of the magnetization transfer ratio (MTR) in the gray matter of patients suffering from mild cognitive impairment. Tambasco et al. (2003) found lower MTR values in the substantia nigra and the red nucleus of patients with Parkinson’s disease, while other brain regions were unchanged. In multiple sclerosis, structural damage of normal appearing gray and white * Corresponding author. Institute for Diagnostic and Interventional Neuroradiology, University Hospital Bern (Inselspital), Freiburgstrasse 4, CH-3010 Bern, Switzerland. Fax: +41 31 632 4872. E-mail address: [email protected] (C. Kiefer). Available online on ScienceDirect (www.sciencedirect.com.) 1053-8119/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.neuroimage.2004.07.066
matter has been demonstrated by MTI (Cercignani et al., 2001). In a first whole-brain MTI study in schizophrenia, Foong (2001) reported significant MTR reductions in the bilateral parietooccipital cortex and the genus of the corpus callosum in chronic schizophrenia. Furthermore, MTI was applied to examine the myelination of white matter fiber tracts in young children (Rademacher et al., 1999). MTI generates contrast dependent upon the phenomenon of magnetization exchange between hydrogen nuclei 1H (protons) bound in semi-solid macromolecules and cellular membranes of brain tissue and free mobile bulk water protons (Wolff and Balaban, 1989). The protons in biological systems can be described as existing in two pools: the free and the bound protons. The most sophisticated model of Balaban and Ceckler (1992) characterizes the free pool as consisting of mobile bulk protons and so-called hydration water, protons connected to the surface of macromolecules via dipole-dipole interactions through space (cross relaxation) (Eng et al., 1991). Protons within the free pool are characterized by a short correlation time s C = 1012 s (hydration water s C = 108 s). This measure is the timeframe for loosing coherence—the protons have less time to interact with each other. In contrast, the bound protons have a correlation time of s C = 106 s. For the restricted protons in the bound pool, this means a very short T2-relaxation time (b0.1 ms) in comparison to the relatively long T2 (NN0.1 ms) of free protons. Thus, even for the shortest echo times (TE approximately 100 As) achievable in MRI using gradient echo sequences (Gatehouse and Bydder, 2003), the magnetization is destroyed. Nonetheless, the bound protons can be used for imaging by taking into account the width and location of the spectral lines of the pools. The free pool has a narrow spectral line (10–100 Hz), whereas the bound pool is characterized by a broad spectral line of 10–50 kHz, but each centered at the same Larmor frequency. This circumstance can be used to presaturate the bound protons by using frequency-selective RF pulses irradiated at a frequency offset D f with respect to the central 1 H Larmor frequency while affecting the dfree poolT due to magnetization transfer. (In the present study, we focused on the exclusive use of Gaussian modulated pre-saturation pulses; the
1094
C. Kiefer et al. / NeuroImage 23 (2004) 1093–1099
utilization of binomial pulses (Henkelman et al., 1993) is not topic of this work) Moreover, diffusion processes are responsible for an effective saturation of all macromolecules. In clinical practice, only a semi-quantitative technique is favored using the magnetization transfer ratio (MTR), which is calculated from the normalized differences of images acquired with and without the MT pre-saturation pulse. The determination of fractional pool sizes and T2-times of bound protons, however, requires an elaborate model to describe the dynamics of the magnetization transfer mechanism. In the present work, a quantitative imaging technique is used that yields all of the observable properties of the binary spin bath model for MT. Adapted from a model of the steady-state behavior of the magnetization during a pulsed MT-weighted imaging sequence (Sled and Pike, 2000a,b), as well as suitable methods in MRI relaxometry (Deichmann et al., 1999; Haase, 1990; MacFall et al., 1987), this approach yields parametric images of the fractional size F of the restricted pool as well as the T2 of the restricted pool and the relaxation times T1 and T2 in the free pool. The present study was conducted on a sample of healthy subjects and closely matched schizophrenic patients to evaluate the possible clinical significance of quantitative MTI for the structural characterization of the hippocampal formation (HF), a brain region that is immensely significant for memory functions and has been shown to be disturbed in numerous neuropsychiatric disorders including schizophrenia. In particular, our study aims at demonstrating that the quantitative MT methodology, additionally to the semiquantitative MTR, is capable to differentiate functionally important subregions of the HF, if the observable properties of the binary spin bath model are included. These parameters comprise the fractional pool size F and the T2 relaxation time of restricted protons.
Materials and methods Anatomical imaging All MRI experiments were performed on a Sonata 1.5T whole body scanner (Siemens, Erlangen, Germany) equipped with a 40 mT/m (200 mT/m-ms) gradient system and a CP standard head coil. The used scanner software release was Syngo MR 2002B (VA21B). Anatomical imaging was obtained using a T1-weighted, sagittal oriented 3D-MPRAGE sequence (TR/TE/TI 2000/3.93/590 ms, matrix 256 256, FOV 256 256 mm, flip angle 158, slab 160 mm) with a 1 mm3 isovoxel resolution. The commercially available software BrainVoyager 2000 (Version 4.9.6) was used for on-line tracing of regions of interest (ROIs) simultaneously in three planes, thus allowing for an exact delineation even of problematic anatomical boundaries (Kalus et al., 2004). Before the tracing procedure, the anatomical 3Disovoxel data set was manually angulated parallel to the long axis of the HF corresponding to the MTI orientation parameters. Every MT slice was carefully adjusted to the analogous obliqueaxial MPRAGE slice by visual inspection. ROI tracing of the HF and its three subdivisions, the hippocampal head (HH), body (HB), and tail (HT), were conducted according to the protocol proposed by Jack et al. (1997) with additional use of the cytoarchitectorial criteria described in the atlases by Duvernoy (1998) Mai et al. (1997). Like Jack et al., we used a calculated estimation for the usually ill-defined border between the HB and
the HT (Duvernoy, 1998): if the number of coronal slices containing the HB is called x, and the number of coronal slices containing the HT is called y, then the total coronal extension of the HB and HT is z = x + y. As the coronal extension of the HB usually amounts approximately the 1.5-fold of the coronal extension of the HT (i. e., x/y = 1.5/1), the slice numbers x and y can be calculated as follows: y = z/2.5 and x = z y. Anatomical ROI tracing was performed by a single person blind to the diagnosis of the participants. It is generally accepted that hippocampal volumes are affected by interindividual variability in the total intracranial volume (Free et al., 1995). Therefore, we used the intracranial volume on the coronal section exactly positioned at the level of the anterior commissure for normalization of the volumetric raw data according to the following formula: normalized volume = (raw volume/intracranial volume) 1000 (Free et al., 1995; Laakso et al., 2001). Statistical assessments were performed based on the normalized ROI volumes, which are reported as dimensionless quantities. MT theory The magnetization of the free pool is described by the Bloch equations while that of the restricted pool are modeled using the Redfield Provotorov theory (Goldman, 1970; Yeung et al., 1994). The fractional saturation of the free pool was calculated according to Bloch neglecting exchange phenomena and T1-relaxation for a range of T2-values (Sled and Pike, 2000a,b). The solution of the Redfield–Provotorov equations requires knowledge of the underlying spin-lattice relaxation time T1 and spin-spin relaxation time T2 of the free water protons. For this purpose, the relaxation times T1 and T2 of the free pool were determined in independent MR experiments. A set of TurboFLASH sequences (TR = 2000 ms (2 conc.), TE = 4.08 ms, a EXC = 58, S = 5 mm (gap 1.6 mm), 10 slices, FOV = 240 mm, matrix 128 128, interpolated to 256 256) with different inversion times (TI = 400, 650, 900, 1200, 1400 ms) has been used for the calculation of the T1 time (Deichmann et al., 1999; Haase, 1990; MacFall et al., 1987). The T2 time has been determined by using a multi-echo T2 weighted spin echo sequence (TR = 2500 ms, S = 3 mm (gap 3.6 mm), 10 slices, FOV = 240 mm, matrix = 256 256, TE = 22, 44, 66, 88 ms) and fitting the measured signal S: S ¼ S0expðTE=T2Þ
ð1Þ
The MT weighted images have been acquired using a set of seven gradient echo FLASH sequences with Gaussian modulated pre-saturation pulses located at frequency offsets Df = 0.25, 0.50, 1.00, 2.00, 4.00, 8.00, 16.00 kHz with respect to the central 1H Larmor frequency (TR = 267 ms, TE = 6 ms, a EXC = 208, a MT = 5408 (Gaussian), S = 5.0 (gap 1.6 mm), 10 slices, FOV = 240 mm, Matrix 128 128, interpolated to 256x256). MTR has been calculated relating the MT images acquired at Df = 1.00 kHz to the data without presaturation (M 0): MTR ¼ 1004ðM0 M ð Df ¼ 1:0ÞÞ=M0 : For the approximate solution of the Redfield–Provotorov system of differential equations and the determination of the T1 and T2 parameters, the nonlinear Levenberg–Marquardt technique has been used. For a detailed description of the MT relevant parameters we refer to the work of Sled et al. (Sled and Pike, 2000a,b; 2001).
C. Kiefer et al. / NeuroImage 23 (2004) 1093–1099
A phantom containing a 2% agar gel solution has been used to calibrate the relaxation and MT parameters by comparing the results with the values from the literature (Sled and Pike, 2001). With the help of the sagittal T1-MPRAGE images, the sequences for the determination of the T1, T2, and the MT parameters were angulated parallel to the long axis of the HF with the same center of slice. The snapshot FLASH sequence for the determination of the T1 values only allows for a minimum gap of 20% to avoid cross-talk of adjacent slices and thus represented the limiting factor with respect to the spatial resolution. To keep the total measurement in a range acceptable to critical head movements especially by schizophrenic patients, our study could not be designed for MR microscopy. Considering the spatial extension of the superior–inferior axis of the hippocampus of about 10 to 15 mm, our procedure guarantees that the major part of the HF is contained in two to three MT slices. Characterization of the control subjects and schizophrenic patients The study was conducted on 14 healthy control subjects without any known neuropsychiatric disorder and 14 sex- and age-matched patients suffering from paranoid schizophrenia according to the criteria of the International Classification of Diseases, Version 10 (ICD-10) (mean duration of disease F SD = 3.31 F 3.68 years). Subjects were included after having given informed consent to the study protocol, which had been approved by the local ethics committee. The psychopathology of schizophrenics was assessed using the Positive and Negative Symptoms Scale (PANSS) (Kay et al., 1987) (Mean total PANSS score F SD = 74.86 F 13.52; mean positive symptoms score F SD = 21.86 F 5.50; mean negative symptoms score F SD = 17.36 F 7.55, mean general psychopathology score F SD = 33.79 F 6.12). All schizophrenic patients were medicated with antipsychotic drugs, while none of the healthy controls took any medicaments.
1095
Results Demographic data The two study groups showed no significant differences in age (controls: mean age F SD = 31.00 F 6.50); schizophrenics: mean age F SD = 31.36 F 10.83), and sex (both groups: 9 males/5 females); all study participants were right-handed according to the Edinburgh Inventory (Oldfield, 1971). Volumetry For the ROI tracing procedure of the HF, the intrarater reliability (one rater, three measurements) was 0.978 for the HH, 0.964 for the HB, and 0.911 for the HT. The interrater reliability (three raters, ten hippocampi each) was 0.969 for the HH, 0.925 for the HB and 0.907 for the HT. One-way analysis of variance (ANOVA revealed a significant effect of the intracranial volume (ICV) at the level of the anterior commissure between the two groups (controls: mean ICV F SD = 12207 mm3 F 786.35; schizophrenics: 11375 mm3 F 712.16) showed a significant difference [ F(1,24) = 8.87, P = 0.007] with a 6.8% reduction in schizophrenics. For the normalized volumes of the hippocampal subregions and the total hippocampus, a repeated-measures mixed-model ANOVA with left/right side as within-subject factor and the diagnostic group as between-subject factor showed a significant main effect for the side [ F(1,26) = 13.61, P = 0.001], while there was no significant between-subject main effect for the diagnostic groups [ F(1,26) = 0.33, P = 0.57] The individual normalized ROI volumes for the hippocampal subregions are shown in Table 1. Furthermore, there were no significant correlations between the normalized volumes and the age and sex of the probands in both groups. Finally, in the schizophrenic group, the different PANSS subscores as well as the duration of illness did not correlate significantly with the volumetric measures. MT parameters
Statistics Statistical assessments were performed using the software package SPSS for Windows (Version 11.0). The intra- and interrater reliabilities of measured volumes were calculated as intraclass correlation coefficients (ICCs) using a two-way mixed effect model. The demographic variables, psychometric data, volumetric measures, and MT parameters were evaluated with the different general linear model (GLM) procedures of SPSS. Correlations between the dependent variables and the demographic and psychometric data were analysed using Pearson’s two-tailed correlation. The level of statistical significance was set at P b 0.05 for all tests.
Two-way ANOVAs factored into left/right side and hippocampal subregions did not show significant main effects of side for any of the MT parameters neither in the control group nor in schizophrenic patients. However, in healthy probands, there were significant within-subject main effects of the hippocampal subregions for T2b [ F(1,13) = 5.77, P = 0.008], T2f [ F(1,13) = 5.44, P = 0.011] and T1 [ F(1,13) = 9.70, P = 0.001]. The appropriate boxplots (Figs. 1–3) and post hoc test results of Bonferroni’s multiple comparisons procedure show that especially the righthemispheric hippocampal subregions contribute to these differences. In contrast to T2b (Fig. 1), T2f (Fig. 2), and T1 (Fig. 3), the main effects of the subregions did not reach significant levels for
Table 1 Normalized volumes (means and standard deviations) in controls and schizophrenic patients
Controls Schizophrenics
Right HH
Left HH
Right HB
Left HB
Right HT
Left HT
Right HC
Left HC
194.19 (34.58) 178.91 (28.50)
176.38 (25.27) 164.41 (37.52)
105.63 (14.93) 109.49 (14.18)
110.51 (14.31) 112.02 (17.42)
62.48 (7.86) 64.36 (9.67)
61.75 (7.36) 63.48 (13.27)
362.30 (44.81) 352.76 (37.92)
348.64 (36.94) 339.91 (50.71)
Values are indicated as dimensionless quantities. HH = hippocampal head, HB = hippocampal body, HT = hippocampal tail, HF = total hippocampal formation.
1096
C. Kiefer et al. / NeuroImage 23 (2004) 1093–1099
Fig. 1. Box plots (indicating the medians, quartiles, and extreme values) of healthy controls for T2b (*0.01 [As]). HH = hippocampal head, HB = hippocampal body, HT = hippocampal tail. Asterisks indicate levels of significance in Bonferroni’s multiple comparisons post hoc tests: * b 0.05, ** b 0.01, *** b 0.005, **** b 0.001.
Fig. 3. Box plots (indicating the medians, quartiles, and extreme values) of healthy controls for T1 ([ms]). HH = hippocampal head, HB = hippocampal body, HT = hippocampal tail. Asterisks indicate levels of significance in Bonferroni’s multiple comparisons post hoc tests: * b 0.05, ** b 0.01, *** b 0.005, **** b 0.001.
the F parameter [ F(1,13) = 1.76, P = 0.193] and the MTR [ F(1,13) = 2.96, P = 0.70]. Mixed ANOVAs with the diagnostic groups as between-subject factor and side and subregions as within-subject factors showed no significant main effects for any of the MT parameters between the proband groups [F parameter: F(1,26) = 0.07, P = 0.794; MTR: F(1,26) = 0.47, P = 0.498; T2b: F(1,26) = 1.88, P = 0.182; T2f: F(1,26) = 0.27, P = 0.607; T1: F(1,26) = 0.34, P = 0.567]. Furthermore, no significant correlations were found between the MT parameters and the age and sex in both study groups. In schizophrenic patients, MT parameters did not correlate significantly neither with the PANSS subscores nor with the duration of disease. To test the reproducibility of quantitative MT parameters, four control subjects were examined at two different points of time. Within-subject ANOVAs showed no significant differences for any of the MT parameters between the two measurements [ F parameter: F(1,23) = 1.43, P = 0.515; MTR: F(1,23) = 0.04, P = 0.851; T2b: F(1,23) = 1.97, P = 0.174; T2f: F(1,23) = 0.98, P = 0.332; T1: F(1,23) = 0.22, P = 0.646].
Discussion
Fig. 2. Box plots (indicating the medians, quartiles, and extreme values) of healthy controls for T2f ([ms]). HH = hippocampal head, HB = hippocampal body, HT = hippocampal tail. Asterisks indicate levels of significance in Bonferroni’s multiple comparisons post hoc tests: * b 0.05, ** b 0.01, *** b 0.005, **** b 0.001.
The present study aimed at the evaluation of differentiated volumetric data of the HF, and for the first time, the calculation of quantitative MT parameters within the hippocampal subregions of control subjects and schizophrenic patients. Generally, normalized ROI volumes as well as all MT parameters showed no significant age- or gender-specific effects. Furthermore, in the schizophrenic group, the duration of disease did not correlate significantly with
C. Kiefer et al. / NeuroImage 23 (2004) 1093–1099
any of the dependent variables. Since all schizophrenic patients were medicated with psychopharmacological drugs during the whole course of their disease, the duration of disease can be taken as a rough indication for the cumulative drug intake. Moreover, none of the scores from the psychopathological tests was significantly correlated with any of the dependant variables. The mean relaxation and MT parameters found in the subregions of the HF (Tables 1 and 2) generally correspond to the values reported in the literature for cerebral gray matter (Sled and Pike, 2004). However, for the first time, we report significant differences of T2b, T2f, and T1 values between the hippocampal subregions. The reported B0 and B1 variations by Sled and Pike (2000a,b) mainly focused on the anterior part of the hippocampus are theoretically associated with a reduced observed T2f value. Because the T2f values for the HH in our study were greater than the values of the HB subregion, we suppose that the significance of the differences between the HH and HB must be even higher if the corrections for B1 and B0 effects were taken into account. The main purpose of our study in our special case is not affected. So we conclude that beside the relaxometry, the quantitative MT parameters instead of the semi-quantitative MT ratio are relevant if alterations within the hippocampal formation are investigated. The fact that the MTR parameter did not reach a significant level reveals that the MTR parameter constitutes only one point within the absorption spectrum, whereas the parameters F and T2b are dependent on the amplitude and the full width at half maximum of the absorption spectrum. Although, at first sight, the MT parameters in this work seem to simply confirm the findings already stated by the results of the relaxometry, they provide further information that is not directly accessible by relaxometry. As described in Materials and methods, the behavior of restricted protons (Redfield–Provotorov model) is different from the protons of the free pool (Bloch model) and cannot be inferred by simply construing the relaxation parameters for the free pool. Pure MT effects have to be strongly distinguished from direct saturation effects. The latter are dependent on T1/T2 but have nothing in common with magnetization transfer (Henkelman et al., 1993). The finding of significant and characteristic differences of quantitative MT parameters between the HF subregions supports the assumption of their structural diversity as the basis for the functional heterogeneity of the hippocampal subregions, which was reported in several recent studies (Hackert et al., 2002; Sperling et al., 2003; Strange and Dolan, 1999). In learning experiments, eventrelated functional MRI studies found the anterior hippocampus to be primarily activated during the processing of novel information,
1097
while familiar stimuli provoked posterior hippocampus activation (Strange and Dolan, 1999). Especially, the encoding of novel facename associations was shown to be associated with selective increases of the fMRI signal within the anterior hippocampus (Sperling et al., 2003). Furthermore, HH volume was reported to be particularly associated with verbal memory performance, while the volumes of the posterior hippocampal subregions were not (Hackert et al., 2002). The neuroanatomical correlates of the functional diversity of hippocampal subregions are still unknown. According to the results of Sled et al., the differences of quantitative MT values in diverse brain areas may be due to their varying composition of gray and white matter (Sled et al., 2004). However, histological studies on the differential cytoarchitectonics and myeloarchitectonics of the hippocampal subregions are needed to prove the promising hypothesis that quantitative MTI will be capable to demonstrate subtle differences of the gray/white matter composition in topographically well-defined regions of the living human brain. Finally, it should be noticed that the differentiation of hippocampal subregions by MTI was only possible from T2f, T2b, and T1, but not the semi-quantitative MTR, which is used as the only parameter in many previous MTI examinations. Our results strongly suggest the employment of further quantitative MT parameters to detect subtle variations of cerebral tissue composition. The group of schizophrenic patients showed marked deviations from the control group in some of the MT values, however, there was no significant main effects for any MT parameter between the two study groups. Although there is multiple evidence for an involvement of the hippocampus in the pathophysiology of schizophrenia, the character, and the dimension of the underlying neuropathological process is still a matter of debate (for review, see Harrison, 2004). Postmortem investigations on the hippocampus in schizophrenia reported different alterations including changes of the spatial orientation of hippocampal pyramidal neurons (Scheibel and Kovelman, 1981), a decrease in hippocampal volume caused by a reduction of the white matter compartment without severe neuron loss (Colter et al., 1987; Heckers et al., 1991), and decreased expressions of diverse synaptic proteins (Eastwood et al., 2001). However, primarily due to the lavishness of the employed histological procedures, none of these studies specified the exact localization of the reported alterations with respect to their localization within the hippocampal subregions. So it can be speculated that the structural changes in the HF of schizophrenics might be extremely subtle and therefore only can be detected by our MT protocol under the condition of correcting for B1, B0, and
Table 2 MT parameter values (means and standard deviations) in controls and schizophrenic patients Controls F *10–4 MTR * 0.1 (%) T2b (As) T2f (ms) T1 (ms)
Schizophrenics
Right HH
Left HH
Right HB
Left HB
Right HT
Left HT
Right HH
Left HH
Right HB
Left HB
Right HT
Left HT
1272.21 (47.79) 330.98 (11.00) 7.98 (0.17) 116.64 (3.83) 1032.97 (38.19)
1249.03 (84.55) 322.02 (13.33) 8.04 (0.28) 127.24 (26.74) 1010.85 (41.31)
1325.31 (62.08) 337.61 (7.19) 8.48 (0.29) 107.26 (3.69) 905.34 (38.80)
1296.61 (140.45) 331.97 (12.13) 8.30 (0.49) 108.33 (7.53) 933.78 (43.48)
1258.75 (108.60) 325.87 (16.60) 8.03 (0.40) 130.53 (29.74) 1045.88 (113.88)
1266.82 (130.30) 327.97 (20.73) 8.02 (0.58) 116.14 (16.29) 1039.57 (164.24)
1253.19 (94.57) 328.18 (10.53) 8.17 (0.95) 121.12 (7.29) 1082.76 (80.21)
1211.22 (93.82) 320.17 (18.75) 8.00 (0.67) 125.16 (12.45) 1054.91 (49.19)
1408.95 (153.47) 346.96 (13.40) 8.65 (0.70) 104.46 (5.60) 894.64 (61.03)
1299.82 (99.62) 336.46 (13.50) 8.26 (0.32) 108.44 (5.46) 934.94 (80.69)
1205.18 (176.25) 325.52 (32.27) 8.43 (0.85) 150.20 (93.75) 1043.61 (173.75)
1339.66 (146.95) 336.42 (22.06) 8.51 (0.64) 114.86 (18.49) 896.23 (164.71)
HH = hippocampal head, HB = hippocampal body, HT = hippocampal tail, HF = total hippocampal formation.
1098
C. Kiefer et al. / NeuroImage 23 (2004) 1093–1099
motion effects. The worst motion artifacts have been considered excluding the affected data from the statistical analysis. In particular, our future MR-protocol will comprise sequences for B0 and B1 field mapping. The neglect of the correction for B0(1) variations and motion is the reason why currently no reliability parameter was defined—except the statistical within-subject ANOVAs mentioned in the work. Nevertheless, the main result of this preliminary study, that is, the differentiation of the hippocampal subregions in control subjects by quantitative MT parameters, was achieved without any B0(1) or motion correction. Previous MTI studies on schizophrenics reported no alterations of the MTR in the thalamus, but in the medial prefrontal cortex and the insula of schizophrenics (Bagary et al., 2002, 2003). In total, the few MTI results in schizophrenia until now support the assumption of a heterogeneous involvement of cerebral gray matter regions. To achieve a final judgment of the sensibility of quantitative MTI for possible hippocampal alterations in schizophrenics, further studies with larger patient samples and the comparison of different forms of the clinically heterogeneous schizophrenic psychoses are needed. MRI-volumetric examinations on the hippocampal subregions in schizophrenia yielded conflicting results. Our finding of no significant volume reductions of any hippocampal subregion is in agreement with the study of Laakso et al. (2001), but is at odds with the results of Szeszko et al. (2002), who found a significant volume reduction of the HH in schizophrenics. A first diffusion tensor imaging (DTI) study on the hippocampal subregions by our group found significant alterations of the inter-voxel coherences for the posterior hippocampus on both sides as well as for the left total HF in paranoid schizophrenics (Kalus et al., 2004). Because this latter study was based on the same ROI tracing protocol as the present examination and also included only paranoid schizophrenics, it can be concluded that the diagnostic information delivered by DTI and by quantitative MTI are different. Concerning the heterogeneous data from different MRI modalities including volumetry, DTI, and MTI, future investigations would profit from combining these techniques to further clarify the obviously subtle neuropathological processes underlying schizophrenic psychoses.
Conclusions In summary, this study demonstrates that quantitative MT parameters based on a two-pool spin model can be used to delineate functionally important subregions within the hippocampus. An MT-based differentiation of hippocampal subregions was only possible by the T2b parameter, but not by the semiquantitative MTR. Considering previous MTI studies and clinical neuroimaging examinations on hippocampal functions, it can be hypothesized that the heterogeneity of quantitative MT values in hippocampal subregions reflect subtle differences in their gray/ white matter composition and density which are not detectable with conventional T1- and T2-weighted MR imaging. However, this assumption has still to be verified by histological studies. Despite marked group differences for single MT parameters, the quantitative MTI protocol used in this study was not able to differentiate exactly between paranoid schizophrenics and normal controls. This finding may be due to technical imperfections that make it impossible at the current state to detect the subtleness of the histopathological alterations found in postmortem studies.
Thus, for a more exact characterization of the hippocampal subregions the further development will be focused on modifying the MT protocol in view of correcting for B0-, B1-, and motionrelated variations. Nevertheless, considering the clear discrimination of hippocampal subregions by quantitative MTI, this methodology appears to be a promising new tool for the differential microstructural evaluation of the HF in Alzheimer’s disease, temporal lobe epilepsy, and other disorders accompanied by impaired memory functions. The total measurement time for the MT and relaxometry part within a complex MRI protocol could be optimized to 25 min and is therefore reasonable for clinical routine as well.
Acknowledgments We thank Mrs. Regula Schweizer and Mrs. Michela Mordasini for their technical support. This work was partially supported by the SNF-Grant 3200-059077.99.
References Bagary, M.S., Foong, J., Maier, M., duBoulay, G., Barker, G.J., Miller, D.H., Ron, M.A., 2002. A magnetization transfer analysis of the thalamus in schizophrenia. J. Neuropsychiatry Clin. Neurosci. 14, 443 – 448. Bagary, M.S., Symms, M.R., Barker, G.J., Mutsarsa, S.H., Joyce, E.M., Ron, M.A., 2003. Gray and white matter abnormalities in first-episode schizophrenia inferred from magnetization transfer imaging. Arch. Gen. Psychiatry 60, 779 – 788. Balaban, R.S., Ceckler, T.L., 1992. Magnetization transfer contrast in magnetic resonance imaging. Magn. Reson. Q. 8 (2), 116 – 137. Cercignani, M., Bozzali, M., Iannucci, G., Comi, G., Filippi, M., 2001. Magnetization transfer ratio and mean diffusivity of normal appearing white and grey matter from patients with multiple sclerosis. J. Neurol. Neurosurg. Psychiatry 70, 311 – 317. Colter, N., Battal, S., Crow, T.J., Johnstone, E.C., Brown, R., Bruton, C., 1987. White matter reduction in the parahippocampal gyrus of patients with schizophrenia. Arch. Gen. Psychiatry 44, 1023. Deichmann, R., Hahn, D., Haase, A., 1999. Fast T1 mapping on a whole body scanner. Med. Risk Med. 42, 206 – 209. Duvernoy, H.M., 1998. The Human Hippocampus. Springer, Berlin. Eastwood, S.L., Cotter, D., Harrison, P.J., 2001. Cerebellar synaptic protein expression in schizophrenia. Neuroscience 105 (1), 219 – 229. Eng, J., et al., 1991. Quantitative 1H magnetization transfer imaging in vivo. Magn. Reson. Med. 17, 304 – 314. Foong, J., 2001. Neuropathological abnormalities in schizophrenia: evidence from magnetization transfer imaging. Brain 124, 882 – 892. Free, S.L., Bergin, P.S., Fish, D.R., Cook, M.J., Shorvon, S.D., Stevens, J.M., 1995. Methods for normalization of hippocampal volumes measured with MR. Am. J. Neuroradiol. 16, 637 – 643. Gatehouse, P.D., Bydder, G.M., 2003. Magnetic resonance imaging of short T2 components in tissue. Clin. Radiol. 58, 1 – 19. Goldman, M., 1970. Spin Temperature and Nuclear Magnetic Resonance in Solids. Oxford Univ. Press, London. Haase, A., 1990. Snapshot FLASH MRI: applications to T1, T2 and chemical shift imaging. Magn. Reson. Med. 13, 77 – 89. Hackert, V.H., den Heijler, T., Oudkerk, M., Koudstaal, A., Hofman, A., Breteler, M.B., 2002. Hippocampal head size associated with verbal memory performance in nondemented elderly. NeuroImage 17, 1365 – 1372. Harrison, P.J., 2004. The hippocampus in schizophrenia: a review of the neuropathological evidence and its pathophysiological implications. Psychopharmacology (Mar. 6, electronic publication ahead of print).
C. Kiefer et al. / NeuroImage 23 (2004) 1093–1099 Heckers, S., Heinsen, H., Geiger, B., Beckmann, H., 1991. Hippocampal neuron number in schizophrenia. A stereological study. Arch. Gen. Psychiatry 48, 1002 – 1008. Henkelman, R.M., Huang, X., Xiang, Q.S., Stanisz, G.J., Swanson, S.D., Bronskill, M.J., 1993. Quantitative interpretation of magnetization transfer. Magn. Reson. Med. 29 (6), 759 – 766. Jack, C.R., Petersen, R.C., Xu, Y.C., Waring, S.C., O’Brien, P.C., Tangalos, E.G., Smith, G.E., Ivnik, R.J., Kokmen, E., 1997. Medial temporal atrophy on MRI in normal aging and very mild Alzheimer’s disease. Neurology 49, 786 – 794. Kabani, N.J., Sled, J.G., Chertkow, H., 2002. Magnetization transfer ratio in mild cognitive impairment and dementia of Alzheimer’s type. NeuroImage 15, 604 – 610. Kalus, P., Buri, C., Slotboom, J., Gralla, J., Remonda, L., Dierks, T., Strik, W.K., Schroth, G., Kiefer, C., 2004. Volumetry and diffusion tensor imaging of hippocampal subregions in schizophrenia. NeuroReport 15, 867 – 871. Kay, S.R., Fitzbein, A., Opler, L.A., 1987. The positive and negative syndrome scale (PANSS) for schizophrenia. Schizophr. Bull. 13, 261 – 276. Laakso, M.P., Tiihonen, J., Syv7lahti, E., Vilkman, H., Laakso, A., Alakare, B., R7kkfl7inen, V., Salokangas, R.K.R., Koivisto, E., Hietala, J., 2001. A morphometric MRI study of the hippocampus in first-episode, neuroleptic-naRve schizophrenia. Schizophr. Res. 50, 3 – 7. MacFall, J.R., Wehrli, F.W., Breger, R.K., Johnson, G.A., 1987. Methodology for the measurement and analysis of relaxation times in proton imaging. Magn. Reson. Imag. 5, 209 – 220. Mai, J.K., Assheuer, J., Paxinos, G., 1997. Atlas of the Human Brain. Academic Press, San Diego. Natt, O., Watanabe, T., Boretius, S., Frahm, J., Michealis, T., 2003. Magnetization transfer MRI of mouse brain reveals areas of high neural density. Magn. Reson. Imag. 21, 1113 – 1120. Oldfield, R.C., 1971. The assessment and analysis of handedness: the Edinburgh inventory. Neuropsychologia 9 (1), 97 – 113. Rademacher, J., Engelbrecht, V., Bqrgel, U., Freund, H.-J., Zilles, K., 1999. Measuring in vivo myelination of human white matter fiber tracts with magnetization transfer MR. NeuroImage 9, 393 – 406.
1099
Scheibel, A.B., Kovelman, J.B., 1981. Disorientation of the hippocampal pyramidal cell and its processes in the schizophrenic patient. Biol. Psychiatry 16, 101 – 102. Sled, J.G., Pike, G.B., 2000a. Correction for B1 and B0 variations in quantitative T2 measurements using MRI. Magn. Reson. Med. 43 (4), 589 – 593. Sled, J.G., Pike, G.B., 2000b. Quantitative interpretation of magnetization transfer in spoiled gradient echo MRI sequences. J. Magn. Reson. 145, 24 – 36. Sled, J.G., Pike, G.B., 2001. Quantitative imaging of magnetization transfer exchange and relaxation properties in vivo using MRI. Magn. Reson. Med. 46 (5), 923 – 931. Sled, J.G., Pike, G.B., 2004. Regional variations in normal brain shown by quantitative magnetization transfer imaging. Magn. Reson. Med. 51, 299 – 303. Sperling, R., Chua, E., Cocchiarella, A., Rand-Giovannetti, E., Poldrack, R., Schacter, D.L., Albert, M., 2003. Putting names to faces: successful encoding of associative memories activates the anterior hippocampal formation. NeuroImage 20, 1400 – 1410. Strange, B.A., Dolan, R.J., 1999. Segregating the functions of human hippocampus. Proc. Natl. Acad. Sci. U. S. A. Neurobiology 96, 4034 – 4039. Szeszko, P.R., Strous, P.R., Goldman, R.S., Ashtari, M., Knuth, K.H., Lieberman, J.A., Bilder, R.M., 2002. Neuropsychological correlates of hippocampal volumes in patients experiencing a first episode of schizophrenia. Am. J. Psychiatry 159, 217 – 226. Tambasco, N., Pelliciolli, G.P., Chiarini, P., Montanari, G.E., Leone, F., Mancini, M.L., Paciaroni, M., Gallai, V., 2003. Magnetization transfer changes of grey and white matter in Parkinson’s disease. Neuroradiology 45, 224 – 230. Wolff, S.D., Balaban, R.S., 1989. Magnetization transfer contrast (MTC) and tissue water proton relaxation in vivo. Magn. Reson. Med. 10, 135 – 144. Yeung, H.N., Adler, R.S., Swanson, S.D., 1994. Transient decay of longitudinal magnetization in heterogeneous spin systems under selective saturation: IV. Reformulation of the spin-bath-model equations by the Redfield–Provotorov theory. J. Magn. Reson., A 106, 37 – 45.
Differentiating hippocampal subregions by means of quantitative magnetization transfer and relaxometry: preliminary results Claus Kiefer,a,* Johannes Slotboom,a Caroline Buri,b Jan Gralla,a Luca Remonda,a Thomas Dierks,b Werner K. Strik,b Gerhard Schroth,a and Peter Kalusa,b a
Institute for Diagnostic and Interventional Neuroradiology, University Hospital Bern, Switzerland University Hospital of Clinical Psychiatry, Bern, Switzerland
b
Received 27 February 2004; revised 5 June 2004; accepted 29 July 2004 Available online 14 October 2004 The hippocampal formation (HF) of healthy control subjects and schizophrenic patients was examined using an MRI experiment that implements sequences for relaxometry and magnetization transfer (MT) quantification. In addition to the semi-quantitative magnetization transfer ratio (MTR), all of the observable properties of the binary spin bath model were included. The study demonstrates that, in contrast to the MTR, quantitative MT parameters (especially the T2 relaxation time of restricted protons, T2b) are capable to differentiate functionally significant subregions within the HF. The MT methodology appears to be a promising new tool for the differential microstructural evaluation of the HF in neuropsychiatric disorders accompanied by memory disturbances. D 2004 Elsevier Inc. All rights reserved. Keywords: Magnetization transfer; Relaxation; Hippocampus; Schizophrenia
Introduction Magnetization transfer imaging (MTI) (Henkelman et al., 1993) is a technique that is very sensitive to subtle neuropathological changes. As reported by Natt et al. (2003), MTI provides morphologic information on brain tissue that is not accessible by conventional T1 and T2 relaxometry. Therefore, MTI has been used for the evaluation of several brain diseases associated with slight structural tissue alterations. Kabani et al. (2002) reported a significant reduction of the magnetization transfer ratio (MTR) in the gray matter of patients suffering from mild cognitive impairment. Tambasco et al. (2003) found lower MTR values in the substantia nigra and the red nucleus of patients with Parkinson’s disease, while other brain regions were unchanged. In multiple sclerosis, structural damage of normal appearing gray and white * Corresponding author. Institute for Diagnostic and Interventional Neuroradiology, University Hospital Bern (Inselspital), Freiburgstrasse 4, CH-3010 Bern, Switzerland. Fax: +41 31 632 4872. E-mail address: [email protected] (C. Kiefer). Available online on ScienceDirect (www.sciencedirect.com.) 1053-8119/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.neuroimage.2004.07.066
matter has been demonstrated by MTI (Cercignani et al., 2001). In a first whole-brain MTI study in schizophrenia, Foong (2001) reported significant MTR reductions in the bilateral parietooccipital cortex and the genus of the corpus callosum in chronic schizophrenia. Furthermore, MTI was applied to examine the myelination of white matter fiber tracts in young children (Rademacher et al., 1999). MTI generates contrast dependent upon the phenomenon of magnetization exchange between hydrogen nuclei 1H (protons) bound in semi-solid macromolecules and cellular membranes of brain tissue and free mobile bulk water protons (Wolff and Balaban, 1989). The protons in biological systems can be described as existing in two pools: the free and the bound protons. The most sophisticated model of Balaban and Ceckler (1992) characterizes the free pool as consisting of mobile bulk protons and so-called hydration water, protons connected to the surface of macromolecules via dipole-dipole interactions through space (cross relaxation) (Eng et al., 1991). Protons within the free pool are characterized by a short correlation time s C = 1012 s (hydration water s C = 108 s). This measure is the timeframe for loosing coherence—the protons have less time to interact with each other. In contrast, the bound protons have a correlation time of s C = 106 s. For the restricted protons in the bound pool, this means a very short T2-relaxation time (b0.1 ms) in comparison to the relatively long T2 (NN0.1 ms) of free protons. Thus, even for the shortest echo times (TE approximately 100 As) achievable in MRI using gradient echo sequences (Gatehouse and Bydder, 2003), the magnetization is destroyed. Nonetheless, the bound protons can be used for imaging by taking into account the width and location of the spectral lines of the pools. The free pool has a narrow spectral line (10–100 Hz), whereas the bound pool is characterized by a broad spectral line of 10–50 kHz, but each centered at the same Larmor frequency. This circumstance can be used to presaturate the bound protons by using frequency-selective RF pulses irradiated at a frequency offset D f with respect to the central 1 H Larmor frequency while affecting the dfree poolT due to magnetization transfer. (In the present study, we focused on the exclusive use of Gaussian modulated pre-saturation pulses; the
1094
C. Kiefer et al. / NeuroImage 23 (2004) 1093–1099
utilization of binomial pulses (Henkelman et al., 1993) is not topic of this work) Moreover, diffusion processes are responsible for an effective saturation of all macromolecules. In clinical practice, only a semi-quantitative technique is favored using the magnetization transfer ratio (MTR), which is calculated from the normalized differences of images acquired with and without the MT pre-saturation pulse. The determination of fractional pool sizes and T2-times of bound protons, however, requires an elaborate model to describe the dynamics of the magnetization transfer mechanism. In the present work, a quantitative imaging technique is used that yields all of the observable properties of the binary spin bath model for MT. Adapted from a model of the steady-state behavior of the magnetization during a pulsed MT-weighted imaging sequence (Sled and Pike, 2000a,b), as well as suitable methods in MRI relaxometry (Deichmann et al., 1999; Haase, 1990; MacFall et al., 1987), this approach yields parametric images of the fractional size F of the restricted pool as well as the T2 of the restricted pool and the relaxation times T1 and T2 in the free pool. The present study was conducted on a sample of healthy subjects and closely matched schizophrenic patients to evaluate the possible clinical significance of quantitative MTI for the structural characterization of the hippocampal formation (HF), a brain region that is immensely significant for memory functions and has been shown to be disturbed in numerous neuropsychiatric disorders including schizophrenia. In particular, our study aims at demonstrating that the quantitative MT methodology, additionally to the semiquantitative MTR, is capable to differentiate functionally important subregions of the HF, if the observable properties of the binary spin bath model are included. These parameters comprise the fractional pool size F and the T2 relaxation time of restricted protons.
Materials and methods Anatomical imaging All MRI experiments were performed on a Sonata 1.5T whole body scanner (Siemens, Erlangen, Germany) equipped with a 40 mT/m (200 mT/m-ms) gradient system and a CP standard head coil. The used scanner software release was Syngo MR 2002B (VA21B). Anatomical imaging was obtained using a T1-weighted, sagittal oriented 3D-MPRAGE sequence (TR/TE/TI 2000/3.93/590 ms, matrix 256 256, FOV 256 256 mm, flip angle 158, slab 160 mm) with a 1 mm3 isovoxel resolution. The commercially available software BrainVoyager 2000 (Version 4.9.6) was used for on-line tracing of regions of interest (ROIs) simultaneously in three planes, thus allowing for an exact delineation even of problematic anatomical boundaries (Kalus et al., 2004). Before the tracing procedure, the anatomical 3Disovoxel data set was manually angulated parallel to the long axis of the HF corresponding to the MTI orientation parameters. Every MT slice was carefully adjusted to the analogous obliqueaxial MPRAGE slice by visual inspection. ROI tracing of the HF and its three subdivisions, the hippocampal head (HH), body (HB), and tail (HT), were conducted according to the protocol proposed by Jack et al. (1997) with additional use of the cytoarchitectorial criteria described in the atlases by Duvernoy (1998) Mai et al. (1997). Like Jack et al., we used a calculated estimation for the usually ill-defined border between the HB and
the HT (Duvernoy, 1998): if the number of coronal slices containing the HB is called x, and the number of coronal slices containing the HT is called y, then the total coronal extension of the HB and HT is z = x + y. As the coronal extension of the HB usually amounts approximately the 1.5-fold of the coronal extension of the HT (i. e., x/y = 1.5/1), the slice numbers x and y can be calculated as follows: y = z/2.5 and x = z y. Anatomical ROI tracing was performed by a single person blind to the diagnosis of the participants. It is generally accepted that hippocampal volumes are affected by interindividual variability in the total intracranial volume (Free et al., 1995). Therefore, we used the intracranial volume on the coronal section exactly positioned at the level of the anterior commissure for normalization of the volumetric raw data according to the following formula: normalized volume = (raw volume/intracranial volume) 1000 (Free et al., 1995; Laakso et al., 2001). Statistical assessments were performed based on the normalized ROI volumes, which are reported as dimensionless quantities. MT theory The magnetization of the free pool is described by the Bloch equations while that of the restricted pool are modeled using the Redfield Provotorov theory (Goldman, 1970; Yeung et al., 1994). The fractional saturation of the free pool was calculated according to Bloch neglecting exchange phenomena and T1-relaxation for a range of T2-values (Sled and Pike, 2000a,b). The solution of the Redfield–Provotorov equations requires knowledge of the underlying spin-lattice relaxation time T1 and spin-spin relaxation time T2 of the free water protons. For this purpose, the relaxation times T1 and T2 of the free pool were determined in independent MR experiments. A set of TurboFLASH sequences (TR = 2000 ms (2 conc.), TE = 4.08 ms, a EXC = 58, S = 5 mm (gap 1.6 mm), 10 slices, FOV = 240 mm, matrix 128 128, interpolated to 256 256) with different inversion times (TI = 400, 650, 900, 1200, 1400 ms) has been used for the calculation of the T1 time (Deichmann et al., 1999; Haase, 1990; MacFall et al., 1987). The T2 time has been determined by using a multi-echo T2 weighted spin echo sequence (TR = 2500 ms, S = 3 mm (gap 3.6 mm), 10 slices, FOV = 240 mm, matrix = 256 256, TE = 22, 44, 66, 88 ms) and fitting the measured signal S: S ¼ S0expðTE=T2Þ
ð1Þ
The MT weighted images have been acquired using a set of seven gradient echo FLASH sequences with Gaussian modulated pre-saturation pulses located at frequency offsets Df = 0.25, 0.50, 1.00, 2.00, 4.00, 8.00, 16.00 kHz with respect to the central 1H Larmor frequency (TR = 267 ms, TE = 6 ms, a EXC = 208, a MT = 5408 (Gaussian), S = 5.0 (gap 1.6 mm), 10 slices, FOV = 240 mm, Matrix 128 128, interpolated to 256x256). MTR has been calculated relating the MT images acquired at Df = 1.00 kHz to the data without presaturation (M 0): MTR ¼ 1004ðM0 M ð Df ¼ 1:0ÞÞ=M0 : For the approximate solution of the Redfield–Provotorov system of differential equations and the determination of the T1 and T2 parameters, the nonlinear Levenberg–Marquardt technique has been used. For a detailed description of the MT relevant parameters we refer to the work of Sled et al. (Sled and Pike, 2000a,b; 2001).
C. Kiefer et al. / NeuroImage 23 (2004) 1093–1099
A phantom containing a 2% agar gel solution has been used to calibrate the relaxation and MT parameters by comparing the results with the values from the literature (Sled and Pike, 2001). With the help of the sagittal T1-MPRAGE images, the sequences for the determination of the T1, T2, and the MT parameters were angulated parallel to the long axis of the HF with the same center of slice. The snapshot FLASH sequence for the determination of the T1 values only allows for a minimum gap of 20% to avoid cross-talk of adjacent slices and thus represented the limiting factor with respect to the spatial resolution. To keep the total measurement in a range acceptable to critical head movements especially by schizophrenic patients, our study could not be designed for MR microscopy. Considering the spatial extension of the superior–inferior axis of the hippocampus of about 10 to 15 mm, our procedure guarantees that the major part of the HF is contained in two to three MT slices. Characterization of the control subjects and schizophrenic patients The study was conducted on 14 healthy control subjects without any known neuropsychiatric disorder and 14 sex- and age-matched patients suffering from paranoid schizophrenia according to the criteria of the International Classification of Diseases, Version 10 (ICD-10) (mean duration of disease F SD = 3.31 F 3.68 years). Subjects were included after having given informed consent to the study protocol, which had been approved by the local ethics committee. The psychopathology of schizophrenics was assessed using the Positive and Negative Symptoms Scale (PANSS) (Kay et al., 1987) (Mean total PANSS score F SD = 74.86 F 13.52; mean positive symptoms score F SD = 21.86 F 5.50; mean negative symptoms score F SD = 17.36 F 7.55, mean general psychopathology score F SD = 33.79 F 6.12). All schizophrenic patients were medicated with antipsychotic drugs, while none of the healthy controls took any medicaments.
1095
Results Demographic data The two study groups showed no significant differences in age (controls: mean age F SD = 31.00 F 6.50); schizophrenics: mean age F SD = 31.36 F 10.83), and sex (both groups: 9 males/5 females); all study participants were right-handed according to the Edinburgh Inventory (Oldfield, 1971). Volumetry For the ROI tracing procedure of the HF, the intrarater reliability (one rater, three measurements) was 0.978 for the HH, 0.964 for the HB, and 0.911 for the HT. The interrater reliability (three raters, ten hippocampi each) was 0.969 for the HH, 0.925 for the HB and 0.907 for the HT. One-way analysis of variance (ANOVA revealed a significant effect of the intracranial volume (ICV) at the level of the anterior commissure between the two groups (controls: mean ICV F SD = 12207 mm3 F 786.35; schizophrenics: 11375 mm3 F 712.16) showed a significant difference [ F(1,24) = 8.87, P = 0.007] with a 6.8% reduction in schizophrenics. For the normalized volumes of the hippocampal subregions and the total hippocampus, a repeated-measures mixed-model ANOVA with left/right side as within-subject factor and the diagnostic group as between-subject factor showed a significant main effect for the side [ F(1,26) = 13.61, P = 0.001], while there was no significant between-subject main effect for the diagnostic groups [ F(1,26) = 0.33, P = 0.57] The individual normalized ROI volumes for the hippocampal subregions are shown in Table 1. Furthermore, there were no significant correlations between the normalized volumes and the age and sex of the probands in both groups. Finally, in the schizophrenic group, the different PANSS subscores as well as the duration of illness did not correlate significantly with the volumetric measures. MT parameters
Statistics Statistical assessments were performed using the software package SPSS for Windows (Version 11.0). The intra- and interrater reliabilities of measured volumes were calculated as intraclass correlation coefficients (ICCs) using a two-way mixed effect model. The demographic variables, psychometric data, volumetric measures, and MT parameters were evaluated with the different general linear model (GLM) procedures of SPSS. Correlations between the dependent variables and the demographic and psychometric data were analysed using Pearson’s two-tailed correlation. The level of statistical significance was set at P b 0.05 for all tests.
Two-way ANOVAs factored into left/right side and hippocampal subregions did not show significant main effects of side for any of the MT parameters neither in the control group nor in schizophrenic patients. However, in healthy probands, there were significant within-subject main effects of the hippocampal subregions for T2b [ F(1,13) = 5.77, P = 0.008], T2f [ F(1,13) = 5.44, P = 0.011] and T1 [ F(1,13) = 9.70, P = 0.001]. The appropriate boxplots (Figs. 1–3) and post hoc test results of Bonferroni’s multiple comparisons procedure show that especially the righthemispheric hippocampal subregions contribute to these differences. In contrast to T2b (Fig. 1), T2f (Fig. 2), and T1 (Fig. 3), the main effects of the subregions did not reach significant levels for
Table 1 Normalized volumes (means and standard deviations) in controls and schizophrenic patients
Controls Schizophrenics
Right HH
Left HH
Right HB
Left HB
Right HT
Left HT
Right HC
Left HC
194.19 (34.58) 178.91 (28.50)
176.38 (25.27) 164.41 (37.52)
105.63 (14.93) 109.49 (14.18)
110.51 (14.31) 112.02 (17.42)
62.48 (7.86) 64.36 (9.67)
61.75 (7.36) 63.48 (13.27)
362.30 (44.81) 352.76 (37.92)
348.64 (36.94) 339.91 (50.71)
Values are indicated as dimensionless quantities. HH = hippocampal head, HB = hippocampal body, HT = hippocampal tail, HF = total hippocampal formation.
1096
C. Kiefer et al. / NeuroImage 23 (2004) 1093–1099
Fig. 1. Box plots (indicating the medians, quartiles, and extreme values) of healthy controls for T2b (*0.01 [As]). HH = hippocampal head, HB = hippocampal body, HT = hippocampal tail. Asterisks indicate levels of significance in Bonferroni’s multiple comparisons post hoc tests: * b 0.05, ** b 0.01, *** b 0.005, **** b 0.001.
Fig. 3. Box plots (indicating the medians, quartiles, and extreme values) of healthy controls for T1 ([ms]). HH = hippocampal head, HB = hippocampal body, HT = hippocampal tail. Asterisks indicate levels of significance in Bonferroni’s multiple comparisons post hoc tests: * b 0.05, ** b 0.01, *** b 0.005, **** b 0.001.
the F parameter [ F(1,13) = 1.76, P = 0.193] and the MTR [ F(1,13) = 2.96, P = 0.70]. Mixed ANOVAs with the diagnostic groups as between-subject factor and side and subregions as within-subject factors showed no significant main effects for any of the MT parameters between the proband groups [F parameter: F(1,26) = 0.07, P = 0.794; MTR: F(1,26) = 0.47, P = 0.498; T2b: F(1,26) = 1.88, P = 0.182; T2f: F(1,26) = 0.27, P = 0.607; T1: F(1,26) = 0.34, P = 0.567]. Furthermore, no significant correlations were found between the MT parameters and the age and sex in both study groups. In schizophrenic patients, MT parameters did not correlate significantly neither with the PANSS subscores nor with the duration of disease. To test the reproducibility of quantitative MT parameters, four control subjects were examined at two different points of time. Within-subject ANOVAs showed no significant differences for any of the MT parameters between the two measurements [ F parameter: F(1,23) = 1.43, P = 0.515; MTR: F(1,23) = 0.04, P = 0.851; T2b: F(1,23) = 1.97, P = 0.174; T2f: F(1,23) = 0.98, P = 0.332; T1: F(1,23) = 0.22, P = 0.646].
Discussion
Fig. 2. Box plots (indicating the medians, quartiles, and extreme values) of healthy controls for T2f ([ms]). HH = hippocampal head, HB = hippocampal body, HT = hippocampal tail. Asterisks indicate levels of significance in Bonferroni’s multiple comparisons post hoc tests: * b 0.05, ** b 0.01, *** b 0.005, **** b 0.001.
The present study aimed at the evaluation of differentiated volumetric data of the HF, and for the first time, the calculation of quantitative MT parameters within the hippocampal subregions of control subjects and schizophrenic patients. Generally, normalized ROI volumes as well as all MT parameters showed no significant age- or gender-specific effects. Furthermore, in the schizophrenic group, the duration of disease did not correlate significantly with
C. Kiefer et al. / NeuroImage 23 (2004) 1093–1099
any of the dependent variables. Since all schizophrenic patients were medicated with psychopharmacological drugs during the whole course of their disease, the duration of disease can be taken as a rough indication for the cumulative drug intake. Moreover, none of the scores from the psychopathological tests was significantly correlated with any of the dependant variables. The mean relaxation and MT parameters found in the subregions of the HF (Tables 1 and 2) generally correspond to the values reported in the literature for cerebral gray matter (Sled and Pike, 2004). However, for the first time, we report significant differences of T2b, T2f, and T1 values between the hippocampal subregions. The reported B0 and B1 variations by Sled and Pike (2000a,b) mainly focused on the anterior part of the hippocampus are theoretically associated with a reduced observed T2f value. Because the T2f values for the HH in our study were greater than the values of the HB subregion, we suppose that the significance of the differences between the HH and HB must be even higher if the corrections for B1 and B0 effects were taken into account. The main purpose of our study in our special case is not affected. So we conclude that beside the relaxometry, the quantitative MT parameters instead of the semi-quantitative MT ratio are relevant if alterations within the hippocampal formation are investigated. The fact that the MTR parameter did not reach a significant level reveals that the MTR parameter constitutes only one point within the absorption spectrum, whereas the parameters F and T2b are dependent on the amplitude and the full width at half maximum of the absorption spectrum. Although, at first sight, the MT parameters in this work seem to simply confirm the findings already stated by the results of the relaxometry, they provide further information that is not directly accessible by relaxometry. As described in Materials and methods, the behavior of restricted protons (Redfield–Provotorov model) is different from the protons of the free pool (Bloch model) and cannot be inferred by simply construing the relaxation parameters for the free pool. Pure MT effects have to be strongly distinguished from direct saturation effects. The latter are dependent on T1/T2 but have nothing in common with magnetization transfer (Henkelman et al., 1993). The finding of significant and characteristic differences of quantitative MT parameters between the HF subregions supports the assumption of their structural diversity as the basis for the functional heterogeneity of the hippocampal subregions, which was reported in several recent studies (Hackert et al., 2002; Sperling et al., 2003; Strange and Dolan, 1999). In learning experiments, eventrelated functional MRI studies found the anterior hippocampus to be primarily activated during the processing of novel information,
1097
while familiar stimuli provoked posterior hippocampus activation (Strange and Dolan, 1999). Especially, the encoding of novel facename associations was shown to be associated with selective increases of the fMRI signal within the anterior hippocampus (Sperling et al., 2003). Furthermore, HH volume was reported to be particularly associated with verbal memory performance, while the volumes of the posterior hippocampal subregions were not (Hackert et al., 2002). The neuroanatomical correlates of the functional diversity of hippocampal subregions are still unknown. According to the results of Sled et al., the differences of quantitative MT values in diverse brain areas may be due to their varying composition of gray and white matter (Sled et al., 2004). However, histological studies on the differential cytoarchitectonics and myeloarchitectonics of the hippocampal subregions are needed to prove the promising hypothesis that quantitative MTI will be capable to demonstrate subtle differences of the gray/white matter composition in topographically well-defined regions of the living human brain. Finally, it should be noticed that the differentiation of hippocampal subregions by MTI was only possible from T2f, T2b, and T1, but not the semi-quantitative MTR, which is used as the only parameter in many previous MTI examinations. Our results strongly suggest the employment of further quantitative MT parameters to detect subtle variations of cerebral tissue composition. The group of schizophrenic patients showed marked deviations from the control group in some of the MT values, however, there was no significant main effects for any MT parameter between the two study groups. Although there is multiple evidence for an involvement of the hippocampus in the pathophysiology of schizophrenia, the character, and the dimension of the underlying neuropathological process is still a matter of debate (for review, see Harrison, 2004). Postmortem investigations on the hippocampus in schizophrenia reported different alterations including changes of the spatial orientation of hippocampal pyramidal neurons (Scheibel and Kovelman, 1981), a decrease in hippocampal volume caused by a reduction of the white matter compartment without severe neuron loss (Colter et al., 1987; Heckers et al., 1991), and decreased expressions of diverse synaptic proteins (Eastwood et al., 2001). However, primarily due to the lavishness of the employed histological procedures, none of these studies specified the exact localization of the reported alterations with respect to their localization within the hippocampal subregions. So it can be speculated that the structural changes in the HF of schizophrenics might be extremely subtle and therefore only can be detected by our MT protocol under the condition of correcting for B1, B0, and
Table 2 MT parameter values (means and standard deviations) in controls and schizophrenic patients Controls F *10–4 MTR * 0.1 (%) T2b (As) T2f (ms) T1 (ms)
Schizophrenics
Right HH
Left HH
Right HB
Left HB
Right HT
Left HT
Right HH
Left HH
Right HB
Left HB
Right HT
Left HT
1272.21 (47.79) 330.98 (11.00) 7.98 (0.17) 116.64 (3.83) 1032.97 (38.19)
1249.03 (84.55) 322.02 (13.33) 8.04 (0.28) 127.24 (26.74) 1010.85 (41.31)
1325.31 (62.08) 337.61 (7.19) 8.48 (0.29) 107.26 (3.69) 905.34 (38.80)
1296.61 (140.45) 331.97 (12.13) 8.30 (0.49) 108.33 (7.53) 933.78 (43.48)
1258.75 (108.60) 325.87 (16.60) 8.03 (0.40) 130.53 (29.74) 1045.88 (113.88)
1266.82 (130.30) 327.97 (20.73) 8.02 (0.58) 116.14 (16.29) 1039.57 (164.24)
1253.19 (94.57) 328.18 (10.53) 8.17 (0.95) 121.12 (7.29) 1082.76 (80.21)
1211.22 (93.82) 320.17 (18.75) 8.00 (0.67) 125.16 (12.45) 1054.91 (49.19)
1408.95 (153.47) 346.96 (13.40) 8.65 (0.70) 104.46 (5.60) 894.64 (61.03)
1299.82 (99.62) 336.46 (13.50) 8.26 (0.32) 108.44 (5.46) 934.94 (80.69)
1205.18 (176.25) 325.52 (32.27) 8.43 (0.85) 150.20 (93.75) 1043.61 (173.75)
1339.66 (146.95) 336.42 (22.06) 8.51 (0.64) 114.86 (18.49) 896.23 (164.71)
HH = hippocampal head, HB = hippocampal body, HT = hippocampal tail, HF = total hippocampal formation.
1098
C. Kiefer et al. / NeuroImage 23 (2004) 1093–1099
motion effects. The worst motion artifacts have been considered excluding the affected data from the statistical analysis. In particular, our future MR-protocol will comprise sequences for B0 and B1 field mapping. The neglect of the correction for B0(1) variations and motion is the reason why currently no reliability parameter was defined—except the statistical within-subject ANOVAs mentioned in the work. Nevertheless, the main result of this preliminary study, that is, the differentiation of the hippocampal subregions in control subjects by quantitative MT parameters, was achieved without any B0(1) or motion correction. Previous MTI studies on schizophrenics reported no alterations of the MTR in the thalamus, but in the medial prefrontal cortex and the insula of schizophrenics (Bagary et al., 2002, 2003). In total, the few MTI results in schizophrenia until now support the assumption of a heterogeneous involvement of cerebral gray matter regions. To achieve a final judgment of the sensibility of quantitative MTI for possible hippocampal alterations in schizophrenics, further studies with larger patient samples and the comparison of different forms of the clinically heterogeneous schizophrenic psychoses are needed. MRI-volumetric examinations on the hippocampal subregions in schizophrenia yielded conflicting results. Our finding of no significant volume reductions of any hippocampal subregion is in agreement with the study of Laakso et al. (2001), but is at odds with the results of Szeszko et al. (2002), who found a significant volume reduction of the HH in schizophrenics. A first diffusion tensor imaging (DTI) study on the hippocampal subregions by our group found significant alterations of the inter-voxel coherences for the posterior hippocampus on both sides as well as for the left total HF in paranoid schizophrenics (Kalus et al., 2004). Because this latter study was based on the same ROI tracing protocol as the present examination and also included only paranoid schizophrenics, it can be concluded that the diagnostic information delivered by DTI and by quantitative MTI are different. Concerning the heterogeneous data from different MRI modalities including volumetry, DTI, and MTI, future investigations would profit from combining these techniques to further clarify the obviously subtle neuropathological processes underlying schizophrenic psychoses.
Conclusions In summary, this study demonstrates that quantitative MT parameters based on a two-pool spin model can be used to delineate functionally important subregions within the hippocampus. An MT-based differentiation of hippocampal subregions was only possible by the T2b parameter, but not by the semiquantitative MTR. Considering previous MTI studies and clinical neuroimaging examinations on hippocampal functions, it can be hypothesized that the heterogeneity of quantitative MT values in hippocampal subregions reflect subtle differences in their gray/ white matter composition and density which are not detectable with conventional T1- and T2-weighted MR imaging. However, this assumption has still to be verified by histological studies. Despite marked group differences for single MT parameters, the quantitative MTI protocol used in this study was not able to differentiate exactly between paranoid schizophrenics and normal controls. This finding may be due to technical imperfections that make it impossible at the current state to detect the subtleness of the histopathological alterations found in postmortem studies.
Thus, for a more exact characterization of the hippocampal subregions the further development will be focused on modifying the MT protocol in view of correcting for B0-, B1-, and motionrelated variations. Nevertheless, considering the clear discrimination of hippocampal subregions by quantitative MTI, this methodology appears to be a promising new tool for the differential microstructural evaluation of the HF in Alzheimer’s disease, temporal lobe epilepsy, and other disorders accompanied by impaired memory functions. The total measurement time for the MT and relaxometry part within a complex MRI protocol could be optimized to 25 min and is therefore reasonable for clinical routine as well.
Acknowledgments We thank Mrs. Regula Schweizer and Mrs. Michela Mordasini for their technical support. This work was partially supported by the SNF-Grant 3200-059077.99.
References Bagary, M.S., Foong, J., Maier, M., duBoulay, G., Barker, G.J., Miller, D.H., Ron, M.A., 2002. A magnetization transfer analysis of the thalamus in schizophrenia. J. Neuropsychiatry Clin. Neurosci. 14, 443 – 448. Bagary, M.S., Symms, M.R., Barker, G.J., Mutsarsa, S.H., Joyce, E.M., Ron, M.A., 2003. Gray and white matter abnormalities in first-episode schizophrenia inferred from magnetization transfer imaging. Arch. Gen. Psychiatry 60, 779 – 788. Balaban, R.S., Ceckler, T.L., 1992. Magnetization transfer contrast in magnetic resonance imaging. Magn. Reson. Q. 8 (2), 116 – 137. Cercignani, M., Bozzali, M., Iannucci, G., Comi, G., Filippi, M., 2001. Magnetization transfer ratio and mean diffusivity of normal appearing white and grey matter from patients with multiple sclerosis. J. Neurol. Neurosurg. Psychiatry 70, 311 – 317. Colter, N., Battal, S., Crow, T.J., Johnstone, E.C., Brown, R., Bruton, C., 1987. White matter reduction in the parahippocampal gyrus of patients with schizophrenia. Arch. Gen. Psychiatry 44, 1023. Deichmann, R., Hahn, D., Haase, A., 1999. Fast T1 mapping on a whole body scanner. Med. Risk Med. 42, 206 – 209. Duvernoy, H.M., 1998. The Human Hippocampus. Springer, Berlin. Eastwood, S.L., Cotter, D., Harrison, P.J., 2001. Cerebellar synaptic protein expression in schizophrenia. Neuroscience 105 (1), 219 – 229. Eng, J., et al., 1991. Quantitative 1H magnetization transfer imaging in vivo. Magn. Reson. Med. 17, 304 – 314. Foong, J., 2001. Neuropathological abnormalities in schizophrenia: evidence from magnetization transfer imaging. Brain 124, 882 – 892. Free, S.L., Bergin, P.S., Fish, D.R., Cook, M.J., Shorvon, S.D., Stevens, J.M., 1995. Methods for normalization of hippocampal volumes measured with MR. Am. J. Neuroradiol. 16, 637 – 643. Gatehouse, P.D., Bydder, G.M., 2003. Magnetic resonance imaging of short T2 components in tissue. Clin. Radiol. 58, 1 – 19. Goldman, M., 1970. Spin Temperature and Nuclear Magnetic Resonance in Solids. Oxford Univ. Press, London. Haase, A., 1990. Snapshot FLASH MRI: applications to T1, T2 and chemical shift imaging. Magn. Reson. Med. 13, 77 – 89. Hackert, V.H., den Heijler, T., Oudkerk, M., Koudstaal, A., Hofman, A., Breteler, M.B., 2002. Hippocampal head size associated with verbal memory performance in nondemented elderly. NeuroImage 17, 1365 – 1372. Harrison, P.J., 2004. The hippocampus in schizophrenia: a review of the neuropathological evidence and its pathophysiological implications. Psychopharmacology (Mar. 6, electronic publication ahead of print).
C. Kiefer et al. / NeuroImage 23 (2004) 1093–1099 Heckers, S., Heinsen, H., Geiger, B., Beckmann, H., 1991. Hippocampal neuron number in schizophrenia. A stereological study. Arch. Gen. Psychiatry 48, 1002 – 1008. Henkelman, R.M., Huang, X., Xiang, Q.S., Stanisz, G.J., Swanson, S.D., Bronskill, M.J., 1993. Quantitative interpretation of magnetization transfer. Magn. Reson. Med. 29 (6), 759 – 766. Jack, C.R., Petersen, R.C., Xu, Y.C., Waring, S.C., O’Brien, P.C., Tangalos, E.G., Smith, G.E., Ivnik, R.J., Kokmen, E., 1997. Medial temporal atrophy on MRI in normal aging and very mild Alzheimer’s disease. Neurology 49, 786 – 794. Kabani, N.J., Sled, J.G., Chertkow, H., 2002. Magnetization transfer ratio in mild cognitive impairment and dementia of Alzheimer’s type. NeuroImage 15, 604 – 610. Kalus, P., Buri, C., Slotboom, J., Gralla, J., Remonda, L., Dierks, T., Strik, W.K., Schroth, G., Kiefer, C., 2004. Volumetry and diffusion tensor imaging of hippocampal subregions in schizophrenia. NeuroReport 15, 867 – 871. Kay, S.R., Fitzbein, A., Opler, L.A., 1987. The positive and negative syndrome scale (PANSS) for schizophrenia. Schizophr. Bull. 13, 261 – 276. Laakso, M.P., Tiihonen, J., Syv7lahti, E., Vilkman, H., Laakso, A., Alakare, B., R7kkfl7inen, V., Salokangas, R.K.R., Koivisto, E., Hietala, J., 2001. A morphometric MRI study of the hippocampus in first-episode, neuroleptic-naRve schizophrenia. Schizophr. Res. 50, 3 – 7. MacFall, J.R., Wehrli, F.W., Breger, R.K., Johnson, G.A., 1987. Methodology for the measurement and analysis of relaxation times in proton imaging. Magn. Reson. Imag. 5, 209 – 220. Mai, J.K., Assheuer, J., Paxinos, G., 1997. Atlas of the Human Brain. Academic Press, San Diego. Natt, O., Watanabe, T., Boretius, S., Frahm, J., Michealis, T., 2003. Magnetization transfer MRI of mouse brain reveals areas of high neural density. Magn. Reson. Imag. 21, 1113 – 1120. Oldfield, R.C., 1971. The assessment and analysis of handedness: the Edinburgh inventory. Neuropsychologia 9 (1), 97 – 113. Rademacher, J., Engelbrecht, V., Bqrgel, U., Freund, H.-J., Zilles, K., 1999. Measuring in vivo myelination of human white matter fiber tracts with magnetization transfer MR. NeuroImage 9, 393 – 406.
1099
Scheibel, A.B., Kovelman, J.B., 1981. Disorientation of the hippocampal pyramidal cell and its processes in the schizophrenic patient. Biol. Psychiatry 16, 101 – 102. Sled, J.G., Pike, G.B., 2000a. Correction for B1 and B0 variations in quantitative T2 measurements using MRI. Magn. Reson. Med. 43 (4), 589 – 593. Sled, J.G., Pike, G.B., 2000b. Quantitative interpretation of magnetization transfer in spoiled gradient echo MRI sequences. J. Magn. Reson. 145, 24 – 36. Sled, J.G., Pike, G.B., 2001. Quantitative imaging of magnetization transfer exchange and relaxation properties in vivo using MRI. Magn. Reson. Med. 46 (5), 923 – 931. Sled, J.G., Pike, G.B., 2004. Regional variations in normal brain shown by quantitative magnetization transfer imaging. Magn. Reson. Med. 51, 299 – 303. Sperling, R., Chua, E., Cocchiarella, A., Rand-Giovannetti, E., Poldrack, R., Schacter, D.L., Albert, M., 2003. Putting names to faces: successful encoding of associative memories activates the anterior hippocampal formation. NeuroImage 20, 1400 – 1410. Strange, B.A., Dolan, R.J., 1999. Segregating the functions of human hippocampus. Proc. Natl. Acad. Sci. U. S. A. Neurobiology 96, 4034 – 4039. Szeszko, P.R., Strous, P.R., Goldman, R.S., Ashtari, M., Knuth, K.H., Lieberman, J.A., Bilder, R.M., 2002. Neuropsychological correlates of hippocampal volumes in patients experiencing a first episode of schizophrenia. Am. J. Psychiatry 159, 217 – 226. Tambasco, N., Pelliciolli, G.P., Chiarini, P., Montanari, G.E., Leone, F., Mancini, M.L., Paciaroni, M., Gallai, V., 2003. Magnetization transfer changes of grey and white matter in Parkinson’s disease. Neuroradiology 45, 224 – 230. Wolff, S.D., Balaban, R.S., 1989. Magnetization transfer contrast (MTC) and tissue water proton relaxation in vivo. Magn. Reson. Med. 10, 135 – 144. Yeung, H.N., Adler, R.S., Swanson, S.D., 1994. Transient decay of longitudinal magnetization in heterogeneous spin systems under selective saturation: IV. Reformulation of the spin-bath-model equations by the Redfield–Provotorov theory. J. Magn. Reson., A 106, 37 – 45.