Movement Disorders Vol. 22, No. 4, 2007, pp. 550 –555 © 2007 Movement Disorder Society
Transcranial Imaging of Substantia Nigra Hyperechogenicity in a Taiwanese Cohort of Parkinson’s Disease Yu-Wen Huang, MD,1 Jiann-Shing Jeng, MD, PhD,1 Chung-Fen Tsai, MD,2 Li-Ling Chen, BS,1 and Ruey-Meei Wu, MD, PhD1* 1
Department of Neurology, National Taiwan University Hospital, College of Medicine, National Taiwan University, Taipei, Taiwan 2 Department of Neurology, Cardinal-Tien Hospital, Taipei, Taiwan
Abstract: Transcranial Doppler imaging (TCDI) has been used as a noninvasive diagnostic tool to differentiate Parkinson’s disease (PD) from atypical parkinsonism by detecting hyperechogenicity in the substantia nigra (SN). To our knowledge, no TCDI data are available for Asian populations, and TCDI sensitivity is uncertain across populations. Early-onset PD (EOPD) represents a specific PD subtype based on clinical features and pathogenic mechanisms. It is not known if EOPD patients have abnormal echogenicity in SN comparable to late-onset PD (LOPD) patients. We assessed the area of SN hyperechogenicity (hyper-SN) and a ratio of hyper-SN over ipsilateral midbrain (S/M ratio) with TCDI in 164 healthy
Taiwanese, 40 EOPD patients, and 40 LOPD patients. The upper 95th percentile values for hyper-SN and S/M ratio were 0.20 cm2 and 0.07. Our results indicate that S/M ratio is a more sensitive measure than hyper-SN in diagnosing PD. Approximately 92.5% of the LOPD patients and 57.5% of the EOPD patients had S/M ratios ⱖ 0.07. Enlarged hyperechogenicity of SN is a common finding in LOPD, but not in EOPD. Ironindependent mechanisms of SN cell degeneration in EOPD distinct from that in LOPD might contribute to the sonographic findings. © 2007 Movement Disorder Society Key words: transcranial doppler image; echogenicity; substantia nigra; midbrain; Parkinson’s disease; Taiwanese.
Transcranial Doppler imaging (TCDI) has long been used as a diagnostic tool for cerebrovascular disorders. Its recent application in patients with extrapyramidal disorders has identified varying degrees of altered echogenicity in basal ganglia.1,2 An enlarged area of hyperechogenic signal in substantia nigra (SN) region is characteristic in Parkinson’s disease (PD) and rarely seen in other atypical parkinsonian syndromes.3 However, sonographic measurement of hyperechogenic areas in SN may be challenged due to the small target size. To date, the use of TCDI for diagnosing PD in Asian populations has not been evaluated. Racial differences in brain size4 and temporal skull thickness5 in Asians may yield different values when using TCDI to measure midbrain.
An early-onset form of PD (EOPD), arbitrarily classified as occurring before age 45 or 50, is recognized as a unique subtype of PD, frequently manifested with dystonia, slow progression, and early appearance of levodopa-related dyskinesia.6 Our recent hospital-based study found that approximately 16% of PD patients diagnosed in our Movement disorders clinic had onset prior to age 50.7 Recent work has shown that a subgroup of these patients has an autosomal recessive juvenile onset PD (AR-JPD) with parkin or PINK1 gene mutations.8,9 Neuropathological reports of several EOPD cases with parkin mutations showed absence of Lewy bodies, and led to speculation that the etiology of ARJPD differs significantly from that of PD.10 Therefore, it was worthwhile to know whether SN hyperechogenicity is commonly observed in EOPD. The present study represents the first comprehensive evaluation of transcranial sonographic data for measuring SN hyperechogenic regions (hyper-SN) and midbrain in a large group of healthy Taiwanese. Furthermore, we investigated the sensitivity of SN hyperechogenicity for
*Correspondence to: Ruey-Meei Wu, Department of Neurology, National Taiwan University Hospital, No. 7 Chung-Shan South Road, Taipei 100, Taiwan. E-mail:
[email protected] Received 14 November 2006; Accepted 19 November 2006 Published online 26 January 2007 in Wiley InterScience (www. interscience.wiley.com). DOI: 10.1002/mds.21372
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diagnosing PD in LOPD and EOPD patients. Many distinguishable features of EOPD were identified by clinical phenotype or genetic etiology. PATIENTS AND METHODS Subjects A total of 282 individuals, including 192 control subjects and 90 Parkinson patients followed up at Movement Disorders Clinic of National Taiwan University, Taipei, Taiwan, were included for this study. All participants gave informed consent according to the Declaration of Helsinki. Volunteers with no evidence of CNS disorders were selected from the hospital’s neurological clinic as control subjects for this TCDI study. The diagnosis of PD was established in accordance with the PD criteria,11 which includes the presence of at least two (possible PD) or three (probable PD) of the four cardinal features (resting tremor, bradykinesia, rigidity, and asymmetric onset), with substantial and sustained response to L-dopa or a dopamine agonist. In addition, PD patients were categorized as LOPD (onset ⱖ 50 years) or EOPD (onset ⬍ 50 years) for further clinical and sonographic studies. All subjects underwent a detail history taking and thorough clinical examinations. Patient evaluations included the Unified PD Rating Scale, Hoehn & Yahr staging, the Folstein Mini Mental State Examination, a standard neurological examination, head CT or magnetic resonance imaging (MRI), and a TCDI examination. Transcranial Doppler Imaging TCDI was performed by one of the authors (Y.W.H. and C.F.T.) who were blind to the clinical information. A color-coded, phased-array ultrasound system equipped with a 2.25 MHz transducer (HP-4500 system) was used to examine echogenicity in the midbrain and SN through the temporal bone with a penetration depth of 16 cm and a dynamic range of 45 dB. As described previously,12 the examined scanning plane was standardized to display the margin of butterfly-shaped hypoechogenic midbrain surrounded by hyperechogenic basal cisterns as clear as possible by mildly adjusting the probe angle. We manually encircled the midbrain outline and the interior hyperechogenic areas of the SN through the echo-tracing method. The midline of midbrain was determined by drawing an intersectional line passing through the intercerebral peduncle and the point of hyperechogenic aqueduct. The respective areas of SN and ipsilateral midbrain were measured bilaterally (shown in Fig. 1). Moreover, considering that the imaged cross-section of midbrain might vary in sequential studies due to shifts in the position and angle of the probe as well as atrophic
FIG. 1. Transcranial Doppler imaging (TCDI) of the midbrain through a temporal bone window in a control subject. We identified the butterfly-shaped, hyperechogenic midbrain (dotted line), and manually encircled the interior hyperechogenic area of substantia nigra (thick solid line) and also ipsilateral midbrain (thin solid line) by the midbrain outline and an intersectional line passing through the aqueduct (arrow) and inter-cerebral peduncle (arrow head). The respective areas were measured bilaterally.
changes in midbrain due to age,13 we simultaneously used the ipsilateral midbrain as an internal control parameter, and calculated a hyper-SN/ipsilateral midbrain ratio (S/M ratio) for further analysis. Statistical Analysis An intra-class correlation coefficient (ICC) was used to determine intra- and inter-observer reliability of midbrain measurements. Reliability coefficients with 95% confidence intervals (CIs) were calculated for 20 controls. The hyper-SN and ipsilateral midbrain areas were recorded, and an S/M ratio was calculated. Data are reported as means and standard deviation. Estimates of sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated for hyper-SN and S/M ratio in LOPD and EOPD, respectively. The Pearson correlation coefficient (␥) was used to evaluate correlations between age and S/M ratio. To give the high specificity of TCDI in diagnosing PD, the top two standard deviations (upper 95th percentile) for hyper-SN and S/M ratio were used as cut-off points for defining abnormal extension of hyperechogenic signals in the SN region (denoting as SN⫹). To evaluate the association between clinical manifestations and hyperSN, LOPD and EOPD patients were classified into two groups depending on presence or absence of SN⫹. Inter-
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FIG. 2. Relationship between S/M ratio and age in 164 control subjects. The Pearson correlation coefficient is 0.546 (P ⬍ 0.001). The S/M ratio represents the area ratio of the substantia nigra (SN) hyperechogenic area and the size of the ipsilateral midbrain detected by transcranial Doppler imaging (TCDI).
group comparisons were conducted by two-sample, unpaired t tests for continuous variables with normal distributions, and 2 tests for discrete variables. The data were analyzed with the SPSS 12.0 statistical package (Chicago, IL). Statistical significance was set at P ⬍ 0.05. RESULTS Data from 20 controls were used to evaluate intraand inter-observer variability. Intra-observer ICCs for hyper-SN, ipsilateral midbrain measurements, and S/M ratios were 0.98 (0.96 – 0.99), 0.79 (0.63– 0.88), and 0.98(0.96 – 0.99) and the corresponding interobserver ICCs were 0.98 (0.96 – 0.99), 0.68 (0.35– 0.86), and 0.96 (0.89 – 0.98).
A total of 192 controls were examined, and 28 were excluded due to insufficient temporal bone windows. The control group, a total of 164 subjects, consisted of 96 men and 68 women (52.4 ⫾ 14.7 years; range 19 – 83). On the right side, hyper-SN was 0.066 ⫾ 0.066 (0 – 0.256) cm2, and the S/M ratio was 0.023 ⫾ 0.023 (0 – 0.091). Corresponding values for the left side were 0.066 ⫾ 0.068 (0 – 0.266) cm2 and 0.023 ⫾ 0.024 (0 – 0.087). There were no significant differences between right and left sides. As shown in Figure 2 there was a positive correlation between S/M ratio and age (␥ ⫽ 0.546, P ⬍ 0.001). This finding is compatible reports of age-related changes in the midbrain.13 The upper 95% values for hyper-SN and S/M ratio were 0.20 cm2 and 0.07. As aforementioned, we defined abnormal extension of SN hyperechogenicity (SN⫹) as any subject with a hyper-SN exceeding 0.20 cm2 or an S/M ratio higher than 0.07 on one or both sides. Fifty LOPD and 40 EOPD patients were examined. Ten LOPD patients were excluded due to poor temporal bone windows. Demographic and sonographic data for 40 LOPD and 40 EOPD patients and their age- and sex-matched controls are shown in Table 1. Both LOPD and EOPD patients had a significantly larger hyper-SN and S/M ratio than controls, but showed no obvious difference in midbrain size. Considering SN⫹, a cutoff of hyper-SN ⱖ0.20 cm2 showed high sensitivity (85%), specificity (85%), PPV (85%), and NPV (85%) in LOPD; and high specificity (100%) and PPV (100%), but relatively low sensitivity (50%) and high NPV (80%) in EOPD. A cutoff of S/M ratio ⱖ 0.07 showed high sensitivity (92.5%), specificity (82.5%), PPV (92%), and NPV (82.1%) in LOPD; and high specificity (97.5%) and PPV (92%), but again, relatively low sensitivity (57.5%) and high NPV (82.1%) in EOPD. The S/N ratio has
TABLE 1. Demographic and sonographic data in late-onset (LOPD) and early-onset (EOPD) Parkinson’s disease patients and controls Variables
LOPD (n ⫽ 40)
Controls (n ⫽ 40)
EOPD (n ⫽ 40)
Controls (n ⫽ 80)
Age (yr) Sex, M/F Onset age (yr) Disease duration (yr) Hoehn & Yahr stage Hyper-SN, right (cm2) Hyper-SN, left (cm2) Midbrain, right (cm2) Midbrain, left (cm2) S/M ratio, right S/M ratio, left
69.0 ⫾ 6.5 25/15 62.5 ⫾ 6.6 6.5 ⫾ 4.9 2.350 ⫾ 1.027 0.274 ⫾ 0.135* 0.276 ⫾ 0.119* 2.847 ⫾ 0.307 2.646 ⫾ 0.295 0.095 ⫾ 0.044* 0.104 ⫾ 0.046*
68.7 ⫾ 6.7 25/15
49.2 ⫾ 7.8 24/16 40.8 ⫾ 8.6 8.4 ⫾ 6.4 2.025 ⫾ 0.947 0.126 ⫾ 0.137* 0.180 ⫾ 0.143* 2.574 ⫾ 0.472 2.612 ⫾ 0.496 0.050 ⫾ 0.053* 0.072 ⫾ 0.055*
48.9 ⫾ 7.8 48/32
0.108 ⫾ 0.069 0.118 ⫾ 0.064 2.713 ⫾ 0.459 2.834 ⫾ 0.445 0.041 ⫾ 0.026 0.042 ⫾ 0.023
0.049 ⫾ 0.049 0.047 ⫾ 0.051 2.708 ⫾ 0.345 2.727 ⫾ 0.409 0.018 ⫾ 0.019 0.017 ⫾ 0.019
Hyper-SN: substantia nigra (SN) area of hyperechogenicity; S/M ratio: Hyper-SN/ipsilateral midbrain area ratio (see Fig. 1). *P ⬍ 0.001, unpaired t test, as compared with age- and gender-matched controls.
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TABLE 2. Comparison of clinical features of PD patients with or without abnormal extension of SN hyperechogenicity (S/M ratio ⱖ 0.07) LOPD Clinical characteristics Age (yr) Age at onset (yr) Duration (yr) Hoehn & Yahr stage L-dopa dose (mg/day) Akinesia-rigidity form Tremor-predominant form Family history Asymmetric symptoms Tremor Dystonia Postural instability Motor fluctuation Dyskinesia
EOPD
S/M ⱖ 0.07 (n ⫽ 37)
S/M ⬍ 0.07 (n ⫽ 3)
P value
S/M ⱖ 0.07 (n ⫽ 23)
S/M ⬍ 0.07 (n ⫽ 17)
P value
69.9 ⫾ 5.9 63.0 ⫾ 6.5 6.9 ⫾ 4.9 2.4 ⫾ 1.1 452.7 ⫾ 247.2 12 (32.4%) 25 (67.6%) 6 (16.2%) 20 (54.1%) 21 (56.8%) 20 (54.1%) 11 (29.7%) 8 (21.6%) 8 (21.6%)
58.0 ⫾ 4.4 56.3 ⫾ 4.0 1.7 ⫾ 0.6 2.0 ⫾ 0 333.3 ⫾ 152.8 1 (33.3%) 2 (66.7%) 1 (33.3%) 3 (100%) 2 (66.7%) 2 (66.7%) 0 (0%) 0 (0%) 0 (0%)
0.001* 0.092 ⬍0.001* 0.551 0.423 0.974 0.974 0.453 0.122 0.738 0.673 0.267 0.368 0.368
51.6 ⫾ 6.8 40.7 ⫾ 8.3 10.8 ⫾ 6.8 2.2 ⫾ 0.9 556.5 ⫾ 313.1 23 (100%) 0 (0%) 7 (30.4%) 8 (34.8%) 16 (69.6%) 9 (39.1%) 5 (21.7%) 16 (67.0%) 14 (60.9%)
45.9 ⫾ 8.2 40.8 ⫾ 9.3 5.2 ⫾ 4.1 1.8 ⫾ 0.9 379.4 ⫾ 301.6 16 (94.1%) 1 (5.9%) 4 (23.5%) 8 (47.1%) 9 (52.9%) 4 (23.5%) 3 (17.7%) 8 (47.0%) 5 (29.4%)
0.023* 0.993 0.004* 0.252 0.080 0.239 0.239 0.629 0.433 0.283 0.298 0.749 0.151 0.049*
S/M ratio: Hyper-SN/ipsilateral midbrain area (see Table 1). *Statistically significant difference between groups, P ⬍ 0.001, unpaired t test. PD, Parkinson’s disease; LOPD, late-onset PD; EOPA, early-onset PD.
higher sensitivity and positive predictive value than that of absolute value of hyper-SN alone. We thus use S/M ratio ⱖ 0.07 as SN⫹ cutoff in further analyses. To evaluate the relationship between clinical manifestation and SN⫹, we compared demographic and clinical features in LOPD and EOPD patients with or without abnormal extension of SN (Table 2). In both LOPD and EOPD groups, patients with SN⫹ were older than those without SN⫹ (LOPD: 69.9 ⫾ 5.9 years vs. 58.0 ⫾ 4.4 years, P ⫽ 0.001; EOPD: 51.6 ⫾ 6.8 years vs. 45.9 ⫾ 8.2 years, P ⫽ 0.023). Patients with SN⫹ also had longer disease duration than those without SN⫹ (LOPD: 6.9 ⫾ 4.9 years vs. 1.7 ⫾ 0.6 years, P ⬍ 0.001; EOPD: 10.8 ⫾ 6.8 years vs. 5.2 ⫾ 4.1 years, P ⫽ 0.004). Other clinical features were not significantly different for LOPD patients (Table 2). However, in EOPD patients, dyskinesia was significantly more frequent in patients with SN⫹ (60.9% vs. 29.4%, P ⫽ 0.049). DISCUSSION The present study is the first to investigate the size of the midbrain and SN hyperechogenic area by TCDI in a large group of Asians. In agreement with previous studies,14 we found that TCDI was a reliable and reproducible method. Our concern that the scanned axial plane of the midbrain would vary on each examination was supported by our finding of relatively lower intra- and interobserver ICCs for midbrain measurements (0.79 and 0.68) than for hyper-SN (0.98 and 0.98) and S/M ratio (0.98 and 0.96). The consistency in S/M ratios indicates that hyper-SN also varies with ipsilateral midbrain size.
S/M ratio was as reliable for assessing SN⫹ as hyper-SN alone. In addition, our LOPD data regarding sensitivity, specificity, PPV and NPV are about the same as that recently described by Prestel et al., who assessed 6 of 35 controls and 36 of 42 LOPD patients and defined SN > 0.2 as an abnormal extension of hyperechogenicity.15 In their study, the sensitivity is 85.7%, specificity 82.9%, positive predictive value (PPV) 85.7%, and negative predictive value (NPV): 82.9%. These findings further support the reliability and reproducibility of TCD study as a useful tool in the diagnosis of sporadic and old-onset Parkinson’s disease. We calculated a smaller hyper-SN in Asian controls (0.20 cm2, 95th percentile) than was reported in Caucasian populations. Berg and coworkers found that the 90th percentile for hyper-SN in their 330 healthy volunteers was 0.25 cm2,14 and the 75th percentile in 93 subjects over 60 years was 0.20 cm2.16 Our data might suggest that there are racial– ethnic differences in hyper-SN in Asians and Caucasians, although the values of controls should be established in individual laboratories. The present study provides a valuable reference for TCDI as an adjuvant tool for diagnosing PD in Asian populations. However, control values should be obtained in each situation, and it should be understood that differences in measuring technique might be associated with differences in sonographic results. In this study, we showed that SN⫹ in PD patients was positively correlated with age and disease duration but not with motor symptoms, disease severity, L-dopa dos-
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age, or L-dopa-related motor complications. This is in accordance with previous reports.17 However, the finding that patients with SN⫹ had longer disease duration than those without SN⫹ is not supported in the literature. A recent paper by Berg’s group revealed that the area of hyper-SN did not change at a five-year follow-up in their 27 Parkinson patients.18 This sonographic finding was thus proposed a trait marker for nigrostriatal vulnerability. The finding that PD patients with SN⫹ had longer disease duration than patients without SN may simply be due to the fact that in patients with shorter disease duration the diagnosis was not entirely clear. It is well known that, even in movement disorder specialist clinics, up to 15–18% of patients with a Parkinsonian syndrome are given the wrong diagnosis (idiopathic PD vs. atypical PD) during lifetime. Therefore, it may well be that there were subjects with atypical PD among the group without SN hyperechogenicity, who were misdiagnosed as idiopathic PD. As diagnostic accuracy increases with duration of the disease, in the group with longer lasting disease progression, SN hyperechogenicity and diagnosis of idiopathic PD may have been more concordant. However, the relationship between the change of hyper-SN area and the course of disease duration need further clarification in larger cohorts of PD with longitudinal follow up. The origin of SN hyperechogenecity is not fully understood. Animal studies19 and postmortem TCDI with histological examination2 suggest that hyperechogenicity is most likely a site of iron deposition undergoing Fenton reactions to form free radicals. The increased iron content in SN in parkinsonian brains is well established, but there are inconsistencies in reports of iron concentration and the time of its emergence in the disease.20 Our data support the notion that SN iron levels reflecting as the hyperechogenicity area increase with age. The frequency of SN⫹ in EOPD patients (57.5%) was significantly lower than in LOPD patients (92.5%; Table 2). This finding might suggest a pathogenic mechanism of nigral cell death that is not attributable to increases of iron in EOPD brains. This assumption is bolstered by cases of parkinsonism induced by MPTP21 and pesticide rotenone22 in young persons and AR-JPD. Among our EOPD patients, 28 were screened for parkin and PINK1 genes. Four patients were positive for parkin mutations on both alleles (all compound-heterozygous). Two patients had alternate allelic deletions of parkin exons 3 and 4,23 one patient had a parkin exon 2 deletion and an exon 7 G284R substitution,7 and one patient harbors a deleted exon 4 and a missense point mutation in C441R. All four patients exhibited expansion of SN⫹ bilaterally. In terms of PINK1 mutations, one
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patient had one mutated allele, and did not display SN⫹. A recent report detected increased hyper-SN in symptomatic parkin mutation carriers.24 In a few autopsied cases with parkin mutation, there was an absence of Lewy bodies10 and aggregates of ␣-synuclein in the cytoplasm that were probably promoted by iron.25 Therefore, we speculate that Lewy bodies are not a major causative factor in hyper-SN in parkin-proven cases. Further studies are necessary to elucidate the role of parkin in the generation of SN hyperechogenicity. PD patients with PINK1 mutations have a loss of presynaptic dopaminergic function,26 although no autopsy studies are yet available. PINK1, a protein kinase with an unknown substrate, appears to protect cells against mitochondrial damage.9 There are still some gaps in our knowledge about the mechanisms of PINK1-related parkinsonism. Further investigation will help clarify the change of SN echogenicity in EOPD patients with PINK1 mutations. In conclusion, our results show that TCDI is reliable for assessing SN echogenicity. Furthermore, we demonstrate the usefulness of S/M ratios for defining SN hyperechogenicity. Assessment of SN hyperechogenicity is useful for the diagnosis of PD in LOPD patients, but should be carefully evaluated in EOPD patients. REFERENCES 1. Becker G, Seufert J, Bogdahn U, Reichmann H, Reiners K. Degeneration of substantia nigra in chronic Parkinson’s disease visualized by transcranial color-coded real-time sonography. Neurology 1995;45:182-184. 2. Berg D, Roggendorf W, Schroder U, et al. Echogenicity of the substantia nigra: association with increased iron content and marker for susceptibility to nigrostriatal injury. Arch Neurol 2002; 59:999-1005. 3. Walter U, Niehaus L, Probst T, Benecke R, Meyer BU, Dressler D. Brain parenchyma sonography discriminates Parkinson’s disease and atypical parkinsonian syndromes. Neurology 2003;60:74-77. 4. Rushton JP, Rushton EW. Brain size, IQ, and racial-group differences: evidence from musculoskeletal traits. Intelligence 2003;31: 139-155. 5. Halsey JH. Effect of emitted power on wave-form intensity in transcranial doppler. Stroke 1990;21:1573-1578. 6. Schrag A, Ben Shlomo Y, Brown R, Marsden CD, Quinn N. Young-onset Parkinson’s disease revisited— clinical features, natural history, and mortality. Mov Disord 1998;13:885-894. 7. Wu RM, Bounds R, Lincoln S, et al. Parkin mutations and earlyonset parkinsonism in a Taiwanese cohort. Arch Neurol 2005;62: 82-87. 8. Kitada T, Asakawa S, Hattori N, et al. Mutations in the parkin gene cause autosomal recessive juvenile parkinsonism. Nature 1998; 392:605-608. 9. Valente EM, Abou-Sleiman PM, Caputo V, et al. Hereditary earlyonset Parkinson’s disease caused by mutations in PINK1. Science 2004;304:1158-1160. 10. Mori H, Kondo T, Yokochi M, et al. Pathologic and biochemical studies of juvenile parkinsonism linked to chromosome 6q. Neurology 1998;51:890-892. 11. Gelb DJ, Oliver E, Gilman S. Diagnostic criteria for Parkinson disease. Arch Neurol 1999;56:33-39.
SUBSTANTIA NIGRA ECHOGENICITY IN PARKINSONISM 12. Becker G, Berg D. Neuroimaging in basal ganglia disorders: perspectives for transcranial ultrasound. Mov Disord 2001;16:23-32. 13. Sohmiya M, Tanaka M, Aihara Y, Hirai S, Okamoto K. Agerelated structural changes in the human midbrain: an MR image study. Neurobiol Aging 2001;22:595-601. 14. Berg D, Becker G, Zeiler B, et al. Vulnerability of the nigrostriatal system as detected by transcranial ultrasound. Neurology 1999;53: 1026-1031. 15. Prestel J, Schweitzer KJ, Hofer A, Gasser T, Berg D. Predictive value of transcranial sonography in the diagnosis of Parkinson’s disease. Mov Disord 2006;21:1763-1765. 16. Berg D, Siefker C, Ruprecht-Dorfler P, Becker G. Relationship of substantia nigra echogenicity and motor function in elderly subjects. Neurology 2001;56:13-17. 17. Berg D, Siefker C, Becker G. Echogenicity of the substantia nigra in Parkinson’s disease and its relation to clinical findings. J Neurol 2001;248:684-689. 18. Berg D, Merz B, Reiners K, Nauman M, Becker G. Five-year follow-up study of hyperechogenicity of the substantia nigra in Parkinson’s disease. Mov Disord 2005;20:383-385. 19. Berg D, Grote C, Rausch WD, et al. Iron accumulation in the substantia nigra in rats visualized by ultrasound. Ultrasound Med Biol 1999;25:901-904.
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20. Berg D, Hochstrasser H. Iron metabolism in Parkinsonian syndromes. Mov Disord 2006;21:1299-1310. 21. Di Monte DA, Lavasani M, Manning-Bog AB. Environmental factors in Parkinson’s disease. Neurotoxicology 2002;23:487502. 22. Betarbet R, Sherer TB, MacKenzie G, Garcia-Osuna M, Panov AV, Greenamyre JT. Chronic systemic pesticide exposure reproduces features of Parkinson’s disease. Nat Neurosci 2000;3:13011306. 23. Wu RM, Shan DE, Sun CM, et al. Clinical, F-18-dopa PET, and genetic analysis of an ethnic Chinese kindred with early-onset parkinsonism and parkin gene mutations. Mov Disord 2002;17: 670-675. 24. Walter U, Klein C, Hilker R, Benecke R, Pramstaller PP, Dressler D. Brain parenchyma sonography detects preclinical parkinsonism. Mov Disord 2004;19:1445-1449. 25. Ostrerova-Golts N, Petrucelli L, Hardy J, Lee JM, Farer M, Wolozin B. The A53T ␣-synuclein mutation increases iron-dependent aggregation and toxicity. J Neurosci 2000;20:6048-6054. 26. Khan NL, Valente EM, Bentivoglio AR, et al. Clinical and subclinical dopaminergic dysfunction in PARK6-linked parkinsonism: an F-18-dopa PET study. Ann Neurol 2002;52:849-853.
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