Meta-analysis of brain iron levels of Parkinson’s disease patients determined by postmortem and MRI measurements

Abstract

Brain iron levels in patients of Parkinson’s disease (PD) are usually measured in postmortem samples or by MRI imaging including R2* and SWI. In this study we performed a meta-analysis to understand PD-associated iron changes in various brain regions, and to evaluate the accuracy of MRI detections comparing with postmortem results. Databases including Medline, Web of Science, CENTRAL and Embase were searched up to 19th November 2015. Ten brain regions were identified for analysis based on data extracted from thirty-three-articles. An increase in iron levels in substantia nigra of PD patients by postmortem, R2* or SWI measurements was observed. The postmortem and SWI measurements also suggested significant iron accumulation in putamen. Increased iron deposition was found in red nucleus as determined by both R2* and SWI, whereas no data were available in postmortem samples. Based on SWI, iron levels were increased significantly in the nucleus caudatus and globus pallidus. Of note, the analysis might be biased towards advanced disease and that the precise stage at which regions become involved could not be ascertained. Our analysis provides an overview of iron deposition in multiple brain regions of PD patients, and a comparison of outcomes from different methods detecting levels of iron.

Full text

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Scientific RepoRts | 6:36669 | DOI: 10.1038/srep36669
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Meta-analysis of brain iron levels
of Parkinson’s disease patients
determined by postmortem and
MRI measurements
Jian-Yong Wang1,2,*, Qing-Qing Zhuang1,*, Lan-Bing Zhu1, Hui Zhu1, Ting Li1, Rui Li2,
Song-Fang Chen1, Chen-Ping Huang2, Xiong Zhang1 & Jian-Hong Zhu2,3
Brain iron levels in patients of Parkinson’s disease (PD) are usually measured in postmortem samples
or by MRI imaging including R2* and SWI. In this study we performed a meta-analysis to understand
PD-associated iron changes in various brain regions, and to evaluate the accuracy of MRI detections
comparing with postmortem results. Databases including Medline, Web of Science, CENTRAL and
Embase were searched up to 19th November 2015. Ten brain regions were identied for analysis based
on data extracted from thirty-three-articles. An increase in iron levels in substantia nigra of PD patients
by postmortem, R2* or SWI measurements was observed. The postmortem and SWI measurements
also suggested signicant iron accumulation in putamen. Increased iron deposition was found in red
nucleus as determined by both R2* and SWI, whereas no data were available in postmortem samples.
Based on SWI, iron levels were increased signicantly in the nucleus caudatus and globus pallidus.
Of note, the analysis might be biased towards advanced disease and that the precise stage at which
regions become involved could not be ascertained. Our analysis provides an overview of iron deposition
in multiple brain regions of PD patients, and a comparison of outcomes from dierent methods
detecting levels of iron.
Iron overload has been implicated in the pathology and pathogenesis of Parkinson’s disease (PD). e substan-
tia nigra, where the selective loss of dopaminergic neurons occurs, is the primary region in the brain known to
deposit iron. Additionally, aberrant iron concentrations have been observed in other brain regions such as red
nuclei, globus pallidus and cortex of PD patients, despite of unknown pathology1–3. Spectroscopic analyses of
postmortem brains display an increased iron levels in the substantia nigra, which has been suggested to correlate
with the severity of PD2,4. In recent decades, advancements in imaging techniques, such as magnetic resonance
imaging (MRI), have contributed to an enhanced understanding of the pathological progression and clinical diag-
nosis of PD. Consequently, iron load may be estimated in a non-invasive manner using R2/R2* relaxometry (with
better results obtained using R2* 5–7) and, more recently, susceptibility-weighted imaging (SWI). Nonetheless,
while largely consistent and reproducible results can be obtained in many experiments these techniques are not
yet fully validated8.
In this study, we extracted results of iron analyses employing postmortem brains and R2* and SWI methods
from the literature, and performed a systematical meta-analysis aiming to 1) conrm the iron overload observa-
tion in the substantia nigra, 2) explore other regions of the brain carrying dierent levels of iron, and 3) evaluate
to what extent these two MRI methods correlate with the measurements of postmortem brains. Meanwhile, as
detailed in the discussion section, several limitations are disclosed in an attempt to fully understand the scope of
this meta-analysis, such that the disease severity was not dierentiated due to insucient information during data
extraction that may aect outcomes of MRI imaging.
1Department of Neurology, the Second Aliated Hospital, Wenzhou Medical University, Wenzhou, Zhejiang 325000,
China. 2Department of Preventive Medicine, Wenzhou Medical University, Wenzhou, Zhejiang 325035, China. 3Key
Laboratory of Watershed Science and Health of Zhejiang Province, Wenzhou Medical University, Wenzhou, Zhejiang
325035, China. *These authors contributed equally to this work. Correspondence and requests for materials should
be addressed to C.P.H. (email: hcp@wmu.edu.cn) or X.Z. (email: zhangxiong98@gmail.com) or J.H.Z. (email:
jhzhu@wmu.edu.cn)
received: 29 February 2016
accepted: 19 October 2016
Published: 09 November 2016
OPEN

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Results
Search Results. e initial search using the keywords as described in the method section returned a total of
4252 articles (Fig.1). A subsequent screening of the titles and abstracts reduced the number to 257. Following an
exhaustive examination of the contents, 224 articles were excluded according to the selection criteria detailed in
the method section. Of the 33 articles being selected that report iron content (summarized in Table1), 11 of them
employed postmortem analyses2,4,9–17, 14 were measured by R2* 3,18–30 and 8 by MRI relaxometry SWI31–38. e
disease comorbidity and diagnostic performance of the cohorts of these 33 studies are summarized in Table S1.
Quality Assessment. Quality assessment by Newcastle-Ottawa Scale suggested four-stars or above out of a
maximum of nine for all of the 33 publications. e detailed quality assessment is listed in Table1.
Postmortem comparison of iron concentration in dened brain regions. Eleven of the manu-
scripts examined iron concentration in seven regions of postmortem brains. e numbers of subjects for each
region were 98 (frontal lobe), 44 (temporal lobe), 117 (nucleus caudatus), 104 (globus pallidus), 173 (substan-
tia nigra), 100 (putamen), and 58 (cerebellum). Although iron concentration was signicantly increased in the
substantia nigra of PD patients (WMD = 39.85, 95% CI, 8.06–71.65, p = 0.01; Fig.2E), signicant heterogeneity
was detected in these cohorts (I2 = 71%; p = 0.0006). Subsequent sensitivity analysis suggested that such heter-
ogeneity was attributed to the study of Griths et al.11. Further analysis that eliminated this study (I2 = 12%;
p = 0.33) also showed a signicant increase of iron concentration in the substantia nigra (WMD = 23.60, 95%
CI = 7.62–39.58, p = 0.004; Fig.2F). Additionally, increased iron levels were observed in the putamen of PD sub-
jects (WMD = 19.30, 95% CI = 7.24–31.36, p = 0.002, I2 = 4%; Fig.2G). No signicant dierences were observed
in other brain regions (Fig.2). e funnel plots analyzing publication bias appeared to be symmetric by visual
inspection (Fig.3).
Figure 1. Flow chart describing the selection process of articles retrieved from initial literature search.
CENTRAL, Cochrane Central Register of Controlled Trials.

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MRI comparison of iron concentration in dened brain regions. Fourteen articles were included in
the R2* subgroup of meta-analyses in seven brain regions. e total subject numbers were 437 (nucleus canda-
tus), 500 (globus pallidus), 631 (substantia nigra), 446 (putamen), 265 (red nucleus), 117 (white matter) and 182
(thalamus). In the substantia nigra of PD subjects, iron content was elevated (WMD = 3.81, 95% CI = 2.59–5.02,
p < 0.00001) despite of a relatively high heterogeneity (I2 = 59%, p = 0.005; Fig.4C). Results of a sensitivity analy-
sis ascribed the heterogeneity to the studies of Ulla et al.25 and Gorell et al.18, as exclusion of them eliminated the
Article
Healthy controls PD patients
PD diagnosis Detection Method type
UPDRS
score
UPDRS
motor score H-Y scale
Disease
duration
Publication
Quality
Assessmentn Agea
Gender
(F/M) n Agea
Gender
(F/M)
Yu et al.17 10 84.6 ± 1.5 6/4 10 82.7 ± 1.7 4/6 UK PD Brain Bank
criteria ICP Postmortem — — — — ******
Loeer et al.12 8 74.6 ± 7.6 4/4 14 74.9 ± 8.7 5/9 Pathological examination COL Postmortem — — — — ****
Griths et al.11 6 83.3 ± 2.1 — 6 83.6 ± 2.4 —Pathological examination AA Postmortem — — — — ******
Dexter et al.234 81.3 ± 1.5 21/13 27 74.9 ± 1.4 11/16 Clinical and pathological
examination ICP Postmortem — — — — *****
Riederer et al.4473 (68–78) 3/1 13 76 (68–82) 7/6 Pathological examination SPH Postmortem — — — — *****
Soc et al.13 875.3 (66–86) 4/4 8 71.3 ± 12.5 4/4 Pathological examination SPH Postmortem — — — 7.5 ± 3.4 ******
Visanji et al.15 362.7 (47–78) 1/2 3 69.3 (56–79) 1/2 Pathological examination AA Postmortem — — — 21 ± 3.8 *****
Wypijewska et al.16 29 61–85 — 17 61–85 — Clinical and pathological
examination MS Postmortem — — — — ******
Galazka-Friedm
et al.10 8 64 ± 6 — 6 70 ± 4 —Clinical and pathological
examination MS Postmortem — — 4–5 4–7 *****
Uitti et al.14 12 70 4/8 9 73 3/6 Pathological examination AA Postmortem — — — — ****
Chen et al.96 — — 10 — — — AA Postmortem — — — — ****
Gorell et al.18 10 60.0 ± 8.7 5/5 13 65.2 ± 12.7 2/11 Clinical diagnosis 3T R2* — — 1.5–3.0 3–13 *****
Graham et al.19 25 64.0 ± 6.6 6/7 21 61.4 ± 7.3 10/11 UK PD Brain Bank
criteria 1.5T R2* — — — 11.1 ± 4.5 ******
Martin et al.20,b 11 55.9 ± 7.3 4/7 22 61.9 ± 9.0 8/14 Published criteria66 3T R2* — 16.7 ± 7.1 — 3.2 ± 1.7 *******
19 60.3 ± 8.4 6/13 — 16.9 ± 7.5 — 2.9 ± 1.6
Du et al.21 29 59.6 ± 6.7 17/12 40 60.7 ± 8.3 17/23 Published criteria66 3T R2* — 23.4 ± 15.2 1.8 ± 0.6 4.2 ± 4.7 ******
Bunzeck et al.22 20 66.0 ± 9.1 10/10 20 66.3 ± 9.0 9/11 Queens Square Brain
Bank criteria67 3T R2* 34.6 ± 17.4 — — — ******
Lee et al.23 21 60.0 ± 6.1 9/12 29 59.1 ± 7.6 12/17 UK PD Brain Bank
criteria 3T R2* — 25.5 ± 9.2 2.05 ± 0.5 2.5 ± 1.9 *******
Lewis et al.323 59.9 ± 7.0 17/12 38 60.6 ± 8.0 17/23 Published criteria66 3T R2* — 23.8 ± 15.4 1.8 ± 0.6 4.4 ± 4.7 *****
Rossi et al.24 21 66 (58–80) 17/4 37 69 (42–86) 18/19 Clinical diagnosis 3T R2* — — — — *****
Ulla et al.25 26 57.0 ± 8.5 17/9 27 60.2 ± 10.7 14/13 PD So ciety Brain Bank68 1.5T R2* — 12.1 ± 8.5 1.9 ± 0.7 5.7 ± 4.4 ******
Rossi et al.26 19 65 (58–80) 15/4 25 73 (44–87) 14/11 Clinical diagnosis 3T R2* — — — — *****
Barbosa et al.27 30 64 ± 7 21/9 20 66 ± 8 8/12 UK PD Brain Bank
criteria 3T R2* — — 2.3 ± 0.6 8.1 ± 4.2 ******
Murakami et al.30 21 69.7 ± 8.6 12/9 21 72.0 ± 7.5 12/9 UK PD Brain Bank
criteria 3T R2* — — 2 (1–3) 2.7 ± 2.3 *****
He et al.29 35 60.5 ± 6.5 14/21 44 58.0 ± 8.8 19/25 UK PD Brain Bank
criteria 3T R2* — 15.6 ± 6.2 1.4 ± 0.5 2.8 ± 1.6 ****
Du et al.28 47 62.2 ± 8.8 24/23 47 65.8 ± 10.1 25/22 UK PD Brain Bank
criteria 3T R2* 39.6 ± 24.8 21.8 + 15.2 — 5.5 ± 4.8 *****
Zhang et al.34 26 57.3 ± 11.6 12/14 40 58.7 ± 12.8 19/21 UK PD Brain Bank
criteria 3T SWI — 19.0 ± 7.8 —3.6 ± 2.9− ******
Jin et al.35 45 55.4 ± 14.9 19/26 45 56.3 ± 10.9 14/31 UK PD Brain Bank
criteria 3T SWI 15.1 ± 9.3 12.0 ± 7.1 — — ******
Wan g et al.32 14 64.3 ± 12.7 7/7 20 67.2 ± 10.7 10/10 Clinical diagnosis 3T SWI — — — 2.8 ± 2.8 ******
Wan g et al.37 44 59.4 ± 11.8 23/21 16 63.3 ± 10.6 7/9 UK PD Brain Bank
criteria 1.5T SWI — — — 2.5 ± 1.7 *****
Han et al.31 20 55.9 ± 6.2 8/12 15 57.4 ± 7.1 8/7 UK PD Brain Bank
criteria 3T SWI 23.0 ± 5.6 — 2.2 ± 0.5 2.5 ± 1.6 *****
Kim et al.36 25 56.2 ± 6.5 13/12 30 57.6 ± 6.8 11/19 UK PD Brain Bank
criteria 3T SWI — 24.5 ± 8.4 1.7 ± 0.5 1.7 ± 1.1 *****
Wu, et al.33 40 66.5 ± 6.0 18/22 54 65.6 ± 5.8 21/33 UK PD Brain Bank
criteria 3T SWI — — ≥ 1.5 — ****
Huang, et al.38 19 65.0 ± 9.0 — 30 68.0 ± 9.0 6/24 — 3T SWI — — — — ****
Table 1. Characteristics of the 33 studies included for meta-analyses. aData in this column are presented
as mean ± SD or Range or Median (Range) or Mean (Range) or the detail ages; bIn this study the patient group
with n = 22 is for mid-brain images including substantia nigra and red nucleus, and the one with n = 19 is for
forebrain images including globus pallidus, putamen, nucleus caudatus, and white matter. UPDRS, Unied
Parkinson’s Disease Rating Scale; H-Y, Hoehn and Yahr; ICP, inductively coupled plasma spectroscopy; COL,
colorimetry; AA, atomic absorption; SPH, spectrophotometry; MS, Mössbauer spectroscopy.

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heterogeneity (I2 = 0%, p = 0.49 ; Fig.4D). Subsequent meta-analysis again demonstrated a signicant increase
of iron concentration in the substantia nigra (WMD = 3.91, 95% CI = 3.05–4.77, p < 0.00001; Fig.4D). Iron con-
centration was signicantly increased in the red nucleus (WMD = 1.93, 95% CI = 0.70–3.17, p = 0.002, I2 = 0%;
Fig.4F), but not in other brain regions (Fig.4). e publication biases were acceptable as determined by funnel
plots (Fig.5).
Eight articles were included in the SWI subgroup of meta-analyses in seven brain regions. e total subject
numbers were 431 (nucleus caudatus), 431 (globus pallidus), 431 (putamen), 306 (thalamus), 465 (substantia
nigra), 465 (red nucleus) and 211 (white matter). A signicant increase in iron concentration was observed in the
substantia nigra (WMD = 6.5, 95% CI = 3.31–9.68, p < 0.0001) with high heterogeneity (I2 = 94%, p < 0.0001;
Fig.6D). Signicant increases in iron concentration were also shown in the nucleus caudatus (WMD = 0.81, 95%
CI = 0.37–1.25, p = 0.0003, I2 = 24%; Fig.6A), putamen (WMD = 1.03, 95% CI = 0.06–2.01, p = 0.04, I2 = 60%;
Fig.6E), and red nucleus (WMD = 0.85, 95% CI = 0.15–1.54, p = 0.02, I2 = 44%; Fig.6H). When the article of
Wang et al.37 was removed based on sensitivity analysis, we still observed an increase of iron concentration in
the putamen (WMD = 0.82, 95% CI = 0.33–1.30, p = 0.001, I2 = 0%; Fig.6F). Signicant heterogeneity (I2 = 87%,
p < 0.00001) was detected in the globus pallidus group (Fig.6B), which was attributed to Han et al.31 as deter-
mined by a sensitivity analysis. Meta-analysis aer exclusion of this paper showed a signicant increase of iron
concentration in the globus pallidus (WMD = 1.76, 95% CI = 0.98–2.54, p < 0.0001, I2 = 0%; Fig.6C). e publi-
cation biases were acceptable as determined by funnel plots (Fig.7).
Structure by structure analyses of results from individual studies and meta-analyses. It is
known that inferences can be particularly prone to Type-I error in studies based on a small number of papers,
especially with a small sample size39. erefore, we herein elaborated on the results reported in each study com-
bining the results of meta-analyses and the methodological factors that could have contributed to discrepancies
in a brain structure-based fashion.
Substantia nigra. As expected, an elevation of iron concentration was found in the substantia nigra in all the
three types of measurements (Table2). is was in line with the majority of the 29 articles we analyzed. Except for
the three that did not show a change in postmortem samples10,12,16, the other 26 articles reported a trend toward
or a statistically signicant increase in iron content in the substantia nigra regardless of the type of measurement
(postmortem, SWI or R2*). As a note, three postmortem iron analyses12,14,16 indicated that the pars compacta and
reticulata were not discriminated during the measurement, while the other six studies did not state the relevant
information to make this determination.
Putamen. Both postmortem and SWI meta-analyses showed an iron overload in PD patients. However, when indi-
vidual articles describing postmortem samples were analyzed, we found that only one study reported a signicant
increase in iron content9, while the other ve were completely negative with mixed trends2,4,11–13. Although the results
of our meta-analysis suggested a signicant increase in iron content in the putamen of PD patients in postmortem
samples, caution should be taken in the interpretation of these results as one positive study9 dominated the other ve
negative ones in the analysis (Fig.2G). For SWI, an iron overload was suggested in the putamen based on both random
Figure 2. Statistical summaries and forest plots of studies comparing iron concentrations by postmortem
analysis. (D,E) Pooled using random-eects models. e others were pooled using xed-eects models.
*Analyzed aer heterogeneity was removed.

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and xed eects models. Results of two independent studies showed elevated iron content in this structure31,37, whereas
the other ve were not signicantly dierent33–36,38. One of the positive studies37 was removed following a sensitivity
analysis, and the remaining one31 drove half of the total eect size thereaer in the xed eects model (Fig.6F). Taken
together, additional studies are needed to conrm iron accumulation in the putamen.
Globus pallidus. For SWI, results of six studies suggested a trend toward, or a signicant, increase in the level of
iron33–38, while one showed a decrease in iron content31, which was later removed based on a sensitivity analysis.
e subsequent meta-analysis returned a signicant increase of iron content in the globus pallidus. However,
Figure 3. Funnel plots that examine possible publication bias in the studies by postmortem analysis.
*Analyzed aer heterogeneity was removed.

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results of either postmortem or R2* meta-analyses did not display signicant dierence, which was in line with
the mixed trends of changes in individual studies.
Nucleus caudatus. Similar to globus pallidus, both postmortem and R2* meta-analyses returned no signicant
dierence with mixed trends in iron content in the individual studies. Results of pooled SWI analysis showed a
signicant increase of iron content in PD patients. ere were six studies that showed a signicant31,37 or a trend
of increase33–36 in iron levels in the nucleus caudatus while only one study suggested a trend of decrease34.
Frontal lobe, temporal lobe and cerebellum. Although postmortem results of these structures were available, the
pooled sample sizes were small (98, 44 and 58, respectively). All the four studies on frontal lobe2,11,12,14 and two
on cerebellum2,14 reported negative results. Although one article reported a signicant decrease of iron levels in
the temporal lobe17, two studies showed no change11,13. Further studies were needed to clarify iron levels in these
structures.
Red nucleus. No available studies using postmortem samples t our criteria. Results of R2* and SWI pooled
analyses suggested an increase of iron levels in the red nucleus. For the R2* analyses, four studies reported a sig-
nicant increase3 or an increasing trend20,27,29, whereas one showed a decreasing trend30. For the SWI analyses,
seven out of eight studies reported no remarkable changes, among which three showed a decreasing trend32,34,38
and four an increasing trend in iron content32,36–38. In comparison, the study that showed signicantly elevated
iron content in PD patients33 drove roughly half of the total eect size (Fig.6H). Noteworthy, two PD groups
(advanced and mild disease stage) were included in this study that had the same control group33. e advanced
PD group was chosen for the current analysis to compare with postmortem samples that are usually obtained at
late stage PD. When the mild group was included, results of SWI meta-analyses were not aected except in the red
nucleus. ere was no signicant increase of iron content detected (Figs S1 and S2), suggesting that the severity of
PD might be a factor aecting iron deposits in the red nucleus. As a note, the mild stage in this study33 was Hoehn
and Yahr scale < 1.5, which appeared milder than normally dened.
alamus and white matter. No qualied study using postmortem samples was available. Results of both R2*
and SWI meta-analyses suggested no association of iron levels with PD in the thalamus and white matter of the
brain. Furthermore, all of the selected individual studies31,34–37 returned negative results.
Discussion
Iron dysregulation is frequently associated with neurodegenerative disorders, including Huntington disease,
Alzheimer’s disease, amyotrophic lateral sclerosis, and frontotemporal lobar degeneration40,41. Nonetheless, it
remains unclear whether such defect is a cause or a consequence of neurodegeneration. A large body of evidence
Figure 4. Statistical summaries and forest plots of studies comparing iron concentrations by MRI R2*
relaxometry. (C) Pooled using random-eects models. e others were pooled using xed-eects models.
*Analyzed aer heterogeneity was removed.

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suggests abnormal iron levels in the brains of PD patients and a role for iron dysregulation in PD pathogene-
sis42–44. Our study represents the rst meta-analysis that systematically assesses iron levels in various brain regions
of PD patients by postmortem measurements and by MRI (R2* and SWI). Our analysis conrms a perturbed iron
homeostasis in the substantia nigra and suggests that an increase in iron levels may also occur in the putamen
and red nucleus (Table2).
Some caveats in regard to the scope of this meta-analysis must be taken into account. First, in the postmortem
analyses dierent iron quantication methods (SPH, AA, COL, ICP and MS) have been used. e dierential
sensitivity and specicity of these methods may contribute to an elevated heterogeneity. Second, disease stage and
Figure 5. Funnel plots that examine possible publication bias in the studies by R2*. *Analyzed aer
heterogeneity was removed.

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age may be two inuencing factors when evaluating iron concentration in the brain40,45,46, which unfortunately
is not addressed in the current study due to incomplete information and limited sample size. For example, the
inclusion of a sub-group of mild-stage PD patients results in a loss of signicance in iron levels in the red nucleus
of SWI meta-analysis.
It is well recognized that iron overload contributes to oxidative stress through Fenton reaction, promoting the
death of dopaminergic neurons in the substantia nigra47. Such iron accumulation is known to be associated with
increased ferritin and neuromelanin iron loads48,49, as well as increased expression of divalent metal transporter 1
that may contribute to PD pathogenesis via its capacity of transporting ferrous iron47. Furthermore, aggregation
of α -synuclein can be accelerated when bound with free iron50. However, it remains unclear whether iron deposit
triggers or accelerates neurodegeneration, or if they are a secondary event due to neuronal degeneration. erefore,
it is important to determine the timing of iron deposit in substantia nigra during the pathogenesis of PD. Because
postmortem measurements are usually made in a very late stage of PD, future longitudinal studies of iron contents
are warranted47. Consistent results obtained from postmortem, R2*, and SWI measurements suggest that longitu-
dinal evaluation of iron content in the substantia nigra can be appropriately made by MRI methods.
It appears that the MRI methods of R2* and SWI do not completely match the postmortem results, presum-
ably the latter being the standard. Iron deposit is detected by SWI in the globus pallidus and nucleus caudatus,
but these are inconsistent with the postmortem observations. Results from R2* studies also suggest an inconsist-
ency in the putamen as both postmortem and SWI eects show an iron overload. Loss of striatal dopamine in
PD is most prominent in sub-regions of the putamen51, which may be associated with an increase in iron levels.
However, this may be a weak argument considering that the postmortem iron increase in this structure is driven
by a single study as noted in the Results. It has previously been proposed that SWI is more specic and precise
than other methods to estimate brain iron content52. Our results suggest that both methods have weakness in
measuring iron content. e iron signal determined by R2* may be disrupted by calcication53 and lipid content54,
and the output value is a weighted summation of magnetic properties from both local and surrounding tissues28.
Intrinsic defects of SWI include a diculty in distinguishing diamagnetic and paramagnetic susceptibility own-
ing to the convoluting eect of the dipole elds55. ere are also limitations of MRI per se, such that myelin, espe-
cially small myelinated bers, cannot be easily distinguishable from iron deposition46, and the phase value of MRI
reects not only non-heme iron deposited in the tissue but also the heme iron in hemosiderin or in circulating
blood56. Microbleeds may also be a confounding factor especially when brain iron content is estimated in older
adults57. Given the MRI phase’s nonlocal behavior, one should pay attention to the signal interference of adjacent
structures. For example, the red nucleus lies adjacent to substantia nigra in the midbrain and is likely high in iron
levels due to its proximity58. In other words, the dierences detected in iron levels in the red nucleus may arise
from the adjacent substantia nigra, instead of from the structure itself. Increased iron levels in red nucleus are
Figure 6. Statistical summaries and forest plots of studies comparing iron concentrations by SWI
relaxometry. (B,D,E) Pooled using random-eects models. e others were pooled using xed-eects models.
*Analyzed aer heterogeneity was removed.

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associated with levodopa-induced dyskinesia of PD3. Future postmortem studies are warranted to conrm iron
deposit in this structure. is is also the case for the putamen and globus pallidus, due to their relative proximity.
Figure 7. Funnel plots that examine possible publication bias in the studies by SWI. *Analyzed aer
heterogeneity was removed.

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Recently, quantitative susceptibility mapping (QSM), a potentially superior method to measuring iron content
in vivo, has been applied to measure PD-related iron deposition and progression28. By this method, Guan et al.59
have recently reported a distinct pattern of iron accumulation according to disease stage, with iron spreading
from the substantia nigra in early stages to the substantia nigra, red nucleus and globus pallidus in later stages.
is could explain the aforementioned discrepancy in the red nucleus when the mild PD group is included, as
well as provide a potential explanation for inconsistent ndings between neuropathology and MRI techniques.
In conclusion, the current meta-analysis corroborates iron overload in substantia nigra and suggests such
iron homeostasis defect in the putamen (by postmortem and SWI, but not R2*) and the red nucleus (by R2* and
SWI; no data by postmortem) of PD patients. Both the R2* or SWI techniques may not authentically reect iron
changes in brain regions other than substantia nigra. Our results oer a comprehensive understanding of iron
loads in dierent brain regions in association with PD, and contribute to the evaluation of measuring accuracy of
iron concentration by MRI methods.
Methods
Literature Search Strategy. Literature related to iron and Parkinson’s disease were searched in four
databases including Medline via PubMed, Web of Science, the Cochrane Central Register of Controlled Trials
(CENTRAL) and Embase via OVID, dated till 19th November 2015. e keywords for iron and Parkinson’s dis-
ease are “iron” or “Fe” and “Parkinson disease”, “Parkinson’s disease”, “Parkinsons disease” or “Parkinsonian”,
respectively.
Study Selection. Based on the keywords, titles and abstracts of the identied publications were screened.
Following an exhaustive examination of the literature contents, articles were included according to our selection
criteria: population (idiopathic PD patients), comparators (individuals free of neurological disorders), outcome
measurement (iron content in brain regions), and language (articles written in English or Chinese). Review arti-
cles, qualitative and semi-quantitative studies were excluded.
Data Extraction. e literature search and data extraction were conducted by two researchers (Qing-Qing
Zhuang and Jian-Yong Wang) independently. In the case of a dispute, a third investigator was included to discuss
and reach an agreement. e following data was extracted: sample size, age, sex, PD diagnosis, iron detection
methods, the type of samples, clinical scores, and iron content or R2* value or phase value in brain regions.
Assessment of the detailed information was listed in Table1. As shown in this table, the disease severity (Hoehn
and Yahr scale) was not provided by all the included studies and the provided else information was also varied in
forms including UPDRS score, UPDRS motor score, and/or disease duration. erefore, we did not include the
disease severity as a source of variance in the analysis.
Iron quantication methods employed in the postmortem study of brain samples included spectropho-
tometry (SPH), atomic absorption (AA), colorimetry (COL), inductively coupled plasma spectroscopy
(ICP) and Mössbauer spectroscopy (MS). To be consistent in brain weights, a conversion of dry weight to
wet weight was applied based on a dry/wet ratio as suggested in previous studies60,61. e SWI signal phase is
orientation-dependent and nonlocal55. As a result, the phase value appears to be either positively or negatively
correlated with iron concentration depending on the orientation relative to the Bo eld62. us, a conversion from
SWI phase value to iron concentration was applied based on formulas suggested in previous studies35,36; that is,
concentration = 397.72 × (phase value) + 3.4097 (extracted from Fig.1 of ref. 36) for the studies of positive set-
ting31,34,36, and concentration = − 128.23 × (phase value) + 3.1897 (extracted from Fig.2 of ref. 35) for the studies
of negative setting32,33,35,37,38.
Postmortem R2* SWI
Change pn Change pn Change pn
Substantia nigra ↑ 0.01 173 ↑ < 10−5631 ↑ < 10−4465
↑
a0.004 161 ↑
a< 10−5556
Putamen ↑ 0.002 100 — 0.74 446 ↑ 0.04 431
↑
a0.001 371
Globus pallidus — 0.72 104 — 0.37 500 — 0.20 431
↑
a< 10−4396
Nucleus caudatus — 0.47 117 — 0.80 437 ↑ 0.0003 431
Frontal lobe — 0.33 98 NA NA NA NA NA NA
Temporal lobe — 0.11 44 NA NA NA NA NA NA
Cerebellum — 0.14 58 NA NA NA NA NA NA
Red nucleus NA NA NA ↑ 0.002 265 ↑ 0.02 465
alamus NA NA NA — 0.81 182 — 0.94 306
White matter NA NA NA — 0.07 117 — 0.93 211
Table 2. A summary of changes in brain iron levels of PD patients based on the current meta-analysis.
↑Increased iron level in PD. —No change of iron level in PD. NA, no data available. aAnalyzed aer heterogeneity
was removed.

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Quality Assessment. e Newcastle-Ottawa Scale63 was employed to assess the quality of the chosen
studies. is tool classied studies in three broad perspectives: selection of the study groups, comparability
of the groups, and ascertainment of either exposure or outcome of interest for the studies. Semi-quantitative
measurement using a star system assesses the quality of study. e highest quality studies can get a maximum
of nine stars.
Statistical Analysis. Eleven postmortem analysis and 22 MRI analysis articles were eventually selected for
our meta-analysis. Means, standard deviations (or standard errors), and the number of samples were extracted
in each study. Meta-analyses were conducted within the studies of the same brain region aer sorting into their
respective quantitative groups of postmortem analysis, R2* and SWI. In the case that the same data appeared in
multiple studies, the data were used only once. All of the analyses were performed using Review Manager 5.2 for
Windows (http://ims.cochrane.org/recman). A two-tailed p value < 0.05 was considered statistically signicant.
Weighted mean dierence (WMD) was regarded as an eect size. Q-statistics and I2 were used for assessing the
heterogeneity64,65. A random eects model was applied when heterogeneity was found by Q-statistics or when
I2 > 50%. A xed eects model was applied otherwise.
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Acknowledgements
e authors appreciate Drs Jennifer Harr and Wen-Hsing Cheng for critical help improving readability and
accuracy of the manuscript. is work was supported by funding from Zhejiang Provincial Natural Science
Foundation (LY16H250003, LY16H260003, and LR13H020002), National Natural Science Foundation of China
(81571087), and Wenzhou Science and Technology Bureau (Y20150005).
Author Contributions
J.Y.W. and Q.Q.Z. performed the data collection, extraction and analyses, L.B.Z., H.Z., T.L., R.L. and S.F.C.
contributed to partial data extraction and interpretation, X.Z., C.P.H. and J.H.Z. designed and supervised the
study, J.Y.W., Q.Q.Z. and J.H.Z. wrote the manuscript. All authors read and approved the nal manuscript.

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Additional Information
Supplementary information accompanies this paper at http://www.nature.com/srep
Competing nancial interests: e authors declare no competing nancial interests.
How to cite this article: Wang, J.-Y. et al. Meta-analysis of brain iron levels of Parkinson’s disease patients
determined by postmortem and MRI measurements. Sci. Rep. 6, 36669; doi: 10.1038/srep36669 (2016).
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