Echogenicity of the Substantia Nigra: Association with Increased Iron

ORIGINAL CONTRIBUTION Echogenicity of the Substantia Nigra Association With Increased Iron Content and Marker for Susceptibility to Nigrostriatal Injury

Daniela Berg, MD; Wolfgang Roggendorf, MD; Ute Schro¨der; Ru¨diger Klein, MD; Thomas Tatschner, MD; Peter Benz, MD; Oliver Tucha, PhD; Michael Preier, MSc; Klaus W. Lange, MD; Karlheinz Reiners, MD; Manfred Gerlach, PhD; Georg Becker, MD

Background: Patients with Parkinson disease charac- Results: Healthy subjects with SN hyperechogenicity ex- teristically exhibit an increased echogenicity of the sub- hibited a significant reduction of the [18F]-dopa uptake, stantia nigra (SN) on transcranial sonography, a new neu- especially in the putamen (Wilcoxon matched pair test: roimaging technique. The same echo feature of the SN left side, P=.006; right side, P=.009), whereas their neu- can be identified in 9% of healthy adults. ropsychological and motor performance were normal. Postmortem studies showed that the echogenicity of the Objective: To evaluate the relevance of the echogenic SN correlated with its iron content. SN in healthy adults. Conclusions: Increased echogenicity of the SN, char- Design: In the first part of the study, 10 healthy subjects acteristically seen in Parkinson disease, is related to a func- younger than 40 years with a distinct SN hyperecho- tional impairment of the nigrostriatal system (even in genicity underwent extensive neurological, motor, neu- young healthy adults) that can be revealed by [18F]- ropsychological, and fluorine 18-dopa positron emission dopa PET studies. Substantia nigra hyperechogenicity is tomographic ([18F]-dopa PET) examinations. Results were related to a higher tissue iron level, which is known to compared with those of 10 subjects with a low echogenic enhance the cells’ generation of reactive oxygen speci- SN. In the second part of the study, the postmortem brains mens. Therefore, we hypothesize that transcranial sonog- of 20 patients without extrapyramidal disorders during their raphy may identify a susceptibility marker for the devel- lifetime were sonographically examined with a particular opment of nigral injury that can be detected early in life, focus on SN echogenicity. Subsequently, one half of the prior to the onset of Parkinson disease. brain was prepared for heavy metal analysis, the other for a histological examination. Arch Neurol. 2002;59:999-1005

URRENT treatment of Par- Recent studies have shown that trans- kinson disease (PD) is cranial sonography (TCS) may identify an From the Department of basically symptomatic. important susceptibility marker for nigral Neurology (Drs Berg and So far, no neuroprotec- injury.3 This is a new neuroimaging tech- Reiners), Division of tive strategy has proved nique that provides a 2-dimensional sono- Neuropathology to be effective in the clinical setting, graphic depiction of the brain parenchyma C 4 (Drs Roggendorf and Klein), although concepts and results of animal through the intact skull. In patients with Institute for Forensic Medicine experiments have been promising.1 One PD, an increase of echogenicity in the SN can (Dr Tatschner), and important reason for the failure of neuro- be depicted using TCS even though results Department of Psychiatry (Dr Gerlach), Bayerische protection may be the fact that nigro- of computed tomographic scans and mag- Julius-Maximilians-Universita¨t striatal degeneration has advanced to a neticresonanceimagingareknowntobenor- 5 Wu¨rzburg, Wu¨rzburg, 60% to 70% reduction of neurons in the mal in these patients. The echo pattern of Germany, the Department of substantia nigra (SN) at the time when the SN found in PD can be identified in ap- Neurology, University of the first clinical symptoms of PD are proximately9%ofhealthyadults.3,5 Toevalu- Homburg, Saar, Germany noticed by the patient.2 It is possible that ate the relevance of this characteristic sono- (Dr Becker and Mrs Schro¨der), at this stage of the disease, drugs with a graphic feature, healthy subjects with SN hy- the Department of potential neuroprotective effect are no perechogenicityunderwentfluorine18-dopa Neuropsychology, University of longer capable of arresting or slowing the positron emission tomography ([18F]-dopa Regensburg, Regensburg, Germany (Drs Tucha and course of degeneration. However, the PET). In this pilot study, subjects with SN Lange and Mr Preier), and speed of neurodegeneration might be hyperechogenicity exhibited a significantly 18 PET-Zentrum Mainz, reduced by the commencement of neuro- reduced [ F]-dopa uptake of the striatum, University of Mainz, Mainz, protective treatment in an earlier (pre- indicating a subclinical impairment of the Germany (Dr Benz). clinical) stage of PD. dopaminergic nigral neurons.3

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©2002 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/26/2021 SUBJECTS AND METHODS (echogenicity) is not quantifiable using ultrasound, the area of hyperechogenic signals in the SN region was encircled and [18F]-DOPA PET STUDIES OF HEALTHY measured. All individuals were assessed independently by VOLUNTEERS WITH A HYPERECHOGENIC SN 2 experienced TCS investigators. The average values of both measurements of each side were used for further analysis. We identified 10 healthy subjects younger than 40 years The TCS examiners were blinded to the results of the PET with a distinct increase in SN echogenicity (mean±SD age, study and the neuropsychological assessments. 26.7±7.6 years; 3 women and 7 men, all right-handed). Iden- Positron emission tomographic scans were per- tification and selection of subjects were performed as de- formed on an ECAT EXACT 47 tomograph (Siemens, Er- scribed previously.3 Subjects were selected from a group langen, Germany), which provided 47 simultaneous planes of 120 members and students of the University of Wu¨ r- with a physical slice thickness of 3.7 mm in a field of view zburg (Wu¨ rzburg, Germany). Subjects with a hyperecho- of 16.2 cm. The thickness of the reoriented slice used in genic SN with an echogenic signal extension of more than the analysis was 6.07 mm. The axial resolution (full with 0.25 cm2 on at least 1 side (prevalence, 9%) and no central half-maximum) was less than 6 mm, and the in-plane reso- nervous system disorders were included in this study. Eleven lution was less than 5.5 mm. Standard techniques as de- subjects fulfilled these criteria, and 10 of them gave their scribed previously were used for the [18F]-dopa PET scans.3 informed consent according to the Declaration of Hel- Activity ratios were calculated as the ratio of the specific sinki11 to participate in further examinations, including a [18F]-dopa uptake in the caudate nucleus and putamen di- thorough neurological examination, comprehensive mo- vided by the mean uptake in the occipital cortex. In addi- tor and neuropsychological testing, and an [18F]-dopa PET tion, influx constants (Ki) were calculated from the right examination. For comparison, we selected 10 healthy con- and left caudate and putamen by using a modified com- trols (3 women, 7 men; mean±SD age, 40±15.5 years) with partment model approach with an occipital nonspecific tis- areas of low SN echogenicity (area of hyperechogenic sig- sue rather than a plasma input function.3 nal of the SN Ͻ 0.2 cm2) matched for sex and IQ who had already undergone the [18F]-dopa PET protocol in the same Motor and Cognitive Examinations nuclear medicine center at the PET Center of the Univer- sity of Mainz (Mainz, Germany) in the last 12 months. The Motor function was assessed using a pegboard examina- selection of the control group was performed according to tion and a series of foot- and finger-tapping tests for a pe- the recommendation of the ethics committee of the Uni- riod of 32 seconds.3,12 versity of Wu¨ rzburg. Following the proposal, we accepted In the neuropsychological assessment, standardized an older age of control subjects because the age-related de- psychometric test procedures were used to measure cline in [18F]-dopa uptake would lessen the differences memory (Digit Span Forward and Digit Span Backward of among a group of subjects with nigral injury and the con- the Wechsler Memory Scale–Revised),13 attention (Alert- trol group. Control subjects also had an [18F]-dopa PET ness and Divided Attention task of the Computerized study, a sonographic examination, and a motor and neu- Neuropsychological Assessment of Attention Deficits),14 ropsychological test battery. and executive functions (S Word Test, H/T Word Test, and the Tower of London Test).13,14 The examiner assess- Neuroimaging ing motor and cognitive performance was unaware of the TCS and PET results. For the TCS examination, we used a color-coded phased- array ultrasound system equipped with a 2.5-MHz trans- ANALYSIS OF THE POSTMORTEM SN AND ducer. The examination was performed through the pre- RELATION OF SN ECHOGENICITY TO auricular acoustic bone window using standard techniques BIOCHEMICAL AND HISTOLOGICAL PARAMETERS as described previously,3-5 with the aim of identifying the SN as clearly as possible within the hypoechogenic mes- In the second part of the study, we compared the echo pat- encephalic brainstem. Because the signal brightness tern of the SN with histological and neurochemical

The cause of the increase in SN echogenicity is un- whether the association between increased SN echo- clear. The similarity in echo pattern of the SN between genicity and nigral neural impairment could be identi- patients with PD and subjects with a subclinical impair- fied in this age group. In the second part, reasons for the ment of the nigrostriatal system has led to the hypoth- increase in tissue echogenicity of the SN were investi- esis that one of the pathological or biochemical hall- gated by studying postmortem brains and correlating ul- marks of PD may also be a causative factor in the increased trasonographic and neurochemical findings. echogenicity seen in healthy volunteers. An increase in 6-8 9 tissue levels of iron and gliosis are 2 of several pro- RESULTS cesses that occur within the SN in the course of PD that may also affect the echogenicity of the tissue.10 [18F]-DOPA PET STUDIES OF HEALTHY Our study was launched to clarify the relation be- VOLUNTEERS WITH AN INCREASED tween increased SN echogenicity and susceptibility to ni- AREA OF SN HYPERECHOGENICITY gral cell injury. In the first part of the study, we assessed nigral function using [18F]-dopa PET in young healthy In the 10 healthy subjects with SN hyperechogenicity, subjects with and without SN hyperechogenicity to test the median extension of hyperechogenic signals was 0.31

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©2002 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/26/2021 findings to identify the cause of the increased echo- hours. Diluted samples were measured with a polarized Ze- genicity. We examined the postmortem brains of 20 pa- man atomic absorption spectrophotometer (Z-8100; Hita- tients (6 women and 14 men; median age, 51 years; range, chi, Tokyo, Japan) according to standard procedures. Con- 38-59 years). None of the patients had experienced any ex- centrations were calculated from external and internal trapyramidal disorder (including PD) prior to death, and standards in the following ranges: 0.5 to 4 mg/mL (iron), no subject had died of a neurological disorder. One pa- 12 to 100 ng/mL (copper), 1.25 to 10 ng/mL (manga- tient had a history of epileptic seizures, and another had a nese), 0.25 to 2 mg/mL (calcium), and 1.25 to 10 ng/mL history of meningoencephalitis. The average period be- (zinc). tween death and autopsy was 48 hours (range, 6-144 hours). After autopsy, an ultrasound examination was per- Histopathological Analysis formed with the unfixed brains immersed in a vessel filled with isotonic sodium chloride. For this examination we used Dissected areas included the neocortex (Brodmann areas the same ultrasound system as for the clinical investiga- 5, 8, 9, 17, 18, 19, 21, 22, and 40), limbic areas (Brod- tions, with a 5-MHz transducer held under the surface of mann area 24 and the hippocampus), striatum, thalamus, the fluid. For ultrasound system indexes, we chose a pen- brainstem (SN and pons with the locus coeruleus and me- etration depth of 4 to 8 cm and a dynamic range of 50 dB dulla), and cerebellum. All paraffin sections were stained with high persistence. Image brightness and time gain com- with hematoxylin-eosin. Sections of the basal ganglia and pensation were adapted as needed for each investigation. SN were stained for iron using the Prussian blue stain. Ad- The brain was placed upside down in the vessel to achieve ditionally, selected areas were silver impregnated using the an optimal picture of the SN. The whole brain was scanned Bielschowsky method and stained for Luxol fast blue. in coronal and axial planes. Attention was focused on the Immunohistochemistry was performed in selected cor- SN region, where hyperechogenic areas were encircled and tical areas (Brodmann areas 9, 21, 22, 24, and 40), the SN, measured. The average area of hyperechogenic signals of the locus coeruleus, and the hippocampus according to a both SNs was related to the neurochemical and histopatho- standard protocol (peroxidase-antiperoxidase method). logical findings. These specimens were incubated with phosphorylation- After the ultrasound examination, all brains were di- dependent anti-␶ antibodies (AT8; dilution, 1:800; Immu- vided midsagitally. The left half was stored at −80°C; the nogenetics, Gent, Belgium),15 ubiquitin antibodies (dilu- other half was fixed for 10 to 14 days in a solution of 4% tion, 1:200; DAKO, Glostrup, Denmark), and ␣-synuclein paraformaldehyde. antibodies (dilution, 1:2000; Chemicon, Temecula, Calif).16 The quality of immunohistochemistry was checked by us- Preparation of Tissue Samples ing control specimens from patients with Lewy body de- for Trace Metal Analysis mentia (for ␣-synuclein antibodies), Alzheimer disease (AT8 and ubiquitin antibodies), and PD. Dissection of the hemisphere was performed when the tem- Biochemical and histological parameters were ob- perature of the brain had risen to −10oC. The brain was coro- tained by investigators blinded to the other results and to nally sectioned into slices approximately 10 mm thick. The those of the ultrasound examination. SN was identified anatomically and dissected. Brain tissue was stored in plastic vessels. STATISTICAL ANALYSIS

Assessment of Total Iron, Copper, Manganese, Descriptive statistics are given as the median with lower Zinc, and Calcium Levels in SN Tissue and upper quartiles (25th and 75th percentiles). Inter- group comparison was performed using the U test, Wil- Brain specimens of approximately 50 mg were weighed and coxon matched pair test, and Kruskal-Wallis test; correla- dried for 2 hours at 105°C. Nitric acid (250 mL, supra- tion analysis was done using the Spearman rank correlation pure) was added, and the mixture was incubated over- and multiple regression analysis. Differences were as- night. The mixture was completely digested at 56°C for 2 sumed to be significant at PϽ.05.

cm2 (range, 0.28-0.35 cm2) for the right SN and 0.31 cm2 dividuals with SN hyperechogenicity compared with the (range, 0.28-0.34 cm2) for the left SN (Figure 1). The controls (Table and Figure 2), although controls were control group had a median echogenic SN area of 0.11 older and were therefore supposed to have a lower [18F]- cm2 (range, 0.08-0.12 cm2) on the right side and 0.14 cm2 dopa uptake because of age-related nigral cell loss. In ad- (range, 0.09-0.15 cm2) on the left side. Differences were dition, the median putamen and caudate Ki values were highly significant (Wilcoxon test: Z=2.8; P=.005). Dif- lower in individuals with enlarged areas of SN hyper- ferences in SN areas between the left and right sides were echogenicity than in controls (Table 1). Differences were not significant in the group of controls (Wilcoxon test: significant for the putamen on both sides (Wilcoxon test: Z=1.4; P=.16) or in the group of subjects with SN hy- left side, P=.04; right side, P=.03) but not for the cau- perechogenicity (Wilcoxon test: Z=1.93; P=.06). date nucleus.

PET Findings Motor and Cognitive Examinations

The [18F]-dopa activity ratios for the putamen and the No significant differences between controls and indi- right caudate nucleus were significantly reduced in in- viduals with more extended areas of SN hyperecho-

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©2002 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/26/2021 probe results in a higher spatial resolution and, conse- A quently, slightly larger hyperechogenic SN areas compared with the transcranial findings. Analysis of the trace metal and calcium tissue levels of the SN with the extent of echogenic signals of the SN showed a positive correlation for iron (Spearman rank correlation: R=0.57; P=.007). Subjects with more extended hyperechogenic signals at the SN had higher iron levels than those with less echogenic SNs (Figure 3). No correlation was found for copper 1 (R=0.34; P=.13), magnesium (R=0.31; P=.18), zinc (R=0.28; P=.21), or calcium (R=0.43; P=.06). Histological examination of the SN revealed increased iron staining in subjects with higher iron levels and larger areas of SN hyperechogenicity (Figure 4A and B). In one subject (age 51 years) with a large hyperechogenic SN area (0.3 cm2) and a high SN iron level (752 ppm), histological analysis revealed Lewy bodies in the locus coeruleus (Figure 4C) and SN. Although ␣ B -synuclein and ubiquitin immunostaining showed several Lewy bodies in the SN (Figure 4D and 4E), they were even more numerous in the locus coeruleus. Evidence of active cell loss was seen including the presence of ex- traneuronal melanin and a slight gliosis. Results of the AT8 immunostaining were normal. No statement regarding a selective reduction of pigmented neurons in the SN could be made because no quantitative morphometric 1 evaluation of the SN was performed. This patient did not have PD or any symptoms attributed to diffuse Lewy body disease17 during his lifetime; therefore, a case of incidental Lewy body disease might be postulated. In no other patient were we able to identify ␣-synuclein-positive Lewy bodies in the SN or in any other brain area. In 3 cases, occasional ␶-positive nerve cells and neurites in the SN and locus coeruleus were seen. In an additional subject, numerous senile plaques were identified in cortical ar- Figure 1. A, Mesencephalic brainstem with normal findings on transcranial eas and the hippocampus region. The other cases re- sonography (axial scanning plane). Number 1 indicates the butterfly-shaped vealed no major gross or histological changes. mesencephalic brainstem surrounded by the hyperechogenic basal cisterns; arrow, the echogenic border zone of the brainstem raphe; and asterisk, the aqueduct. B, Subject with hyperechogenic substantia nigra on the ipsilateral COMMENT (arrowheads) and contralateral (encircled) side, depicted within the peduncles of the mesencephalic brainstem (1). Arrow indicates the Increased echogenicity of the SN is the characteristic ul- brainstem raphe; asterisk, the aqueduct. trasound feature of PD. Our findings indicate that the same echo pattern of the SN detected in young healthy sub- genicity were detected on pegboard and finger-tapping jects may be associated with an impairment of the ni- tests (Wilcoxon test: PϾ.05) or regarding cognitive func- grostriatal system as revealed by PET. Because the stria- tioning, problem solving, verbal memory, or attention tal [18F]-dopa uptake is related to the number and (Wilcoxon test: PϾ.05). metabolism of the nigral dopaminergic neurons,18 a reduced [18F]-dopa tracer uptake in subjects with SN hy- POSTMORTEM ANALYSIS OF THE SN AND perechogenicity reflects nigral cell damage or a preex- CORRELATION OF SN ECHOGENICITY WITH isting reduced cell count. These findings corroborate the TRACE METAL CONTENT AND RELATION data from our pilot study.3 However, subjects included TO HISTOLOGICAL PARAMETERS in the present study were even younger. Both the echogenic SN sonographic phenotype and nigral dysfunc- Sonographic examination of the SN in the 20 postmor- tion can therefore be depicted at a young age. Despite tem brains revealed a median extension of the hyperecho- the reduction in striatal [18F]-dopa uptake, results of neu- genic signals originating from the SN of 0.18 cm2 (range, rological and neuropsychological examinations of the 0.11-0.25 cm2; left side, 0.17 cm2 [range, 0.11-0.23 cm2]; young subjects in the present study were normal. right side, 0.18 cm2 [range, 0.11-0.26 cm2]). In 3 brains Our data do not indicate that subjects with SN hy- the extension of hyperechogenic signals was much higher perechogenicity and reduced [18F]-dopa uptake have pre- than the threshold level of 0.25 cm2 (range, 0.30-0.32 cm2); clinical PD because it is unknown whether nigral injury in another 3 brains it was close to this threshold (range, will continue in some of these subjects in the future. How- 0.24-0.27 cm2). The higher frequency of the ultrasound ever, it may be tempting to consider SN hyperecho-

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©2002 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/26/2021 [18F]-Dopa Ratios and Ki Values for the Caudate Nucleus and Putamen of Individuals With a Hyperechogenic Substantia Nigra Area Greater Than 0.25 cm2 and Control Individuals*

Left Side Right Side

SN+ CO Z ValuesP SN+ CO Z Values P [18F]-dopa uptake, caudate 2.14 2.30 2.29 .02 2.12 2.31 2.80 .005 (2.03; 2.20) (2.20; 2.38) (2.07; 2.14) (2.27; 2.38) [18F]-dopa uptake, putamen 2.10 2.36 2.7 .006 2.11 2.38 2.6 .009 (2.04; 2.14) (2.25; 2.46) (2.06; 2.15) (2.28; 2.45) Ki values, caudate 0.010 0.010 1.47 .13 0.010 0.010 0.56 .57 (0.007; .010) (.008; 0.012) (0.008; 0.011) (0.008; 0.011) Ki values, putamen 0.008 0.009 1.98 .04 0.008 0.009 2.08 .03 (0.007; 0.009) (0.009; 0.010) (0.007; 0.010) (.008; .010)

*All values are expressed as medians (25th percentile; 75th percentile). Comparison between both groups using the Wilcoxon matched pair test (Z values and P values) is given for each side separately. Significant differences are highlighted in boldface. [18F]-dopa indicates fluorine 18-dopa; Ki, influx constant; SN+, a hyperchogenic substantia nigra area greater than 0.25 cm2; and CO, control individuals.

2.8 1100

900 2.6

700 2.4 500 2.2

Iron Level, ppm 300

F]-Dopa Uptake Ratios 2.0 100 18 [

1.8 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 Area of SN, cm2 1.6 CO SN + CO SN + CO SN + CO SN + Figure 3. Correlation between substantia nigra (SN) iron level (given as Caudate (Right) Caudate (Left) Putamen (Right) Putamen (Left) micrograms per gram of dry-weight SN tissue) and area of hyperechogenic signals originating from the SN in 20 postmortem brains. The figure Figure 2. Fluorine 18-dopa ([18F]-dopa) activity ratios as measured by represents a regression analysis and demonstrates that SN positron emission tomography in the caudate nucleus and putamen of hyperechogenicity is related to increased iron level (regression analysis: subjects with a hyperechogenic substantia nigra area greater than 0.25 cm2 R=0.57; F=8.59; P=.008). The patient with an intense iron stain and Lewy (SN+) and control subjects (CO). Median values (25th percentile and 75th bodies in the SN is marked with an asterisk. percentile) are also presented. The Wilcoxon matched pair test showed a significant difference between CO and SN+ groups for both the caudate nucleus and putamen on either side. genicity. Other brain structures with physiologically high amounts of iron, such as the globus pallidus or red genicity in healthy adults to represent a susceptibility nucleus, usually appear as unechogenic or slightly echo- marker for nigral injury because of the similarity of the genic structures on TCS.4 Furthermore, the physiologi- echo feature compared with PD and the association with cally iron-rich SN normally reflects only a slightly echo- an altered nigral function disclosed by [18F]-dopa PET genic signal. Therefore, factors such as iron-binding studies. proteins may be important in creating increased SN echo- Increased echogenicity of the SN may be associ- genicity. ated with higher tissue iron levels, as seen in the post- Additionally, one postmortem study demonstrated mortem examination. The finding of a correlation be- an increased SN echogenicity, high SN iron levels, and tween tissue iron content and echogenicity is supported Lewy bodies in the SN and locus coeruleus, classifying by an animal experiment revealing a dose-dependent in- this case as one of incidental Lewy body disease.23 This crease in SN echogenicity after the stereotactic injection single-case observation of an association among SN hy- of various concentrations of iron into the SN.10 In- perechogenicity, iron accumulation, and Lewy body for- creased iron content may explain not only the increase mation must be replicated and confirmed in a larger in echogenicity but also nigral cell injury as demon- series, particularly because it contrasts the findings of strated by PET findings.19 From in vitro and in vivo ex- Dexter et al,24 who reported normal SN iron levels in pa- periments, it is known that iron may catalyze free radi- tients with incidental Lewy body disease. cal formation, leading to an impairment of cellular The prevalence rate of subjects with SN hyperecho- function by a cascade of events including damage to the genicity is similar to that of incidental Lewy body dis- mitochondrial electron transport system, the induction ease,25,26 which is suggested to be the preclinical form of of proteases, and increased membrane lipid peroxida- PD disclosed only by histopathological examinations.27 tion.6-8,20-22 However, increased iron content alone can- However, the prevalence rate of 9% for the phenotype not be the reason for the observed increased SN echo- of SN hyperechogenicity is much greater than the esti-

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C D

Figure 4. A, Substantia nigra with moderate iron deposits (arrows) (Prussian blue stain, original magnification ϫ200). B, Substantia nigra with marked increase of iron, either diffuse or globular (Prussian blue stain, original magnification ϫ 200). C, Locus coeruleus with occasional Lewy bodies (arrow) and numerous Lewy neurites. In addition, a mild gliosis and free neuromelanin can be seen (␣-synuclein antibodies [peroxidase antiperoxidase method], original magnification ϫ250). D and E, Substantia nigra with intracellular Lewy bodies (D, Nissl stain with cresyl violet, original magnification ϫ400. E, ␣-Synuclein antibodies, original magnification ϫ450).

mated risk of PD.28 Therefore, SN hyperechogenicity, Accepted for publication August 21, 2001. which may be related to increased iron content, might This study was supported by the Deutsche For- be only 1 of several factors playing a role in the initia- schungsgemeinschaft BE 1774/4-1 and Siemens AG, Erlan- tion of neuronal loss in the SN. Other factors such as en- gen, Germany; Orion Pharma GmbH, Hamburg, Ger- dotoxins or exotoxins29,30 are probably necessary to in- many; Schering Deutschland GmbH, Berlin, Germany; and duce or accelerate the degenerative process leading to SmithKline Beecham, Munich, Germany. clinical PD. According to the differences in the preva- We gratefully acknowledge K. V. Toyka, MD, and P. lence rates, it is clear that if at all, only a minority of sub- Riederer, PhD, for their continuous support, and W.-D. jects with SN hyperechogenicity will proceed to PD. How- Rausch, PhD, for providing the trace metal analyses. ever, recent findings provide evidence of an association Corresponding author and reprints: Daniela Berg, MD, between SN hyperechogenicity and motor impairment Department of Neurology, University of Wu¨rzburg, Josef- in elderly patients, indicating that the factors causing this Schneider-Str 11, 97080 Wu¨ rzburg, Germany (e-mail: echo feature may gain functional relevance during a pa- [email protected]). tient’s lifetime. In a cross-sectional study including 93 subjects older than 60 years without prediagnosed ex- REFERENCES trapyramidal disorders, subjects with more extended hyperechogenic signals at the SN showed a more severe slow- 1. Langston JW, Irwin I, Langston EB, Forno LS. Pargyline prevents MPTP- ing of motor function even though most of them did not induced parkinsonism in primates. Science. 1984;225:1480-1482. 31 fulfill the diagnostic criteria for PD. 2. Bernheimer H, Birkmayer W, Hornykiewicz O, Jellinger K, Seitelberger F. Brain Our findings suggest that an increase in SN echo- dopamine and the syndromes of Parkinson and Huntington: clinical, morpho- genicity, which appears to be related to an increase in logical and neurochemical correlations. J Neurol Sci. 1973;20:415-455. 3. Berg D, Becker G, Zeiler B, et al. Vulnerability of the nigrostriatal system as de- tissue iron content, may point toward a susceptibility to tected by transcranial ultrasound. Neurology. 1999;53:1026-1031. nigrostriatal injury. This pattern of SN hyperecho- 4. Bogdahn U, Becker G, Schlachetzki, eds. Echoenhancers and Transcranial Color genicity, similar to that found in PD, can be detected early Duplex Sonography. Berlin, Germany: Blackwell Science; 1998. in life. To further elucidate the significance of this find- 5. Becker G, Seufert J, Bogdahn U, Reichmann H, Reiners K. Degeneration of sub- ing and the relationship between SN hyperechogenicity stantia nigra in chronic Parkinson’s disease visualized by transcranial color- coded real-time sonography. Neurology. 1995;45:182-184. and PD, follow-up studies with PET should clarify whether 6. Sofic E, Riederer P, Heinsen H, et al. Increased iron (III) and total iron content in the functional impairment of the nigrostriatal system will post mortem substantia nigra of parkinsonian brain. J Neural Transm. 1988;74: progress in any of our subjects. 199-205.

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©2002 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/26/2021 7. Dexter DT, Sian J, Jenner P, Marsden CD. Implications of alterations in trace el- 20. Jenner P, Dexter DT, Sian J, Schapira AHV, Marsden CD. Oxidative stress as a ement levels in brain in Parkinson’s disease and other neurological disorders cause of nigral cell death in Parkinson’s disease and incidental Lewy body dis- affecting the basal ganglia. Adv Neurol. 1993;60:273-281. ease. Ann Neurol. 1992;32:S82-S87. 8. Gerlach M, Ben-Shachar D, Riederer P, Youdim MBH. Altered brain metabolism 21. Youdim MBH, Ben-Shachar D, Eshel G, Finberg JPM, Riederer P. The neurotox- of iron as a cause of neurodegenerative diseases? J Neurochem. 1994;63:793- icity of iron and nitric oxide: relevance to the etiology of Parkinson’s disease. 807. Adv Neurol. 1993;60:259-266. 9. McGeer PL, Itagaki S, Akiyama H, McGeer EG. Rate of cell death in parkinson- 22. Gerlach M, Double K, Riederer P, et al. Iron in the Parkinsonian substantia nigra. ism indicates active neuropathological process. Ann Neurol. 1988;24:574-576. Mov Disord. 1997;12:258-260. 10. Berg D, Grote C, Rausch W-D, et al. Iron accumulation of the substantia nigra in 23. Gibb WR, Lees AJ. The relevance of the Lewy body to the pathogenesis of rats visualized by ultrasound. Ultrasound Med Biol. 1999,25:901-904. idiopathic Parkinson’s disease. J Neurol Neurosurg Psychiatry. 1988;51:745- 11. International Committee of Medical Journal Editors. Statements from the Van- 752. couver Group. BMJ. 1991;302:1194. 24. Dexter DT, Sian J, Rose S, et al. Indices of oxidative stress and mitochondrial 12. Vingerhoets FJ, Schulzer M, Calne DB, Snow BJ. Which clinical sign of Parkin- function in individuals with incidental Lewy body disease. Ann Neurol. 1994;35: son’s disease best reflects the nigrostriatal lesion? Ann Neurol. 1997;41:58-64. 38-44. 13. Lezak MD. Neuropsychological Assessment. 3rd ed. New York, NY: Oxford Uni- 25. Fearnley JM, Lees A. Ageing and Parkinson’s disease: substantia nigra regional versity Press; 1995. selectivity. Brain. 1991;114:2283-2301. 14. Zimmermann P, Fimm B. A Computerized Neuropsychological Assessment of 26. Fearnley J, Lees A. Pathology of Parkinson’s disease. In: Calne DB, ed. Neuro- Attention Deficits. Freiburg, Germany: Institute of Psychology, University of degenerative Diseases. Philadelphia, Pa: WB Saunders Co; 1994:545-554. Freiburg; 1989. 27. Forno LS. Concentric hyalin intraneuronal inclusions of Lewy type in the brains 15. Goedert M, Jakes R, Vanmechelen E. Monoclonal antibody AT8 recognizes tau of elderly persons (50 incidental cases): relationship to parkinsonism. J Am Geri- protein phosphorylated at both serine 202 and threonine 205. Neurosci Lett. 1995; atr Soc. 1969;17:557-575. 189:167-170. 28. Golbe LI. The genetics of Parkinson’s disease: a reconsideration. Neurology. 1990; 16. Spillantini MG, Schmidt ML, Lee VM, Trojanowski JQ. ␣-Synuclein and Lewy bod- 40(suppl l3):7-14. ies. Nature. 1997;388:839-840. 29. Iwin I, Langston JW. Endogenous toxins as potential etiologic agents in Parkin- 17. McKeith IG, Galasko D, Kosoka K, et al. Consensus guidelines for the clinical and son’s disease. In: Ellenberg JH, Koller WC, Langston JW, eds. Etiology of Par- pathologic diagnosis of dementia with Lewy bodies (DLB): report of the consor- kinson’s Disease. New York, NY: Marcel Dekker Inc; 1995:319-366. tium on DLB international workshop. Neurology. 1996;47:1113-1124. 30. Spencer PS, Butterfield PG. Environmental agents and Parkinson’s disease. In: 18. Vingerhoets FJ, Snow BJ, Lee CS, Schulzer M, Mak E, Calne DB. Longitudinal Ellenberg JH, Koller WC, Langston JW, eds. Etiology of Parkinson’s Disease. New fluorodopa positron emission tomographic studies of the evolution of idio- York, NY: Marcel Dekker Inc; 1995:319-366. pathic parkinsonism. Ann Neurol. 1994;36:759-764. 31. Berg D, Siefker C, Ruprecht-Do¨rfler P, Becker G. Relationship of substantia ni- 19. Dunnett SB, Bjo¨klund A. Prospects for new restorative and neuroprotective treat- gra echogenicity and motor function in elderly subjects. Neurology. 2001;56: ments in Parkinson’s disease. Nature. 1999;339(suppl):A32-A39. 13-17.

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