Quantitative mapping of T1 and T2* discloses nigral and brainstem pathology in early Parkinson's disease

NeuroImage 51 (2010) 512–520

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Quantitative mapping of T1 and T2* discloses nigral and brainstem pathology in early Parkinson's disease Simon Baudrexel a,b,⁎, Lucas Nürnberger a, Udo Rüb c, Carola Seifried a, Johannes C. Klein a, Thomas Deller c, Helmuth Steinmetz a, Ralf Deichmann b, Rüdiger Hilker a a b c

Department of Neurology, University Hospital, Goethe University Frankfurt am Main, Schleusenweg 2-16, D-60528 Frankfurt am Main, Germany Brain Imaging Center, Goethe University Frankfurt am Main, Schleusenweg 2-16, D-60528 Frankfurt am Main, Germany Institute of Clinical Neuroanatomy, Dr. Senckenberg Anatomy, Neuroscience Center, Goethe University Frankfurt am Main, Theodor Stern Kai 7, D-60590 Frankfurt am Main, Germany

a r t i c l e

i n f o

Article history: Received 28 September 2009 Revised 26 February 2010 Accepted 2 March 2010 Available online 6 March 2010 Keywords: Parkinson's disease Quantitative MRI T1 T2*

a b s t r a c t Quantitative magnetic resonance imaging is a promising in vivo imaging technique revealing insights into different aspects of brain morphology in neurodegenerative diseases based on the determination of physical tissue parameters. Using combined T1- and T2*-mapping, we investigated changes of local relaxation times in the midbrain and lower brainstem of 20 patients with early Parkinson's disease (PD) compared to 20 healthy controls. Voxelwise statistical parametric mapping disclosed a widespread reduction of midbrain T1 values contralateral to the clinically more severely affected limbs. Within the SN, the T1 decrease matched the known pattern of selective neuronal loss as examined in various post-mortem studies, suggesting that T1 is a marker for PD related tissue pathology. However, the spatial extent of T1 reductions exceeded the SN and reached non-dopaminergic areas in the pontomesencephalic junction potentially involved in early nonmotor symptoms of PD. In contrast, T2*-mapping revealed a bilateral decrease of T2* values restricted to the SN, indicating a local increase in total iron content. We conclude that, particularly in longitudinal studies, quantitative T1 may be a valuable marker for the monitoring of progressive neuronal loss in PD, whereas nigral T2* reductions might be more closely associated with an increased general vulnerability for the development of the disorder. © 2010 Elsevier Inc. All rights reserved.

Introduction Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by asymmetric onset of motor symptoms due to a substantial damage of dopaminergic neurons in the substantia nigra pars compacta (SNc) (Hoehn and Yahr, 1967). Although the biochemical mechanisms causing selective cell death in PD are still unresolved, the formation of Lewy bodies and Lewy neurites accompanied by microglia activation and proliferation is generally considered to play a key role in PD pathology (Braak et al., 1998; Croisier and Graeber, 2006; Gibb and Lees, 1988; Imamura et al., 2003; McGeer and McGeer, 2008). Furthermore, the iron content is typically increased in the SNc of PD patients, which may also contribute to neuronal degeneration by the production of reactive oxygen species (Dexter et al., 1991; Sofic et al., 1991). Currently, much effort is being undertaken to visualize PD related changes in tissue architecture in vivo by using novel conventional or quantitative magnetic resonance imaging (qMRI) techniques ⁎ Corresponding author. Department of Neurology and Brain Imaging Center, Goethe University Frankfurt am Main, Schleusenweg 2-16, D-60528 Frankfurt am Main, Germany. E-mail address: [email protected] (S. Baudrexel). 1053-8119/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.neuroimage.2010.03.005

(Ordidge et al., 1994; Seppi and Schocke, 2005). In contrast to conventional imaging, qMRI aims for an unbiased quantification of distinct physical tissue properties, remarkably increasing intra- and inter-individual comparability of images and, thus, allowing the objective measurement of disease related brain changes. Longitudinal (T1) and transversal relaxation times (T2, T2* and T2’) are important parameters frequently assessed with qMRI. So far, a reduction in T2* and T2’ in the lateral SNc, likely caused by an increase in tissue iron content, was shown consistently in patients suffering from early PD stages (Gorell et al., 1995; Graham et al., 2000; Martin et al., 2008; Wallis et al., 2008). Only few studies have investigated T1-based techniques in PD (Hu et al., 2001; Hutchinson and Raff, 1999, 2008), describing a lateral-to-medial gradient of signal loss within the substantia nigra (SN) which matched the spatial gradient of nigral cell loss known from histological studies (Damier et al., 1999; Fearnley and Lees, 1991). Recently, a reduced SN volume was derived from manually segmented quantitative T1-maps (Menke et al., 2009). In this study, we used combined quantitative T1- and T2*-mapping to investigate two important issues that have not yet been addressed in imaging of early PD: (1) It is currently not known whether changes in midbrain and lower brainstem areas other than the SN can be detected using T1- or T2*-imaging, potentially reflecting morphological substrates of non-motor PD symptoms such as depression and

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sleep disturbance (Chaudhuri and Naidu, 2008; Grinberg et al., 2009). As T1 relaxation times of gray matter are typically longer than those of white matter (Wansapura et al., 1999) and PD is associated with gray matter loss (Fearnley and Lees, 1991), we expected a decrease in T1 in affected brain regions. (2) It is still controversial, whether iron increase represents an independent pathomechanism or just an epiphenomenon that is in some way linked to cell death (Götz et al., 2004). To address this question, we explored how alterations in T1 (as a potential marker for gray matter loss) and T2* (as a marker of iron accumulation) relate to each other using left–right asymmetry of motor symptoms as an indicator for side-specific disease state.

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of two T1-weighted data sets using an RF-spoiled 3D gradient echo (GE) sequence (TR = 7.6 ms, TE = 2.4 ms, matrix size 256*224*160, resolution 1 × 1 × 1 mm3, band width 206 Hz/pixel), with different flip angles (4° and 18°). T1-maps were subsequently obtained from contrast differences according to Deoni et al. (2005). To account for spatial inhomogeneities in RF transmission, signal amplitude maps were corrected on the basis of an additionally acquired B1-field map (Yarnykh, 2007). To improve stability and accuracy, the RF phase increment was optimized and additional corrections for the effects of incomplete spoiling were applied according to the literature (Preibisch and Deichmann, 2009). Scan duration for T1-mapping of the whole brain amounted to 14 min.

Methods T2*-mapping

Study Subjects Twenty right-handed patients (mean age 62.2 ± 10.2 years, mean disease duration 4.9 ± 2.4 years) diagnosed with PD in Hoehn and Yahr stage I (n = 7) and II (n = 13) were recruited from our Movement Disorders outpatient clinic (see Table 1 for epidemiological and clinical patient characteristics). All study subjects fulfilled the standard UK Brain Bank criteria for PD (Hughes et al., 1992), none had a history of head injury, stroke or other neurological diseases. PD was absent in the family history of all patients. The severity of motor symptoms was assessed by an experienced neurologist (RH) using part III of the Unified Parkinson Disease Rating Scale (UPDRS) 12 h after the cessation of all PD medication (practically defined drug offstate) yielding an average score of 18.3 ± 6.1. The lateralization of motor signs was assessed by UPDRS III items 20–26 (limb evaluation), yielding a sum score of 9.0 ± 2.9 for the more and 2.7 ± 2.1 for the less affected body side. For all subjects, the clinically more affected body side matched the self-reported side of symptom onset. PD type was classified as akinetic-rigid in 15 and tremor-dominant in five patients. Twelve patients received a combined medication with low or moderate doses of levodopa and dopamine agonists, four patients received dopamine agonists and one patient received a levodopa monotherapy, two patients were drug-naive. Twenty age and gender matched healthy controls (mean age 62.3 ± 10.8 years) in good general health, with normal findings in standard neurological examination and without a history of neurological or psychiatric diseases were recruited from a volunteer database. In accordance with the Declaration of Helsinki, all individuals gave written informed consent to participate in the study, which was approved by the local Ethics Committee. MR imaging T1- and T2*-imaging was carried out on a 3 T MR scanner system (Magnetom Trio, Siemens Medical Solutions, Erlangen, Germany) in a single session using an 8-channel phased-array head coil for signal reception and a whole body coil for radio frequency (RF) transmission. T1- and T2*-maps were computed offline using custom built programs written in MATLAB (MATLAB, The MathWorks Inc., Natick, MA, USA). T1-mapping T1-mapping was performed as described recently (Preibisch and Deichmann, 2009). In brief, the technique is based on the acquisition

T2*-mapping was carried out using a multi-echo GE technique with the following parameters: TE = [10, 16, 22, 28, 34, 40, 46, 52] ms, TR = 3 s, flip angle = 30°, in-plane resolution 1 x 1 mm2, slice thickness 2 mm, no slice gap, exponential slice-excitation RF pulse of 2 ms duration, band width 300 Hz/pixel (Baudrexel et al., 2009). Fifty axial slices covering basal ganglia, midbrain, pons and rostral medulla oblongata were acquired with a total scan duration of 13 min. B0-gradient field maps were calculated from phase data of the first two echoes (Preibisch et al., 2008). A typical problem with T2* imaging is that macroscopic static magnetic field inhomogeneities accelerate signal decay resulting in T2* values which are systematically too small. Thus, for each TE, signal amplitudes were multiplied by a correction factor depending on the respective TE value and the local field inhomogeneity gradient (Baudrexel et al., 2009). T2*-maps were subsequently obtained by monoexponentially fitting of the corrected signal amplitudes. Data analysis No study subject showed major structural brain abnormalities, particularly no lacunar brainstem infarction. While all T1-maps could be subjected to further statistical analysis, T2* data from three patients and five healthy controls had to be removed due to marked movement artefacts. Data postprocessing Data postprocessing was performed with SPM5 (Wellcome Department of Imaging Neuroscience, Institute of Neurology, UCL, London, UK, available at http://www.fil.ion.ucl.ac.uk/spm) and with custom built programs written in MATLAB. It consisted of the following steps: 1. Segmentation. T1-maps were used for calculation of individual probability maps of gray matter, white matter and cerebrospinal fluid (CSF) using the standard segmentation routine implemented in SPM5. 2. Coregistration. To account for possible head movements between scans, each T2*-map was spatially realigned to the individual T1map prior to further processing. 3. Flipping. All images were flipped with respect to the z-y-plane (left–right), resulting in an additional (mirrored) data set.

Table 1 Demographic and clinical characteristics of patients with PD and healthy control subjects. Group (n)

Mean age (years)

Gender (M/F)

Mean disease duration (years)

H&Y stage (I/II)

PD type akinetic-rigid/ tremor dominant

Mean UPDRS III

UPDRS III, limb evaluation more affected side

UPDRS III, limb evaluation less affected side

PD (20) Controls (20)

62.2 ± 10.2 62.3 ± 10.8

12/8 12/8

4.0 ± 2.3 –

7/13 –

15/5 –

18.3 ± 6.1 –

9.0 ± 2.9 –

2.7 ± 2.1 –

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4. Normalization. T1-maps were spatially normalized into MNI-space using the Montreal T1(152)-template and the SPM5 normalization routine. T2* and tissue probability maps were co-normalized using the same transformation parameters. All images were spatially resampled with 1 mm isotropic resolution. Normalized images were checked by visual inspection to rule out possible registration errors. For the mirrored data set, the procedure was repeated in the same manner. 5. Smoothing. All images were smoothed with a 2.5 mm isotropic Gaussian kernel. 6. Masking. Brainstem and midbrain were segmented manually on the basis of the mean T1-image (z-level of the most cranial [caudal] plane = −4 [−64], coordinates in MNI-space) using the imagingsoftware Vinci (Vinci, MPIfnF, Cologne, Germany). All images were realigned perpendicular to the long axis of the brainstem and displayed in radiological convention.

corrected for multiple comparisons. Using the a priori approach suggested by Friston (1997), uncorrected cluster levels (p b 0.05) were considered significant only if the respective cluster overlapped with the SN. To provide reference values of nigral T1 and T2* relaxation times in PD patients and healthy controls, elliptical ROIs of size 4 × 4 × 2.5 mm3 were placed symmetrically in several areas of interest in the lateral SN. The positioning of all ROIs was performed on mean T1maps relatively to well-known landmarks and has been validated by the use of an anatomical atlas (Afshar et al., 1978): ROIs designated “rostral SN” in the following were positioned at the intercollicular level, anterior to the medial lemniscal system (ML) and 1 mm lateral to the border of the red nucleus (RN, see Fig. 3B). ROIs designated “caudal SN” were positioned in nigral gray matter at the level of the pontomesencephalic junction anterior to the ML, where cell loss was shown to be most pronounced in PD patients (Damier et al., 1999) (see Fig. 3E).

Statistical analysis

Results

Data were analyzed with voxel-by-voxel and region-of-interest (ROI) based statistics. We used a paired t-test design for the assessment of intra-individual side asymmetries in T1 and T2*. Group differences between patients and controls were calculated with two sample t-test statistics. Since we hypothesized reduced longitudinal and transverse relaxation times in diseased brain areas, the corresponding one-sided t-test was used. All patients were pooled as if primarily affected on the right body side by using the flipped images of patients affected on the left body side. Thus, the most prominent pathologic findings were expected to be located in the left brainstem and midbrain which is for simplicity referred to as the contralateral side. Distinctive features had to be considered with respect to each design. (a) Paired t-test design: To account for possible confound of the data by disease independent left–right gradients, an equal number of patients predominantly affected on the right side as on the left side were included in the study. However, this condition was not fully matched for the T2* analysis in which three patients and five healthy controls had to be removed due to movement artefacts. Thus, to account for this asymmetry in the design, the most affected body side was used as an additional binary covariate in the T2* analysis. The analysis was performed in the same manner for the group of healthy control participants. (b) Two sample t-test design: Gender and age were used as covariates in the group analysis, since they have been shown to affect MRI relaxation times (Bottomley et al., 1984; Siemonsen et al., 2008). Again, to account for disease independent side asymmetries in T1 and T2*, for each subject affected on the left body side, the flipped image of the corresponding age and gender matched control participant was used. In the T2* analysis, the affected body side was used as an additional binary covariate for the same reason as described in (a). To avoid edge-effects corrupting statistical analysis due to arbitrary fractions of CSF contributing to T1 and T2* at the brainstem borders, further analysis was restricted to voxels for which the probability of belonging to gray or white matter was larger than 0.8 for all subjects, both in the respective flipped and unflipped smoothed tissue probability maps. The resulting volume of interest was symmetrical with respect to the mid-sagittal plane and consisted of ∼18000 voxels. Since previous imaging studies on PD using T2*-mapping revealed rather small effects of interest with large inter-individual variability even on a ROI-basis (Graham et al., 2000; Martin et al., 2008; Wallis et al., 2008), we applied a lenient threshold of p b 0.05 (uncorrected) for the display of t-maps. Cluster significance levels were assessed with random permutation analysis using SnPM5b, a freely available SPMtoolbox for non-parametric statistical mapping (http://www.fil.ion.ucl. ac.uk/spm) (Nichols and Holmes, 2002). The calculation consisted of 5000 random permutations with voxel level threshold p = 0.05. Clusters were considered significant if passing the threshold p b 0.05,

Anatomy of the substantia nigra as revealed by T1- and T2*-mapping Single subject and mean T1- and T2*-maps of three transverse sections (I-III) through the midbrain are shown in Fig. 1C–F. For comparison, stereotactic drawings based upon probabilistic data from 22 hemibrainstems (Fig. 1A) and corresponding histological slices (Fig. 1B) taken from an anatomical atlas (Afshar et al., 1978) were added. In the T1-maps (Fig. 1C and D), the SN was clearly visible as a two-layered arc of gray matter at the intercollicular level (I), which narrowed inferior to the RN (II, inferior collicular level) and extended into the pontomesencephalic junction (III) (Menke et al., 2009; Oikawa et al., 2002). Anatomically, large portions of the SN are located inferior to the RN and strictly anterior to the ML (Afshar et al., 1978), the latter being clearly visible in T1-maps on single subject and group level. As has been shown by Oikawa and colleagues, a T2*-based anatomical distinction between the SNc and the iron rich SN pars reticularis (SNr) is not reliably possible (Oikawa et al., 2002). For this reason, in the following we will refer to the SN as a whole, which is clearly visible in the T1-maps, corresponding in shape and position to histological findings.

Side asymmetries in T1 and T2* (contra- versus ipsilateral) Significantly reduced T1 values were found contra- versus ipsilateral to the most affected body side in a huge cluster of 1800 voxels comprising large portions of the midbrain and extending into the midpontine level (Fig. 2, p = 0.01, corrected cluster level). The contralateral SN was covered by this cluster with its lateral portions at the intercollicular level and completely at the level of the caudal midbrain. An asymmetric T1 decrease was also observed in the caudal midbrain dorsal to the ML and lateral to the decussatio of the superior cerebellar peduncles (see Figs. 2C–E and 1B–D for comparison) in a T1-hypointense appearing zone. In this area, the PPN (Zrinzo et al., 2008) and the reticular cuneiform nucleus are located (see Supplementary Fig. S1, Naidich et al., 2009). Furthermore, at the cranial pontine and midpontine level, T1-asymmetries were found in the dorsal pons in areas close to the midline (Fig. 2F and G), covering parts of the pontine reticular formation (see Supplementary Fig. S1). In striking contrast, the corresponding analysis for the detection of side asymmetries in T2* did only reveal a small cluster of 149 voxels, which was mainly located in the midbrain dorsal to the ML (p = 0.07, uncorrected cluster level). The respective T1 and T2* analysis in healthy controls did not show any significant side asymmetries, even at uncorrected voxel level (p b 0.05), arguing against a systematic bias of the data due to residual non-disease related gradients in T1 or T2*.

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Fig. 1. Transverse sections through the midbrain perpendicular to the long axis of the brainstem (cut levels are indicated top right). Row I: intercollicular/caudal rubral level (19 mm rostral to fastigial floor line, FFL). Row II: inferior collicular level (16 mm rostral to FFL). Row III: level of the pontomesencephalic junction (14 mm rostral to FFL). Columns A and B: adapted from Afshar et al. (1978): Column A presents probabilistic stereotactic drawings based upon data from 22 hemibrainstems, lines indicate N70 % probability. The SN appears in black, the medial lemniscus (ML) in yellow, the RN in red and the decussatio of the superior cerebellar peduncles (DSCP) in green. Column B presents photomicrographs of modified Mulligan stain sections of a single specimen. Please note: Stereotactic drawings and photomicrographs have been rotated 180° for the display in radiological convention. Column C and D: Mean T1- (C) and single subject T1-maps (D). T1-maps are displayed with inverted contrast to achieve a similar contrast as in T1-weighted imaging. Column E and F: Mean T2*- (E) and single subject T2*-maps (F). Please note: all images are displayed in radiological convention. Mean images were calculated using the respective relaxation time maps from all subjects. Top left: adapted from Naidich et al. (2009): Photograph of a sagittal section through the midbrain. The brainstem displayed on the photograph was rotated to fit the long brainstem axis orientation. Top right: sagittal section through the mean T1-map. Cut levels of axial slices are indicated I–III. Abbreviations: AQ, aqueduct; DSCP, decussatio of the superior cerebellar peduncles; ML, medial lemniscus; PT, pyramidal tract; RN, red nucleus; SN, substantia nigra; X, (blue) area of the pedunculopontine and reticular cuneiform nucleus. Illustrations in rows I–III (column 1 and 2) reprinted from Afshar et al. (1978) with permission from Lippincott Williams & Wilkins. Illustration top left reprinted from Naidich et al. (2009) with permission from Springer Wien New York.

Group differences in T1 and T2* (PD vs. controls) Reduced relaxation times in PD patients compared to healthy controls are displayed in Fig. 3. We found a cluster of 400 voxels with significantly decreased T1 in the contralateral SN (p = 0.02, uncorrected cluster level). Cross sections at the intercollicular level revealed a T1 decrease in the lateral portion of the SN, whereas the nucleus was almost completely affected at the level of the pontomesencephalic junction. Noticeably, this cluster was considerably smaller but overlapped the area of reduced midbrain T1 in the comparison of brain sides depicted in Fig. 2 completely. In contrast, T2* analysis revealed two bilateral clusters of signal decrease strictly located within each SN of PD patients (contralateral cluster size: 197, p = 0.04; ipsilateral cluster size 107, p = 0.14; each uncorrected cluster level). Fig. 4 provides a coronal view of the key results of Figs. 2 and 3, further showing that the T2* decrease (Fig. 4C) was located more medially as compared to the decrease of T1 (Fig. 4A and B). The ROI analysis confirmed the major findings of the voxel-based analysis and showed a contralateral decrease of nigral T1 values, pronounced in the caudal SN, along with a bilateral T2* decrease in the rostral SN, only (Table 2). Regional T1 values did not significantly correlate with UPDRS III scores, although there was a tendency toward smaller T1 values with higher motor ratings in all ROIs under investigation (Table 2). Box-plots of contralateral nigral T1 value distributions in patients and healthy

controls are shown in Fig. 5. Receiver operating characteristics (ROC) analysis yielded a significant AUC for the differentiation between PD and healthy controls by T1 values for the caudal SN only (AUC = 0.75, p b 0.05, threshold T1 = 1123 ms, sensitivity = 0.71, specificity = 0.8). Discussion Unilateral decrease in T1 The unilateral onset of motor symptoms is characteristic for PD and side differences often persist during the entire course of the disease (Hoehn and Yahr, 1967). According to clinical symptoms, the most prominent qMRI finding in this study is a midbrain and pontine T1 decrease strictly contralateral to the clinically most affected body side. Within the affected SN, the pattern of T1 decrease matched the known caudal-to-rostral and lateral-to-medial gradient of nigral cell loss as has been demonstrated by post-mortem studies (Damier et al., 1999; Fearnley and Lees, 1991). In view of the congruence of our findings with these histopathological studies and with the clinical preponderance of motor symptoms at the contralateral body side, it is likely that the T1 decrease is a marker for PD related pathology. Biophysically, T1 is the time constant of the energy transfer from free water protons to the surrounding matter (spin-lattice relaxation), its value is depending on the molecular composition of brain tissue. T1 is

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Fig. 2. Voxelwise t-maps thresholded at p b 0.05 (uncorrected voxel level) indicating an asymmetric decrease in T1 (column 1 and 3) and T2* (column 2 and 4) contralateral to the most affected body side in PD patients. T-maps are projected onto the respective mean relaxation time maps over all study subjects (T1-maps are displayed with inverted contrast). (A–G) Transaxial sections through the midbrain and pons in descending order as indicated on the sagittal section (top left). Abbreviations: DSCP, decussatio of the superior cerebellar peduncle; MCP, medial cerebellar peduncle; ML, medial lemniscus; RF, pontine reticular formation; RN, red nucleus; SCP, superior cerebellar peduncle; SN, substantia nigra; X, area of the pedunculopontine and reticular cuneiform nucleus.

thus very sensitive to changes in the gray-to-white matter ratio. Gray matter loss constitutes the principle final pathway in neurodegenerative disorders and is most likely the major determinant of the T1 decrease observed. Although a considerably increased number of activated microglia is a further typical histopathological finding in PD (Gerhard et al., 2004; Imamura et al., 2003; McGeer and McGeer, 2008), its contribution to the changes in quantitative human T1-maps of the brain is currently unknown. Since a significant negative correlation of T1 values with the number of activated microglia has been recently shown in a mouse model of focal ischemia (Aoe et al., 2006), one might speculate that microglial activation also contributes to the T1 decreases in our PD patients. The mean nigral ROI values reported here are in excellent agreement with values reported in a previous study (1147 ± 50 ms) (Gelman et al., 2001) at 3 T, constituting an intermediate value between typical white and gray matter T1 values (Preibisch and Deichmann, 2009), probably due to partial voluming. In another study (Menke et al., 2009), values as low as 874 ± 42 ms were reported, rather corresponding to the white matter T1. The reason for this

discrepancy may be due to methodological differences between the T1-mapping techniques used. A more thorough discussion would be beyond the scope of this paper. In contrast to the results presented here, Vymazal et al. (1999) did not find significant T1 changes in the SN in a PD population of 23 subjects conducted at 1.5 T. However, most probably due to the increased noise level at low field strengths, the standard deviation of T1 values was relatively large in this study (133 ms), and a rather coarse spatial resolution of 1 × 2 × 5 mm3 had to be used which may have masked significant effects. It is important to note that T1 alterations in PD patients were restricted to the brainstem contralateral to the clinically most affected body side, although 13 patients also suffered from slight opposite motor symptoms. The clinical asymmetry of motor signs is thus reflected in corresponding lateralized T1 decreases. One possible explanation for this phenomenon is a later onset and less pronounced degeneration in the ipsilateral mesencephalon. However, a nigrostriatal dopaminergic cell loss of around 50% at the time of symptoms onset has been hypothesized in a previous post-mortem study

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Fig. 3. T-maps thresholded at p b 0.05 (uncorrected voxel level) indicating a decrease in T1 (column 1 and 3) and T2* (column 2 and 4) in PD vs. controls, projected onto the respective mean relaxation time maps (T1-maps are displayed with inverted contrast). (A–G) Transaxial sections at the same levels as in Fig. 2. Reference values from ROIs drawn in panels B and E are provided in Table 2.

(Fearnley and Lees, 1991). Therefore, the absence of ipsilateral T1 alterations despite the presence of very mild clinical signs might also reflect a limited sensitivity of the T1 signal for the detection of early brain pathology in PD. On the other hand, post-mortem dopaminergic cell counts demonstrated that the number of surviving neurons in the SN contralateral to the initially affected body side was on average 25% lower as compared to the opposite side even 11.9 years after symptoms onset (Kempster et al., 1989). Therefore, a side preponderance of dopaminergic degeneration seems to exist which is also a common finding in asymmetrical striatal radiotracer uptake reduction in PET or SPECT of PD patients, to a certain degree correlating with the asymmetry in motor performance (Morrish et al., 1995). The pathophysiological base for the lateralization of motor and even non-motor-symptoms, such as sympathetic skin response (Fusina et al., 1999) or pain threshold (Djaldetti et al., 2004), is currently unknown (Djaldetti et al., 2006). Theoretical explanations range from genetically determined side differences in the total number of nigral dopaminergic neurons to an increased speed of cell loss due to a higher inherent vulnerability of one SN as compared to the SN at the opposite side (Djaldetti et al., 2006).

Despite significant mean differences of T1 values in the ROI analysis, there is considerable overlap of individual T1 values between patients and healthy controls. Consequently, the ROC analysis revealed a limited diagnostic power of midbrain T1-mapping for the differentiation of PD and healthy controls on an individual level. Despite significant advances in MRI techniques, for example diffusion tensor imaging (Menke et al., 2009; Scherfler et al., 2006; Vaillancourt et al., 2009), radionuclid-based brain imaging remains the gold standard for the early diagnosis of PD in the clinical setting (Brooks, 2000; Hu et al., 2001; Hutchinson and Raff, 2007). Furthermore, our T1 values did not significantly correlate with UPDRS motor scores; this was also reported by Menke et al. (2009). Aside from a relatively large inter-individual variability of absolute T1 values, the lack of statistical significance may be also explained by the relatively uniform disease severity in our PD study cohort. In contrast, previous T1-weighted MRI studies demonstrated a significant correlation of the aforementioned lateral-to-medial gradient of SN signal loss with UPDRS motor scores (Hutchinson and Raff, 2000) and with striatal 18-Fluorodopa uptake rates in PET (Hu et al., 2001), arguing for a relationship between PD stage, striatal dopaminergic depletion and nigral T1 reduction.

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Fig. 5. Box-plots of contralateral caudal and rostral SN ROI T1 values in patients and healthy controls.

Fig. 4. Thresholded t-maps from Figs. 2 and 3 in coronal view (section through the RN). (A) Paired t-test for an asymmetric decrease in T1 contralateral to the most affected body side. (B and C) Two-sample t-test indicating a decrease in T1 (B) and T2* (C) as compared to healthy controls.

We found contralateral T1 reductions in the midbrain of PD patients clearly exceeding the borders of the SN. Specifically, the T1 decreases overlapped with some of the non-dopaminergic nuclei of the midbrain and of the pontine reticular formation, indicating the involvement of non-dopaminergic brainstem regions with possible pathophysiological impact for the development of autonomic or psychiatric symptoms in the course of PD (Chaudhuri et al., 2006; Sullivan et al., 2007). According to Braak's hypothesis, PD is initiated

by some unknown pathogen entering the CNS from the autonomic peripheral nervous system via the dorsal motor nucleus of the vagus and subsequently ascends in cranial direction (Braak et al., 2003). However, we could not detect a decrease in T1 in the lower brainstem and the vagal area, most likely due to the small spatial extent of this nucleus and due to spatial normalization errors, which are typically more pronounced in the caudal brainstem (Napadow et al., 2006). Pulsatile motion of the brain related to the cardiac and respiratory cycle is another serious source of variability, deteriorating MR-image quality in this region, possibly contributing to false negative results (Enzmann and Pelc, 1992; Harvey et al., 2008). Low signal-to-noise ratios are, thus, a general problem in brainstem MRI and this has to be considered in the choice of statistical thresholds in voxel-based analysis approaches. An additional point meriting discussion refers to the inter-individual anatomical variability of brainstem fiber tracts and nuclei, which, in contrast to cortical structures, have less extensively been investigated so far. The clear assignment of a certain T1-hypointense area to a specific subarea of the SN or the reticular formation solely based on the gray-to-white matter contrast is impossible without the use of defined anatomical landmarks. This problem underpins the need of a probabilistic MRI-atlas of the brainstem of that kind that already exists for the brain hemispheres in order to improve the interpretation of functional and structural brainstem MRI data in general (Eickhoff et al., 2005). Bilateral decrease in T2* T2* reductions indicating a general increase in tissue iron content were mainly restricted to the SN and showed a bilateral pattern (Fig. 3 and Table 2). These results excellently agree with various histological (Dexter et al., 1991; Sofic et al., 1991) and imaging studies of the SN

Table 2 T1 and T2* relaxation times in ROIs from Fig. 3B (rostral lateral SN) and 3E (caudal SN). T1 ROI Rostral SN Contralateral

T2*

Patients T1 [ms] ± 1 SD

Controls T1 [ms] ± 1 SD

p-valuea

Correlation with UPDRS IIIb

Patients T2* [ms] ± 1 SD

Controls T2*[ms] ± 1 SD

p-valuea

Correlation with UPDRS IIIb

1128 ± 60.4

1160 ± 50.5

0.04c

r = − 0.31 (p = 0.18) r = − 0.30 (p = 0.21)

35.7 ± 5.7

39.5 ± 4.6

0.02c

36.8 ± 4.8

40.9 ± 4.2

0.01

c

r = − 0.10 (p = 0.70) r = 0.20 (p = 0.43)

49.9 ± 10.6

48.2 ± 7.9

0.69

51.0 ± 10.4

46.2 ± 10.1

0.90

Ipsilateral

1152 ± 55.2

1155 ± 59.6

0.43

Caudal SN Contralateral

1104 ± 54.4

1158 ± 59.5

0.002d

Ipsilateral

1159 ± 79.7

1161 ± 62.1

0.47

a b c d

r = − 0.31 (p = 0.19) r = − 0.27 (p = 0.25)

Decreased relaxation times in PD vs. controls (one sided two-sample t-test). Pearson's coefficient of correlation between ROI values and total UPDRS III motor scores. p b 0.05. α b 0.05, corrected for multiple comparisons (Bonferroni).

r = − 0.04 (p = 0.88) r = 0.07 (p = 0.79)

S. Baudrexel et al. / NeuroImage 51 (2010) 512–520

using quantitative T2* or T2’ MRI (Graham et al., 2000; Martin et al., 2008; Wallis et al., 2008). Interestingly, T2* and T2’ did not correlate with disease duration and the correlation with disease severity was rather small in these cross-sectional studies (R2 b 0.3), suggesting that the amount of nigral iron content remains relatively stable during the course of the disease. Additional evidence supporting this theory was provided by a transcranial ultrasound study showing that the area of SN-hyperechogenicity, which is assumed to be associated with tissue iron levels (Berg et al., 2002), did not change over a five year follow-up period in PD patients (Berg et al., 2005). Thus, it has been suggested that an increase in nigral iron content may reflect a marker for disease susceptibility or vulnerability, rather than disease progression (Berg et al., 2002). This interpretation implicates that alterations in nigral iron levels may be present many years before PD becomes symptomatic. Two aspects of our data corroborate this view: (1) Brainstem areas outside the SN affected by a decrease in T1 did not show significant reductions in T2*, suggesting that increased iron deposition in these regions is not a prerequisite for PD related neuronal loss. (2) The pattern of nigral T2* decrease was different from that seen in T1mapping in the sense that T2* reductions were located more medially as compared to the T1 reductions (see Fig. 4). Technical or statistical limitations resulting in a decreased sensitivity for the detection of T2* as compared to T1 reductions are unlikely, since we failed to demonstrate a T1, but not a T2* decrease in the ipsilateral SN, which is in good agreement with the literature (Gorell et al., 1995; Martin et al., 2008; Wallis et al., 2008). A possible masking of extranigral T2* alterations due to drug-induced changes of cerebral blood flow is also rather unlikely: All methods used for this study are based on gradient echoes, so flow effects should not yield major signal losses (in contrast to spin echoes, where moving spins do not necessarily experience the slice-selective refocusing pulse). In addition, global excitation pulses were used for the T1-mapping sequence, so steady state conditions were constantly maintained for the magnetisation of moving spins. A possible confound is due to the fact that a medication induced CBF increase of about 10% (Hirano et al., 2008) may yield an increased wash-out of deoxyhemoglobin and thus a T2* increase, yielding false negative results. However, it can be shown that this effect would yield very minor T2* changes of less than 1 ms: even a relatively strong CBF increase of about 25–40% induced by hypercapnia has been shown to yield a signal increase of only 2% in functional imaging data (Corfield et al., 2001). Assuming a signal dependence I ∼ exp(-TE/T2*), a TE of 40 ms and a T2* of 50 ms, this signal increase would correspond to a T2* increase of only 1.3 ms for the hypercapnic CBF increase, so changes of less than 1 ms can be predicted for a drug related CBF increase. Taken together, our findings suggest that the pathophysiological process resulting in neuronal loss in PD may differ from the mechanisms causing increased iron deposition. However, it has to be emphasized that it is not necessarily the total amount of iron but rather its physical state, especially the amount of highly reactive unbound Fe2+, which might contribute to nigral cell loss by the production of reactive oxygen species via Fenton's reaction (Berg and Hochstrasser, 2006; Sofic et al., 1991). The exact mechanisms that couple neuronal iron metabolism with a selective vulnerability of the SN are not fully understood. Aside from cell loss due to increased oxidative stress, biochemical studies suggest that the formation of Lewy bodies is facilitated by the presence of iron (Munch et al., 2000; Uversky et al., 2001). Conclusions This combined quantitative T1 and T2* MRI study at 3 T field strength demonstrated distinct alterations in the tissue morphology of the rostral brainstem in patients with early-stage PD. Decreased T1 values likely reflecting gray matter loss were found contralateral to the most affected body side and extended from the SN down to the

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caudal midbrain and to the midpontine level. Within the SN, the T1 decrease matched the known pattern of dopamine cell degeneration from various post-mortem studies, suggesting that T1 is a marker for PD related neuronal loss. Longitudinal studies are required to further validate the capability of this technique to monitor PD progression. In contrast, mapping of T2* revealed a bilateral increase in tissue iron content in PD patients which was spatially restricted to the SN. We conclude that decreased nigral T2* values more likely reflect increased disease vulnerability rather than progressive neuronal degeneration. Acknowledgments This study was supported by the Bundesministerium für Bildung und Forschung (Brain Imaging Center Frankfurt, DLR 01GO0203) and the Deutsche Forschungsgemeinschaft (ZA 233/1-1). Simon Baudrexel was partly funded by the Interdisciplinary Center for Neuroscience Frankfurt (ICNF), Frankfurt am Main. We thank Florian Beissner for his assistance in the revision of the figures. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.neuroimage.2010.03.005. References Afshar, F., Watkins, E.S., Yap, J.C., 1978. Stereotaxic Atlas of the human brainstem and cerebellar nuclei. Raven Press, New York, A Variability Study. Aoe, H., Takeda, Y., Kawahara, H., Tanaka, A., Morita, K., 2006. Clinical significance of T1weighted MR images following transient cerebral ischemia. J. Neurol. Sci. 241, 19–24. Baudrexel, S., Volz, S., Preibisch, C., Klein, J.C., Steinmetz, H., Hilker, R., Deichmann, R., 2009. Rapid single-scan T2*-mapping using exponential excitation pulses and image-based correction for linear background gradients. Magn. Reson. Med. 62, 263–268. Berg, D., Hochstrasser, H., 2006. Iron metabolism in Parkinsonian syndromes. Mov. Disord. 21, 1299–1310. Berg, D., Roggendorf, W., Schroder, U., Klein, R., Tatschner, T., Benz, P., Tucha, O., Preier, M., Lange, K.W., Reiners, K., Gerlach, M., Becker, G., 2002. Echogenicity of the substantia nigra: association with increased iron content and marker for susceptibility to nigrostriatal injury. Arch. Neurol. 59, 999–1005. Berg, D., Merz, B., Reiners, K., Naumann, M., Becker, G., 2005. Five-year follow-up study of hyperechogenicity of the substantia nigra in Parkinson's disease. Mov. Disord. 20, 383–385. Bottomley, P.A., Foster, T.H., Argersinger, R.E., Pfeifer, L.M., 1984. A review of normal tissue hydrogen NMR relaxation times and relaxation mechanisms from 1– 100 MHz: dependence on tissue type, NMR frequency, temperature, species, excision, and age. Med. Phys. 11, 425–448. Braak, H., de Vos, R.A., Jansen, E.N., Bratzke, H., Braak, E., 1998. Neuropathological hallmarks of Alzheimer's and Parkinson's diseases. Prog. Brain Res. 117, 267–285. Braak, H., Del Tredici, K., Rüb, U., de Vos, R.A., Jansen Steur, E.N., Braak, E., 2003. Staging of brain pathology related to sporadic Parkinson's disease. Neurobiol. Aging 24, 197–211. Brooks, D.J., 2000. Morphological and functional imaging studies on the diagnosis and progression of Parkinson's disease. J. Neurol. 247 (Suppl. 2), II11–II18. Chaudhuri, K.R., Naidu, Y., 2008. Early Parkinson's disease and non-motor issues. J. Neurol. 255 (Suppl 5), 33–38. Chaudhuri, K.R., Healy, D.G., Schapira, A.H., 2006. Non-motor symptoms of Parkinson's disease: diagnosis and management. Lancet Neurol. 5, 235–245. Corfield, D.R., Murphy, K., Josephs, O., Adams, L., Turner, R., 2001. Does hypercapniainduced cerebral vasodilation modulate the hemodynamic response to neural activation? Neuroimage 13, 1207–1211. Croisier, E., Graeber, M.B., 2006. Glial degeneration and reactive gliosis in alphasynucleinopathies: the emerging concept of primary gliodegeneration. Acta Neuropathol. 112, 517–530. Damier, P., Hirsch, E.C., Agid, Y., Graybiel, A.M., 1999. The substantia nigra of the human brain: II. Patterns of loss of dopamine-containing neurons in Parkinson's disease. Brain 122, 1437–1448. Deoni, S.C., Peters, T.M., Rutt, B.K., 2005. High-resolution T1 and T2 mapping of the brain in a clinically acceptable time with DESPOT1 and DESPOT2. Magn. Reson. Med. 53, 237–241. Dexter, D.T., Carayon, A., Javoy-Agid, F., Agid, Y., Wells, F.R., Daniel, S.E., Lees, A.J., Jenner, P., Marsden, C.D., 1991. Alterations in the levels of iron, ferritin and other trace metals in Parkinson's disease and other neurodegenerative diseases affecting the basal ganglia. Brain 114, 1953–1975. Djaldetti, R., Shifrin, A., Rogowski, Z., Sprecher, E., Melamed, E., Yarnitsky, D., 2004. Quantitative measurement of pain sensation in patients with Parkinson disease. Neurology 62, 2171–2175.

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