Abstract

Signal abnormalities of the substantia nigra and the olfactory tract detected either by diffusion tensor imaging, including measurements of mean diffusivity, a parameter of brain tissue integrity, and fractional anisotropy, a parameter of neuronal fibre integrity, or transcranial sonography, were recently reported in the early stages of Parkinson’s disease. In this study, changes in the nigral and olfactory diffusion tensor signal, as well as nigral echogenicity, were correlated with clinical scales of motor disability, odour function and putaminal dopamine storage capacity measured with 6-[18F] fluorolevodopa positron emission tomography in early and advanced stages of Parkinson’s disease. Diffusion tensor imaging, transcranial sonography and positron emission tomography were performed on 16 patients with Parkinson’s disease (mean disease duration 3.7 ± 3.7 years, Hoehn and Yahr stage 1 to 4) and 14 age-matched healthy control subjects. Odour function was measured by the standardized Sniffin’ Sticks Test. Mean putaminal 6-[18F] fluorolevodopa influx constant, mean nigral echogenicity, mean diffusivity and fractional anisotropy values of the substantia nigra and the olfactory tract were identified by region of interest analysis. When compared with the healthy control group, the Parkinson’s disease group showed significant signal changes in the caudate and putamen by 6-[18F] fluorolevodopa positron emission tomography, in the substantia nigra by transcranial sonography, mean diffusivity and fractional anisotropy (P < 0.001, P < 0.01, P < 0.05, respectively) and in the olfactory tract by mean diffusivity (P < 0.05). Regional mean diffusivity values of the substantia nigra and the olfactory tract correlated significantly with putaminal 6-[18F] fluorolevodopa uptake (r = −0.52, P < 0.05 and r = −0.71, P < 0.01). Significant correlations were also found between nigral mean diffusivity values and the Unified Parkinson’s Disease Rating Scale motor score (r = −0.48, P < 0.01) and between mean putaminal 6-[18F] fluorolevodopa uptake and the total odour score (r = 0.58; P < 0.05) as well as the Unified Parkinson’s Disease Rating Scale motor score (r = −0.53, P < 0.05). This study reports a significant association between increased mean diffusivity signal and decreased 6-[18F] fluorolevodopa uptake, indicating that microstructural degradation of the substantia nigra and the olfactory tract parallels progression of putaminal dopaminergic dysfunction in Parkinson’s disease. Since increases in nigral mean diffusivity signal also correlated with motor dysfunction, diffusion tensor imaging may serve as a surrogate marker for disease progression in future studies of putative disease modifying therapies.

Introduction

Parkinson’s disease is characterized pathologically by the loss of dopamine neurons in the substantia nigra pars compacta in association with intracellular Lewy body inclusions. By the time a clinical diagnosis is made, extrapolation from autopsy studies estimate up to 60% cell loss in the lateral ventral tier of the substantia nigra pars compacta (Fearnley and Lees, 1991). The neurodegenerative process within the substantia nigra cannot be depicted by imaging techniques in vivo. However, radiotracer-based modalities such as 6-[18F] fluorolevodopa ([18F] DOPA) PET and dopamine transporter single photon emission computed tomography (SPECT) were shown to facilitate the visualization of dopamine terminal function from nigral projections in the striatum and the correlation of these signals with Parkinson’s disease-related motor disturbances (Brooks et al., 1990; Seibyl et al., 1995). Longitudinal measurements of putaminal [18F] DOPA storage revealed rates of progression that followed a negative exponential curve that were reported to be remarkably consistent with clinicopathological observations (Hilker et al., 2005; Greffard et al., 2006). Nevertheless, the use of such imaging markers to follow disease progression and study putative neuroprotective agents has remained controversial as the imaging paradigm may directly be affected by confounding pharmacological or regulatory influences of dopaminergic treatment. Recently, advances in MRI modalities have allowed the visualization of structural integrity in the brain tissue of patients with Parkinson’s disease. Diffusion tensor imaging is a unique form of MRI contrast that enables the measurement of direction of diffusing water molecules within the entire brain. Within fibre tracts, motion of water molecules perpendicular to the main axonal direction is restricted to a greater extent than is diffusion along the main axis, resulting in an anisotropically shaped space termed fractional anisotropy. In addition, mean diffusivity reflects the total magnitude of diffusion and hence provides information about alterations in the extracellular volume of both grey and white matter (Basser and Pierpaoli, 1996; Le Bihan, 2003). Both the fractional anisotropy and mean diffusivity signals are shown to be sensitive enough to detect tissue alterations in the substantia nigra and olfactory tract of patients with Parkinson’s disease (Scherfler et al., 2006; Vaillancourt et al., 2009).

Signal abnormalities in the substantia nigra have also been identified by transcranial B-mode sonography in up to 90% of patients with Parkinson’s disease and at least 10% of subjects without clinically overt parkinsonian features (Becker et al., 1995; Berg et al., 2001, 2011). The size of the hyperechogenic substantia nigra appears to remain stable over the course of the disease (Berg et al., 2005; Mahlknecht et al., 2012), however, it has also been reported to predict the rate of disease progression (Schweitzer et al., 2006).

In the present study, we aimed to characterize diffusion tensor imaging signal alterations in the substantia nigra and olfactory tracts of patients in early and advanced stages of Parkinson’s disease and sought to correlate microstructural abnormalities with measures of motor disability, olfactory dysfunction, nigral transcranial sonography and degree of putaminal dopamine terminal function determined with [18F] DOPA PET.

Materials and methods

Subjects

A total of 21 patients were recruited from referrals to the Movement Disorders Clinic in the Department of Neurology at Innsbruck Medical University. Only patients fulfilling the established UK Parkinson’s Disease Society Brain Bank clinical diagnostic criteria were eligible for the study (Gibb and Lees, 1988). Motor disability related to parkinsonism was assessed in all patients in OFF drug states using part III of the Unified Parkinson’s Disease Rating Scale (UPDRS) and disease stages were classified according to the Hoehn and Yahr rating scale (Hoehn and Yahr, 1967; Fahn et al., 1987; Table 1).

Table 1

Demographic data of patients with Parkinson’s disease and healthy control subjects

 Parkinson’s disease Controls 
n = 16 n = 14 
Female/male (n10/6 8/6 
Age at scan (years)   
Mean ± SD 68.1 ± 6.1 (54.8–76.3) 67.3 ± 3.7 (60–74.4) 
Disease duration (years)   
Mean ± SD 3.7 ± 3.7 (0.1–15.2)  
UPDRS motor score   
Mean ± SD 20 ± 10.3 (8–42)  
Hoehn and Yahr staging   
Mean ± SD 2.3 ± 1 (1–4)  
Total odour score   
Mean ± SD 19.2 ± 4.4 (12.3–28.5)*** 34.3 ± 2.2 (31.3–37.3) 
Odour threshold   
Mean ± SD 3.8 ± 2.6 (1–9.8)*** 7.3 ± 2.2 (4.3–12) 
Odour discrimination   
Mean ± SD 8 ± 1.9 (5–13)*** 13 ± 1.4 (10–15) 
Odour identification   
Mean ± SD 7.4 ± 2.9 (3–13)*** 13.9 ± 0.9 (12–15) 
 Parkinson’s disease Controls 
n = 16 n = 14 
Female/male (n10/6 8/6 
Age at scan (years)   
Mean ± SD 68.1 ± 6.1 (54.8–76.3) 67.3 ± 3.7 (60–74.4) 
Disease duration (years)   
Mean ± SD 3.7 ± 3.7 (0.1–15.2)  
UPDRS motor score   
Mean ± SD 20 ± 10.3 (8–42)  
Hoehn and Yahr staging   
Mean ± SD 2.3 ± 1 (1–4)  
Total odour score   
Mean ± SD 19.2 ± 4.4 (12.3–28.5)*** 34.3 ± 2.2 (31.3–37.3) 
Odour threshold   
Mean ± SD 3.8 ± 2.6 (1–9.8)*** 7.3 ± 2.2 (4.3–12) 
Odour discrimination   
Mean ± SD 8 ± 1.9 (5–13)*** 13 ± 1.4 (10–15) 
Odour identification   
Mean ± SD 7.4 ± 2.9 (3–13)*** 13.9 ± 0.9 (12–15) 

Values represent the means ± 1 SD and the data range in brackets.

*** P < 0.001 versus healthy control subjects.

Exclusion criteria included a Mini-Mental State Examination score of <28, signs of vertical gaze palsy or cerebellar symptoms, no visible butterfly-shaped midbrain on transcranial sonography or evidence of severe white matter, vascular or space-occupying lesions within the cerebrum at T1- and T2-weighted conventional MRI. Two patients with marked cerebral microangiopathy, including subcortical and brainstem lesions and three patients with no detectable bone window were excluded such that 16 patients with Parkinson’s disease (10 females; mean age 68.1 ± 6.1 years; Table 1) were included in the study. This group included a minimum of four patients each in Hoehn and Yahr stages I, II and III and was thus representative of the earlier phases of disease progression in Parkinson’s disease. Nine patients were taking dopamine agonists, 12 were receiving levodopa therapy (minimum 300 mg daily, maximum 1100 mg daily) and six were being treated additionally with amantadine and/or rasagiline. Patients were on dopaminergic medication at the time of olfactory function testing and transcranial sonography since no modifying effect of levodopa on either olfaction or midbrain echogenicity has been reported (Doty et al., 1992; Berg et al., 2005). Antiparkinsonian medication was stopped at least 12 h before [18F] DOPA PET and 4 h before diffusion tensor imaging.

Fourteen age- and gender-matched healthy individuals with no signs of parkinsonism, olfactory dysfunction or hyperechogenicity of the substantia nigra served as a control group. The study was approved by the Ethics Committee of the Innsbruck Medical University. Subjects’ written informed consent was obtained according to the Declaration of Helsinki.

Olfactory function testing

Olfactory threshold, odour discrimination and odour identification were investigated in three separate subtests using standardized and commercially available Sniffin’ Sticks (Hummel et al., 1997; Burghart Messtechnik). Results of the three subtests were presented as the (i) composite threshold; (ii) discrimination and identification score, which was calculated from the sum of odour threshold; and (iii) discrimination and identification measures (Kobal et al., 2000). The interstimulus interval was at least 20 s to prevent olfactory desensitization. During the examination the patients wore blindfolds.

The olfactory threshold subtest consisted of 16 Sniffin’ Stick triplets with different concentrations of n-butanol. Three pens were presented in a randomized order, with two containing the solvent and the third the odorant at a specific dilution. Subjects were asked to identify the pen containing the odorant. Presentation of the sticks occurred until the odorant had been successfully discriminated in two successive trials, which triggered a reversal of the staircase (Stiasny-Kolster et al., 2005). Threshold was defined as the mean of at least four out of seven staircase reversal points (Ehrenstein and Ehrenstein, 1999). For the odour discrimination task, triplets of pens were presented in a randomized order, with two containing the same odorant and the third a different odorant. Subjects were asked to determine which of three odour-containing pens smelled differently. The maximum score for correct discrimination was 16. Odour identification was assessed by means of 16 common odours. Using a multiple choice task, identification of individual odorants was performed from a list of four descriptors. The pens contained familiar fragrances, such as orange, leather, cinnamon, peppermint, banana, lemon, liquorice, turpentine, garlic, coffee, apple, clove, pineapple, rose, aniseed and fish. Again, the subjects’ scores ranged from 0 to 16, with the highest score indicating the correct identification of each odour.

Imaging

Magnetic resonance imaging

All measurements were performed on a 1.5 Tesla whole-body magnetic resonance scanner (Magnetom Avanto, Siemens) with the help of an eight-channel head coil. The MRI protocol comprised a coronal T1-weighted MPRAGE 3D (repetition time, 1600 ms; echo time, 3.44 ms; inversion time, 800 ms; slice thickness, 1.2 mm; matrix, 256 × 224 pixels; number of excitations, 1; flip angel, 15°; field of view 220 × 192.5 mm), a transversal double-echo fast spin echo sequence with T2 and proton density contrast (repetition time, 3270 ms; echo time 1–2, 12 and 85 ms; slice thickness, 3–5 mm; spacing between slices, 3.6–6 mm; matrix, 256 × 200; number of excitations, 1; flip angle, 150°; field of view 220 × 171.88 mm; echo train length, 5), and a transversal diffusion-weighted echoplanar imaging sequence with diffusion-sensitizing gradients in 12 directions and b-factors of 0 and 1000 s/mm2 [repetition time, 6000 ms; echo time, 94 ms; slice thickness, 3 mm; spacing between slices, 0.75 mm; matrix, 128 × 128 (k − s pace interpolation to 256 × 256); number of excitations, 1; flip angle, 90°; field of view 230 × 230 mm; parallel imaging factor, 2].

Maps of mean diffusivity and fractional anisotropy were automatically calculated by the magnetic resonance scanner. For region of interest analysis of the substantia nigra, mean diffusivity and fractional anisotropy maps, as well as the mean b–1000 images and proton density images were co-registered with the ‘Pipeline Affine’ algorithm implemented in the software package 3D Slicer (Pieper et al., 2004). Fractional anisotropy maps provided an excellent delineation between substantia nigra and crura cerebri, whereas the proton-density weighted image as well as the b−1000 images permitted an accurate delineation of the posterior border. Mean diffusivity, fractional anisotropy, b−1000 and the co-registered proton images were synchronized with the help of the ‘Sync Windows’ tool (by Joachim Walter at biz.uni-muenchen.de) available through ImageJ (Adachi et al., 1999; Oikawa et al., 2002; Abramoff et al., 2004) and segmentation of the substantia nigra was performed as shown in Fig. 1. For analysis of the mean diffusivity signal within the olfactory tracts, regions of interest previously identified by the software package statistical parametric mapping (Friston et al., 1995) and published by our group were applied (Scherfler et al., 2006; Fig. 2). In order to transform regions of interest of the olfactory tracts from statistical parametric mapping space to the individuals’ mean diffusivity image space the following steps were performed: (i) individual mean diffusivity images were co-registered onto the individual T1-weighted image; (ii) the individual T1-weighted image was normalized to the T1-weighted template provided by statistical parametric mapping; and (iii) the obtained deformation fields were inverted and applied to the regions of interest of the olfactory tracts (Ashburner et al., 2000; Fig. 2). This method was preferred to a rate-driven approach in order to guarantee the highest level of accuracy in positioning of previously identified regions of interest in statistical parametric mapping space. The mismatch of the inversion in the deformation fields procedure was 1.4 % ± 0.7, which did not affect the reported between-group significance level of mean diffusivity measures in the mean region of interest values of the olfactory tracts.

Figure 1

The substantia nigra was segmented on the fractional anisotropy maps (B), on the trace images (C), and on the proton-density weighted images (D) after co-registration. Segmentation was performed with the help of the window synchronization tool of ImageJ permitting simultaneous drawing in all activated windows. The fractional anisotropy maps provided an excellent delineation between substantia nigra and crura cerebri, whereas the proton-density weighted image as well as the b−1000 images permitted an accurate delineation of the posterior border. The substantia nigra was divided in three levels [upper (1), middle (2) and lower (3)]. At the lower level, we positioned two separate regions of interest in the medial and the lateral part of the substantia nigra. After segmentation, the regions of interest were applied to the fractional anisotropy maps and the co-registered mean diffusivity maps (A).

Figure 1

The substantia nigra was segmented on the fractional anisotropy maps (B), on the trace images (C), and on the proton-density weighted images (D) after co-registration. Segmentation was performed with the help of the window synchronization tool of ImageJ permitting simultaneous drawing in all activated windows. The fractional anisotropy maps provided an excellent delineation between substantia nigra and crura cerebri, whereas the proton-density weighted image as well as the b−1000 images permitted an accurate delineation of the posterior border. The substantia nigra was divided in three levels [upper (1), middle (2) and lower (3)]. At the lower level, we positioned two separate regions of interest in the medial and the lateral part of the substantia nigra. After segmentation, the regions of interest were applied to the fractional anisotropy maps and the co-registered mean diffusivity maps (A).

Figure 2

Regions of interest within the olfactory tract superimposed on a mean diffusivity image of a patient with Parkinson’s disease (A) and a healthy control subject (B). Regions of interest represent voxel clusters previously obtained from a separate voxel-based group analysis of patients with Parkinson’s disease and healthy controls (Scherfler et al., 2006).

Figure 2

Regions of interest within the olfactory tract superimposed on a mean diffusivity image of a patient with Parkinson’s disease (A) and a healthy control subject (B). Regions of interest represent voxel clusters previously obtained from a separate voxel-based group analysis of patients with Parkinson’s disease and healthy controls (Scherfler et al., 2006).

6-[18F] fluorolevodopa positron emission tomography

[18F]DOPA acquisition was performed in patients and control subjects with a GE Advance positron emission tomograph with an axial field of view of 15 cm. Emission scan data were acquired in a high sensitivity, 3D mode giving mean reconstructed full-width at half-maximum transaxial and axial spatial resolutions of 5.2 and 4.7 mm, respectively. A correction for tissue attenuation of 511 KeV gamma radiation was measured with a 5 min 3D transmission scan performed before tracer injection and acquired using a retractable [86Ge] source. Image reconstruction was performed with a FORE 3D rebinning algorithm on a SUN Ultra Sparc 60 II workstation (Sun Microsystems). To minimize artefacts arising from head motion, subjects were positioned within a moulded head holder such that their orbitomeatal line was parallel to the transaxial plane of the tomograph. Head position was carefully monitored with a video camera and by direct observation throughout. Each patient had their antiparkinsonian medication stopped at least 12 h before PET examination. Patients were pretreated with 150 mg carbidopa to block peripheral DOPA-decarboxylase activity and 200 mg entacapone, a catechol-O-methyltransferase inhibitor, to prolong the availability of [18F] DOPA within the blood system. Following a transmission scan, [18F] DOPA (183.8 ± 20.2 MBq in 10 ml of normal saline solution, IASOdopa®, Iason) was infused intravenously as a bolus over 30 s. Scanning began 30 s before tracer injection with a protocol of 26 time frames over 94 min 30 s (1 × 30 s; 4 × 1 min; 3 × 2 min; 3 × 3 min; and 15 × 5 min). Following correction for decay, parametric images of [18F] DOPA Ki influx rate constant values, representing the rate of [18F] DOPA uptake and storage as [18F]DOPA, were generated using the Patlak graphical approach with a cerebellar cortex reference input function (Patlak et al., 1983; Gunn et al., 1997).

Co-registration of parametric DOPA Ki maps was achieved by generating individual integrated ‘add images’ (combined time frames 1–26 of the dynamic data set) and estimating the rigid body transformation parameters for spatial co-registration to the structural volumetric MRIs using the normalized mutual information co-registration tool of SPM8 (Collignon et al., 1995). Obtained transformation parameters were applied to DOPA Ki maps. Finally, the caudate and putamen of each side was outlined on the volumetric MRI and superimposed onto the co-registered DOPA Ki maps (Rorden and Brett, 2000). Caudate and putaminal asymmetry indices were calculated, reflecting the percentage difference between the PET signal in the respective region of higher tracer-uptake compared with the contralateral region.  

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Additionally, the hemispheric ratio between caudate and putaminal PET signal was measured.

Transcranial sonography

Transcranial sonography was performed using a 2.5 MHz transducer (Logic 7; General Electric). The penetration depth was 16 cm, and the dynamic range was between 45 and 50 dB. The image brightness and time gain were adapted as needed for best visualization. The examination was performed from both sides using the temporal approach. The images of the transcranial sonography examinations were stored digitally and used for the segmentation procedure. All patients with Parkinson’s disease and healthy control subjects were examined by the same experienced sonographer. Areas of echogenicity were manually encircled on digitally stored images and the total area of echogenic signals in the substantia nigra was measured (Berg et al., 1999a; Stockner et al., 2007).

Statistical analysis

Two-tailed unpaired Student’s t-tests were applied for comparison of clinical data, mean regional [18F] DOPA PET, transcranial sonography, mean diffusivity and fractional anisotropy values. The relationship between parameters obtained by the imaging modalities listed was investigated using Pearson’s correlation statistics. The Spearman Rank test was used to correlate ordinal clinical data with imaging parameters. Data were tabulated and analysed using a commercial software package (SPSS for Windows 17.0).

Results

Clinical variables

There was no significant difference in age and sex distribution between the Parkinson’s disease and control cohorts (Table 1). The Hoehn and Yahr stages were identified as: Stage I in four patients; Stage II in five patients; Stage III in five patients; and Stage IV in two patients. The mean duration of illness was 3.7 years [± 1 standard deviation (SD) 3.7, range 0.1–15.2] and the median UPDRS motor score was 20 (± 1 SD 10.3, range 8–42). The average scores of the olfactory task performance are listed in Table 1. Measures of odour discrimination, identification and threshold were significantly decreased in the group of patients with Parkinson’s disease compared with the control group (P < 0.001, respectively).

Region of interest analysis of 6-[18F] fluorolevodopa positron emission tomography

Regional mean [18F] DOPA Ki measures are detailed in Table 2. When comparing patients with Parkinson’s disease to control subjects, we found significant decreases in [18F] DOPA uptake in the caudate (P < 0.001) and putamen (P < 0.001) of the Parkinson’s disease group. Both the putamen asymmetry index and the caudate to putamen ratio of [18F] DOPA Ki were significantly increased in the group of patients with Parkinson’s disease (P < 0.001).

Table 2

Mean regional putamen (anterior, posterior) and caudate [18F] DOPA Ki min−1 values in patients with Parkinson’s disease and healthy control subjects

 Parkinson’s disease Controls 
n = 16 n = 14 
Caudate, mean ± SD 0.0154 ± 0.0023*** 0.0219 ± 0.0015 
(0.0102–0.0197) (0.0197–0.0252) 
Putamen, mean ± SD 0.0112 ± 0.0021*** 0.0229 ± 0.0019 
(0.0066–0.0163) (0.0203–0.0273) 
Asymmetry index % caudate, mean ± SD 8.5 ± 7.2* 3.7 ± 2.2 
(1–27.6) (0.5–7.6) 
Asymmetry index % putamen, mean ± SD 12.6 ± 8.2*** 2.8 ± 1.7 
(3–35.7) (0.5–7.1) 
Ratio caudate/putamen 1.4 ± 0.2*** 0.96 ± 0.4 
(1.1–1.8) (0.88–1) 
 Parkinson’s disease Controls 
n = 16 n = 14 
Caudate, mean ± SD 0.0154 ± 0.0023*** 0.0219 ± 0.0015 
(0.0102–0.0197) (0.0197–0.0252) 
Putamen, mean ± SD 0.0112 ± 0.0021*** 0.0229 ± 0.0019 
(0.0066–0.0163) (0.0203–0.0273) 
Asymmetry index % caudate, mean ± SD 8.5 ± 7.2* 3.7 ± 2.2 
(1–27.6) (0.5–7.6) 
Asymmetry index % putamen, mean ± SD 12.6 ± 8.2*** 2.8 ± 1.7 
(3–35.7) (0.5–7.1) 
Ratio caudate/putamen 1.4 ± 0.2*** 0.96 ± 0.4 
(1.1–1.8) (0.88–1) 

Values represent the means ± 1 SD and the data range in brackets.

*** P < 0.001, * P < 0.05 versus healthy control subjects.

Significant correlations were found between disease duration and both mean caudate and putamen [18F] DOPA Ki values (r = −0.75; P < 0.001 and r = −0.68; P < 0.01, Pearson correlation, respectively). Mean putaminal Ki values correlated significantly with the total odour score (r = 0.58; P < 0.05, Spearman’s rank correlation), the Hoehn and Yahr stage (r = −0.5, P < 0.05, Spearman’s rank correlation) and the UPDRS motor score (r = −0.53, P < 0.05, Spearman’s rank correlation). Decreased odour identification ability correlated significantly with decreases of [18F] DOPA uptake in the putamen (P < 0.05; r = 0.52). Significant correlations were also found between mean putaminal Ki and nigral measures of mean diffusivity (r = −0.44, P < 0.05, Pearson correlation) which increased when combining mean diffusivity values of the middle and medial caudal section of the substantia nigra (r = −0.52, P < 0.05, Pearson correlation, correlation slope: y = −15 × x + 1, Fig. 3A). Moreover, mean putaminal Ki values correlated with mean diffusivity values of the olfactory tract (r = −0.71, P < 0.01, Pearson correlation, correlation slope: y = −55 × x + 1.7, Fig. 3B). No correlations were found between caudate and putamen [18F] DOPA Ki values and measures of fractional anisotropy and transcranial sonography of the substantia nigra.

Figure 3

Correlation between mean diffusivity values of the ipsilateral and contralateral middle and medial caudal section of the substantia nigra and the corresponding [18F] DOPA mean putamen Ki values in patients with Parkinson’s disease (A). Correlation between mean diffusivity values of both olfactory tracts and the mean putaminal [18F] DOPA Ki values in patients with Parkinson’s disease (B). Correlation between the UPDRS motor score and the mean diffusivity values of the middle and medial caudal section of the substantia nigra (C) and the mean diffusivity values of both olfactory tracts (D) in patients with Parkinson’s disease. OT = olfactory tract; SN = substantia nigra.

Figure 3

Correlation between mean diffusivity values of the ipsilateral and contralateral middle and medial caudal section of the substantia nigra and the corresponding [18F] DOPA mean putamen Ki values in patients with Parkinson’s disease (A). Correlation between mean diffusivity values of both olfactory tracts and the mean putaminal [18F] DOPA Ki values in patients with Parkinson’s disease (B). Correlation between the UPDRS motor score and the mean diffusivity values of the middle and medial caudal section of the substantia nigra (C) and the mean diffusivity values of both olfactory tracts (D) in patients with Parkinson’s disease. OT = olfactory tract; SN = substantia nigra.

Region of interest analysis of diffusion tensor imaging

Regional mean diffusivity and fractional anisotropy measures are detailed in Table 3. When comparing patients with Parkinson’s disease and control subjects, we found significant increases of substantia nigra mean diffusivity values in the group of patients with Parkinson’s disease (P < 0.01). In a detailed analysis of the upper, middle and lower level of the substantia nigra, significant changes in mean diffusivity were apparent in the middle level (P < 0.01) and the medial portion of the lower level (P < 0.01). No significant changes were found in the upper level. Significant decreases of fractional anisotropy values were evident in the medial portion of the lower level in the substantia nigra of the Parkinson’s disease group compared with the control group (P < 0.05). The region of interest analysis of the olfactory tract showed significant mean diffusivity signal increases of the Parkinson’s disease group when compared with the control group (P < 0.05).

Table 3

Mean diffusivity, fractional anisotropy and echogenicity values in patients with Parkinson’s disease and healthy control subjects

 Parkinson’s disease Controls 
n = 16 n = 14 
Mean diffusivity   
Substantia nigra, mean ± SD 0.79 ± 0.06** 0.73 ± 0.03 
(0.71–0.89) (0.68–0.77) 
 upper level, mean ± SD 0.74 ± 0.06 0.71 ± 0.04 
(0.68–0.89) (0.66–0.79) 
 middle level, mean ± SD 0.79 ± 0.05** 0.73 ± 0.04 
(0.70–0.86) (0.65–0.81) 
 lower level medial portion, mean ± SD 0.93 ± 0.12** 0.77 ± 0.10 
(0.83–1.14) (0.56–0.92) 
Olfactory tract, mean ± SD 1.13 ± 0.16* 1 ± 0.1 
(0.94–1.49) (0.8–1.09) 
Fractional anisotropy   
Substantia nigra, mean ± SD 0.57 ± 0.05 0.59 ± 0.05 
(0.48–0.65) (0.49–0.66) 
 lower level medial portion, mean ± SD* 0.53 ± 0.07* 0.59 ± 0.09 
(0.41–0.65) (0.5–0.82) 
Echogenicity   
substantia nigra, mean ± SD 0.24 ± 0.06*** 0.07 ± 0.03 
(0.1–0.31) (0.02–0.11) 
 Parkinson’s disease Controls 
n = 16 n = 14 
Mean diffusivity   
Substantia nigra, mean ± SD 0.79 ± 0.06** 0.73 ± 0.03 
(0.71–0.89) (0.68–0.77) 
 upper level, mean ± SD 0.74 ± 0.06 0.71 ± 0.04 
(0.68–0.89) (0.66–0.79) 
 middle level, mean ± SD 0.79 ± 0.05** 0.73 ± 0.04 
(0.70–0.86) (0.65–0.81) 
 lower level medial portion, mean ± SD 0.93 ± 0.12** 0.77 ± 0.10 
(0.83–1.14) (0.56–0.92) 
Olfactory tract, mean ± SD 1.13 ± 0.16* 1 ± 0.1 
(0.94–1.49) (0.8–1.09) 
Fractional anisotropy   
Substantia nigra, mean ± SD 0.57 ± 0.05 0.59 ± 0.05 
(0.48–0.65) (0.49–0.66) 
 lower level medial portion, mean ± SD* 0.53 ± 0.07* 0.59 ± 0.09 
(0.41–0.65) (0.5–0.82) 
Echogenicity   
substantia nigra, mean ± SD 0.24 ± 0.06*** 0.07 ± 0.03 
(0.1–0.31) (0.02–0.11) 

Values represent the means ± 1 SD and the data range in brackets.

* P < 0.05, ** P < 0.01; and *** P < 0.001 versus healthy control subjects.

Significant correlations were found between mean diffusivity values of the middle and medial portion of the lower section of the substantia nigra and the UPDRS motor score (r = −0.48, P < 0.01, Spearman’s rank correlation, correlation slope: y = 0.0033 × x + 0.79, Fig. 3C) and the mean diffusivity values of the olfactory tract and the UPDRS motor score (r = 0.59, P < 0.01, Spearman’s rank correlation, correlation slope: y = 0.0087 × x + 0.96, Fig. 3D). No significant correlations were found between nigral fractional anisotropy values and parameters for motor and olfactory function as well as striatal [18F] DOPA PET or nigral transcranial sonography measures. Correlations between nigral diffusion tensor imaging and striatal [18F] DOPA measures are listed in the respective [18F] DOPA PET section.

Region of interest analysis of transcranial sonography

Mean echogenic areas of the substantia nigra are listed in Table 3. When comparing patients with Parkinson’s disease and control subjects, we found a significant enlargement of nigral hyperechogenic area in the Parkinson’s disease group (P < 0.001). No significant correlations were found between nigral transcranial sonography measures and parameters for motor and olfactory function as well as striatal [18F] DOPA PET or nigral diffusion tensor imaging measures.

Discussion

We have shown for the first time a significant correlation between microstructural alterations of the substantia nigra, measured by diffusion tensor imaging, and motor dysfunction determined by the UPDRS motor score as well as decreased putaminal [18F] DOPA uptake, a surrogate marker of dopaminergic terminal function in patients with Parkinson’s disease.

In line with our results, nigral mean diffusivity and/or fractional anisotropy signal abnormalities have been reported previously in patients with early stage Parkinson’s disease (Yoshikawa et al., 2004; Vaillancourt et al., 2009; Péran et al., 2010). Concomitant fractional anisotropy decreases and mean diffusivity increases are thought to be the correlate of neuronal and myelin damage leading to depletion of restricting barriers to water molecular motion. As a result, anisotropy, which is a measure of the degree of alignment of cellular structures within fibre tracts, decreases, and the magnitude of diffusion, a measure of extracellular fluid volume, increases. This antipodal behaviour of fractional anisotropy and mean diffusivity coefficients was observed in brain areas known to be affected by neurodegenerative processes in patients with multiple system atrophy and Alzheimer’s disease (Schocke et al., 2004; Stebbins et al., 2009; Tha et al., 2010). Consistent with the study by Péran et al. (2010), we found significant fractional anisotropy and mean diffusivity signal alterations in the medial and caudal portion of the substantia nigra. This finding also correlates with neuropathological observations documenting more prominent dopaminergic neuron loss in the caudal segments of the substantia nigra (Fearnley and Lees, 1991). Diverging from histopathological investigations, we were not able to localize additional microstructural alterations in the ventrolateral tier of the substantia nigra, which is most likely due to the small size of this structure being beyond the spatial resolution allowed by diffusion weighting gradients obtained from a 1.5T MRI. Since large diffusion weighting gradients have been reported to be highly sensitive to subject motion, it was suggested that fast single-shot echo-planar imaging sequences were used to maximize the signal-to-noise ratio and the accuracy of diffusion measurements, however, at the expense of spatial resolution. In our study, a slice thickness of 3 mm allowing analysis of the diffusion tensor imaging signal from the substantia nigra on three slices was considered to be an acceptable trade-off between spatial resolution and a reasonable acquisition time of 100 s in patients with Parkinson’s disease. However, partial volume effects, particularly in the area of the caudal and rostral section of the substantia nigra may have added to variability in the diffusion tensor imaging signal and need to be considered when interpreting correlations between nigral mean diffusivity signal increases and decreased putaminal [18F] DOPA uptake. Higher resolution can be obtained either by using more sensitive radiofrequency coils or higher field strength at the expense of higher magnetic susceptibility effects and the increased echo–planar image distortion, which could in turn be reduced by parallel imaging methods. In this respect, further studies are warranted to evaluate the effects of higher field magnets on the quantitative measurements of mean diffusivity and fractional anisotropy of the substantia nigra in patients with Parkinson’s disease and healthy subjects (Alexander et al., 2006).

Focal mean diffusivity signal increases of the caudal section of the substantia nigra were also correlated with increases in motor disability, overlapping partially with a recently published study reporting fractional anisotropy values of the entire substantia nigra to be correlated with the UPDRS motor score (Zhan et al., 2012). A certain variability of nigral mean diffusivity and fractional anisotropy findings in Parkinson’s disease among recent publications may result from: (i) different algorithms applied to calculate those indices; (ii) the field strength of the magnet and its corresponding spatial resolution as well as signal-to-noise behaviour; and (iii) the method of substantia nigra delineation and partition used. Pronounced mean diffusivity increases in combination with relatively extenuated alterations of the fractional anisotropy signal as observed in the substantia nigra in our study relate to an increase in water diffusion and only minor deformation of the shape of the extracellular fluid space. In line with this observation, mean diffusivity increases were encountered in brain areas known to consist predominately of neuronal nuclei, whereas fractional anisotropy decreases adjacent to mean diffusivity increases suggest damage in connecting axons secondary to the structural damage (Erbetta et al., 2009). The significant relationship between microstructural damage and Parkinson’s disease severity supports evidence from animal studies that mean diffusivity increases presumably reflect nigral pathology (Boska et al., 2007). This interpretation is further corroborated by a significant association of increased mean diffusivity signal and decreased putaminal [18F] DOPA uptake. The [18F] DOPA influx constant measures terminal aromatic DOPA-decarboxylase activity and was also shown to correlate with the degree of motor disability as determined by the UPDRS motor score and the number of remaining nigral dopaminergic cells in both human and MPTP-lesioned monkey autopsy studies (Brooks et al., 1990; Pate et al., 1993; Snow et al., 1993; Vingerhoets et al., 1997).

Mean putaminal Ki values were also significantly correlated with the total odour score and impairments of odour identification capability, suggesting the olfactory deficit in Parkinson’s disease to be progressive. However, an increasing impairment of olfactory function with disease progression has only been demonstrated for odour discrimination (Tissingh et al., 2001; Boesveldt et al., 2008). In cross-sectional clinical investigations, odour detection and identification deficits were found to be unrelated to disease duration or severity (Ward et al., 1983; Doty et al., 1988). This was further corroborated by an earlier dopamine transporter binding study of patients with Parkinson’s disease reporting no relation between whole striatal dopamine transporter binding loss with measures of odour detection, recognition memory and identification (Lehrner et al., 1995). In line with our results, significant correlations between decreases of odour identification capability and putaminal dopamine transporter binding were consistently identified in two studies (Siderowf et al., 2005; Berendse et al., 2011). Reasons for diverging results may be due to the observation that only studies analysing the caudate and putamen separately succeeded to identify a relation between dopaminergic function and olfactory impairment in Parkinson’s disease. In addition, floor effects of the olfactory dysfunction were suggested to mask any relation with disease duration in late stage Parkinson’s disease (Siderowf et al., 2005). Overall, our data support the concept of parallel progression of both odour deficits and putaminal dopaminergic dysfunction in the early motor disease stages of Parkinson’s disease and this was further substantiated by a highly significant relation between mean diffusivity changes in the olfactory tract and bulb and the reduction of putaminal DOPA storage capacity. In accordance with our data, significant associations of decreases in olfactory performance including olfactory identification testing and measurements of olfactory tract/bulb degradation have repeatedly been reported using diffusion tensor and volumetric imaging studies in patients with Parkinson’s disease (Scherfler et al., 2006; Rolheiser et al., 2011; Wang et al., 2011; Brodoehl et al., 2012). In the present study, however, we could not identify a clear-cut threshold differentiating all patients from controls, which was most likely due to the limited spatial resolution separating the tissue compartment of the surrounding CSF compartment. In this respect, further studies are warranted to enhance the spatial accuracy and signal-to-noise performance of diffusion tensor imaging to depict small tissue changes of the olfactory tract and bulb.

In contrast with mean diffusivity measures, substantia nigra echogenicity did not correlate with the motor part of the UPDRS score and the mean diffusion tensor imaging values of the substantia nigra. This is in accordance with previous studies showing that substantia nigra echogenicity neither correlates with the measures of clinical severity nor with markers of putaminal dopaminergic terminal function visualized by SPECT or PET (Berg et al., 2001; Spiegel et al., 2006; Behnke et al., 2009). Based on animal and post-mortem studies, the paradigm of hyperechogenicity of the substantia nigra in Parkinson’s disease was suggested to relate to increased iron depositions in tissue (Berg et al., 1999b, 2002; Zecca et al., 2005). Until now, however, it has remained unclear how these echosignal changes correspond to the progressive degeneration of presynaptic dopaminergic nerve terminals. Our findings underline the current concept that the parameter of hyperechogenicity does not parallel the extent of the underlying pathological process of the substantia nigra in a quantitative manner. However, as a qualitative feature, transcranial sonography of the substantia nigra has attracted considerable attention as a potential modality in the work-up of patients presenting with a parkinsonian syndrome (Gaenslen et al., 2008; Berardelli et al., 2013).

Conclusion

We report a significant association between increased mean diffusivity signal and decreased [18F] DOPA uptake, indicating that microstructural degradation of the substantia nigra and the olfactory tract parallels progression of putaminal dopaminergic dysfunction in Parkinson’s disease. Nigral mean diffusivity signal increases also correlate with motor dysfunction, suggesting a need for further evaluation of diffusion tensor imaging as an objective marker for disease progression that would be not be affected by pharmacological treatment (Degirmenci et al., 2007).

Funding

This work was supported by grants of the Innsbruck Medical University (MFI 6169; IFTZ-2007152).

Abbreviations

    Abbreviations
  • Ki

    influx rate constant

  • SPECT

    single photon emission computed tomography

  • UPDRS

    Unified Parkinson’s Disease Rating Scale

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Author notes

*These authors contributed equally to this work.