Journal of Alzheimer’s Disease xx (20xx) x–xx 1

DOI 10.3233/JAD-181296
IOS Press

1 Repetitive Transcranial Magnetic

2 Stimulation Induced Hypoconnectivity
Within the Default Mode Network Yields

f
3

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4 Cognitive Improvements in Amnestic Mild
5 Cognitive Impairment: A Randomized

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6 Controlled Study

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7 Hailun Cuia,1 , Rujing Rena,1 , Guozhen Linb , Yang Zoua , Lijuan Jiangc , Zhengde Weid , Chunbo
8 Lic,e,f,2,∗ and Gang Wanga,2,∗
9
a Department of Neurology & Institute of Neurology, Ruijin Hospital, Shanghai Jiao Tong University School of
10 Medicine, Shanghai, China
b Department of Psychiatry, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
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11

12
c Shanghai Key Laboratory of Psychotic Disorders, Shanghai Mental Health Center, Shanghai Jiao Tong

13 University School of Medicine, Shanghai, China

14
d Key Laboratory of Brain Function and Disease, Chinese Academy of Sciences, School of Life Sciences, University

15 of Science & Technology of China, Hefei, Anhui, China

e Brain Science and Technology Research Center, Shanghai Jiao Tong University, Shanghai, China
d

16

17
f CAS Center for Excellence in Brain Science and Intelligence Technology (CEBSIT), Chinese Academy of Science,

Shanghai, China
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18

19 Handling Associate Editor: Jin-Tai Yu

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Accepted 15 April 2019

20 Abstract.
21 Background: Repetitive transcranial magnetic stimulation (rTMS) is thought to be effective in alleviating cognitive symptoms
co

22
24
in patients with amnestic mild cognitive impairment (aMCI), but the mechanisms related to network modification are poorly
23
25
understood.
Objective: Here we tested rTMS efficacy and explored the effect of rTMS-induced changes in the default mode network
(DMN) and their predictive value for treatment response.
Un

1 These authors contributed equally to this work.

2 These authors contributed equally to this work as correspond-
ing authors.
∗ Correspondence to: Gang Wang, MD, PhD, Department of
Neurology& Institute of Neurology, Ruijin Hospital, Shanghai
Jiao Tong University School of Medicine, Shanghai, China.
Tel.: +86 021 64454473; Fax: +86 021 64454473; E-mail:
wg11424@rjh.com.cn. and Chunbo Li, MD, Shanghai Key Lab-
oratory of Psychotic Disorders, Shanghai Mental Health Center
(SMHC), Shanghai Jiao Tong University School of Medicine,
Shanghai, China. Tel.: +86 021 34773243; Fax: +86 021 64387986;
E-mail: licb@smhc.org.cn.

ISSN 1387-2877/19/$35.00 © 2019 – IOS Press and the authors. All rights reserved
 2 H. Cui et al. / Hypoconnected DMN Improves Cognition in aMCI

26 Methods: Twenty-one subjects clinically diagnosed with aMCI were recruited to complete a 10-session randomized and sham-
27 controlled rTMS treatment targeting the right dorsolateral prefrontal cortex. Resting-state functional magnetic resonance
28 imaging in tandem with neuropsychological assessments were administered before and after the intervention. Changes in
29 functional connectivity of the DMN and relevant brain regions, as well as the correlations between baseline functional
30 connectivity and clinical rating scales were calculated in order to elucidate the mechanism of treatment response to rTMS
31 therapy.
32 Results: Compared to the sham group, the rTMS group achieved improvement of neuropsychological performance and
33 significant functional connectivity changes within the DMN. Group × Time interactions were found between posterior cin-
gulate gyrus and right fusiform gyrus (F(1,19) = 17.154, p = 0.001), and also left anterior cingulate gyrus (F(1,19) = 3.908,

f
34

35 p = 0.063), showing an rTMS-induced deactivation of functional connectivity within the DMN. Baseline functional con-

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36 nectivity analysis of seeds within the DMN in the rTMS group revealed negative correlation with AVLT-Recognition score
37 changes.
38 Conclusion: rTMS-induced hypoconnectivity within DMN is associated with clinical cognitive improvements in patients
39 with aMCI. Further, pre-rTMS baseline activity of the DMN at rest may be a predictor for favorable rTMS treatment
40 response.

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Keywords: Alzheimer’s disease, amnestic mild cognitive impairment, default mode network, repetitive transcranial magnetic
41 stimulation, resting-state functional MRI
42

Trial Registration: Chinese Clinical Trial Registry (ChiCTR); ChiCTR-IPR-16009063. Trial Name: Repetitive transcra-

tho
43

44
nial magnetic stimulation on cognition in mild cognitive impairment and early stage of Alzheimer’s disease. URL:
45
http://www.chictr.org.cn/showproj.aspx?proj = 15273.

INTRODUCTION diseases (such as refractory major depressive disor-

26
Au 54

der, movement disorders, and chronic pain) [8–10], 55

27 Mild cognitive impairment (MCI) has been sug- and has been shown to indeed influence intrinsic 56

28 gested to represent a distinct status intermediate brain connectivity through functional magnetic res- 57

29 between prodromal Alzheimer’s disease (AD) [1–3], onance imaging (fMRI) [11–13]. It is hypothesized 58

30 and more severe, debilitating, cognitive impair- that the memory improvement observed in patients 59
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31 ment. Among variable presentations, amnestic MCI with MCI and AD undergoing rTMS treatment could 60

(aMCI), characterized by impaired episodic memory be attributed to the reversal of neural connectivity
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32 61

33 retention and retrieval, is of special interest both for changes which coincide with dysfunction; however, 62

34 its high risk of progression to AD and its potential to little is known concerning exact pattern(s) of func- 63

35 be a treatment window in which symptomatic relief tional rewiring of the brain connectome underlying 64

36 can be achieved [4, 5]. In the face of unfavorable off- any mechanism of improvement [7, 14, 15]. To 65
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37 target effects of the currently available medications tackle this question, it is very important to iden- 66

38 for AD symptoms, with little alteration of disease tify the commonly disturbed network(s) influencing 67

39 course or effect on higher-order brain dysfunction, brain dysfunction, if not also upstream of neuronal 68

40 clinicians are turning to non-invasive physiotherapies deterioration and death in AD patients, before any 69

which hold promise to defer AD morbidity through therapeutic intervention. In aMCI and AD, the con-
co

41 70

42 functional neural regulation. The most promising and sensus of literature in the field is that the most 71

43 well-tolerated means of AD intervention in recent prominently affected resting-state network with dis- 72

44 literature is the technique of repetitive transcranial ease alteration of measurable baseline activity is the 73
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45 magnetic stimulation (rTMS) [6, 7]. default mode network (DMN) [16, 17]. 74

46 The magnetic field produced by rTMS penetrates Hyperactivated at rest while deactivated through 75

47 the scalp and generates a sufficient electromotive task-related events, the DMN is presumed to be 76

48 force likely to depolarize superficial axons and involved in passively controlled states during which 77

49 influence neural activation patterns [8–10]. The the mind is internally oriented toward free thinking, 78

50 potential effects of rTMS to modify neuronal plastic- past reminiscence, and envisioning the future rather 79

51 ity across entire functional brain circuits and thereby than goal-directed tasks [18]. The earliest evidence 80

52 affect cortical excitability has promoted the appli- of its role in AD pathogenesis came from the obser- 81

53 cation of rTMS in a variety of neuropsychological vation of hypometabolism affecting regions that are 82
 H. Cui et al. / Hypoconnected DMN Improves Cognition in aMCI 3

83 central components of the DMN, such as the pos- (rsfMRI) examination. 135

84 terior cingulate gyrus (PCG) [16, 19]. It is believed

85 that the PCG is among the earliest of regions that have METHODS 136

86 abnormal metabolism if not frank circuit disruption

87 during the prodromal stage of AD with deposition of Subjects 137

88 amyloid, tau protein, and atrophy of this brain region

89 [20–22]. The PCG is also one of the earliest regions All aMCI subjects (n = 25) were recruited from the 138

90 within the DMN able to predict cognitive decline, via memory clinic of Ruijin Hospital, Shanghai Jiao Tong 139

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91 observation of metabolism and regional blood flow, University School of Medicine. Inclusion criteria are 140

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92 either of which can be used to evaluate the probability as follows: 1) age between 50–85 years old, no gen- 141

93 for MCI to develop into AD [23, 24]. In addition to der limitation, right-handed; 2) 6 years of education 142

94 the PCG, other major nodes within the DMN include at least; 3) met the previously published NIA-AA 143

95 the medial prefrontal cortex, lateral inferior parietal criteria (2011) for “MCI due to AD”, with memory 144

96 lobes, and medial temporal structures, which are all deficits as the only complaint of cognitive impair- 145

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97 interconnected and may be affected pathologically ment, reported by the patient, caregiver, or physician; 146

98 [25, 26]. Clinical trials applying rTMS to AD or MCI 4) Clinical Dementia Rating scale (CDR) memory 147

99 have found positive effects on memory, behavior, and score of 0.5; 5) Hachinski Ischemia Score (HIS) less 148

100 executive functions; however, there have been lim- than 4; 6) absence of dementia according to the DSM- 149

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101 ited studies revealing any possible mechanisms and IV for AD; 7) stable medication for at least 3 months. 150

102 therapeutic effects that rTMS exerts on brain func- Significant neurological or psychiatric diseases that 151

103 tional connectivity of the DMN to date [7, 27–29]. might result in cognitive dysfunction, unstable sys- 152

104 Measurable changes in regions distant from target temic condition, and MRI/rTMS contraindications 153

105 sites of rTMS have been demonstrated previously to (such as metal implants or claustrophobia) were 154
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106 represent modulation of neural activity. Thus, it is regarded as disqualified subjects and excluded. 155

107 reasonable to hypothesize that functional connectiv- The complete procedures of both rTMS and 156

108 ity within the DMN could be affected by targeting fMRI acquisition were approved by the Institutional 157

109 elsewhere using rTMS and that it may be possible to Review Board of Ruijin Hospital, Shanghai Jiao Tong 158

110 reveal mechanisms of cognitive improvement follow- University School of Medicine and conducted as the 159

ing rTMS from the perspective of internal network

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111 institutional guidelines required. Written informed 160

112 modulation. consents from all the subjects were obtained before 161
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113 In the present study, we analyzed the rTMS- participation. 162

114 induced alterations in neural network functional

115 connectivity within the DMN by targeting the right Study design 163

116 dorsal lateral prefrontal cortex (DLPFC), a widely

117 used stimulation site which is implicated in sev- This was a prospective, double-blind, placebo- 164
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118 eral brain networks concerning cognitive, affective controlled pilot study carried out from October 165

119 and sensory processing [30–34]. Direct targeting 2016 to June 2017. All subjects involved were 166

120 of DLPFC has previously been proven to affect randomly assigned to two groups (1:1) following 167

121 episodic memory ability, likely through underlying simple randomization procedures (computerized ran- 168
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122 functional pathways that connect to the subcorti- dom numbers), namely the rTMS group and the 169

123 cal memory related system, and therefore DLPFC sham group. Each subject received a 10-session 170

124 is one reasonable site to elicit therapeutic effects daily treatment for approximately 2 weeks (two 5- 171

125 in diseases showing memory impairments includ- consecutive-sessions with a 2-day break in between) 172
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126 ing MCI and AD [7, 27, 35]. We hypothesized that and a two-month follow-up neuropsychological 173

127 DMN functional connectivity changes are involved reassessment. A senior technician blinded for study 174

128 in the therapeutic mechanisms of rTMS and could design and neuropsychological assessments was des- 175

129 help explain individual responses through base- ignated to be the constant performer of the TMS 176

130 line functional connectivity assessment. To test this treatment (both for rTMS and sham group) using a 177

131 hypothesis, we tested the effectiveness of rTMS to MagPro R30 (Magventure, Denmark) with a figure- 178

132 improve functional memory in aMCI patients and of-eight coil model MCF-B70 (97 mm in diameter). 179

133 validated resulting changes in the DMN through Each daily stimulation session consisted of thirty 180

134 resting-state functional magnetic resonance imaging trains of 5 s stimuli delivered at 10 Hz spaced-out by 181
 4 H. Cui et al. / Hypoconnected DMN Improves Cognition in aMCI

182 25 s of no stimulation (total number of stimuli: 1500). as the within-subjects factor. All analyses were 232

183 For subjects of the rTMS group, rTMS was delivered performed using SPSS software. Two-sided proba- 233

184 with an intensity of 90% of the resting motor thresh- bility values of p < 0.05 were considered statistically 234

185 old (RMT) rightly over the right DLPFC. RMT was significant. 235

186 defined as the lowest stimulus intensity exerted by

187 single-pulse TMS over the optimal location on the
188 scalp (the motor “hotspot”, defined as the optimal fMRI data acquisition and prepossessing 236

189 area for stimulating the thumb with a visual twitch),

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190 and was able to induce a minimum of 50 mv of Each subject obtained MRI data in two sessions 237

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191 motor evoked potential (MEP) in five out of ten trials that occurred at baseline (pre-rTMS, less than a 238

192 recorded by the electrode pad located at the contralat- week before the treatment started) and afterwards 239

193 eral target muscle of the left first dorsal interosseous (less than 2 hours after the treatment finished). Dur- 240

194 muscle (FDI). The right DLPFC is located 5.5 ing the scanning, subjects were instructed to lay 241

195 cm anteriorly to the hotspot of each subject [36]. on their backs with their heads fastened in foam 242

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196 Subjects of the sham group received the same record- padding and wearing earplugs for noise reduction. 243

197 ing sound with the coil 90◦ perpendicular to the MRI data were acquired using a 3.0-T Siemens Verio 244

198 skull, with no practical magnetic field penetrated the MRI scanner (Erlangen, Germany) with a 12-channel 245

199 brain. head coil. Three-dimensional T1-weighed anatom- 246

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ical images were scanned along the sagittal plane 247

200 Neuropsychological assessment and statistical with parameters as follows: repetition time/echo 248

201 analysis time (TR/TE) = (1900 ms/3.43 ms), field of view 249

(FOV) = 240 × 240 mm2 , FOV phase = 100%, voxel 250

202 General cognitive status and the treatment response size = 0.9 × 0.9 × 10.0 mm3 , slice thickness = 1 mm, 251
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203 were assessed by a researcher concealed from the and slice number = 160 slices. rsfMRI images were 252

204 allocation sequence using a multiple of neuropsycho- obtained using a gradient-recalled echo-planar imag- 253

205 logical scales at baseline (pre-intervention), 2 weeks ing (EPI) sequence with the following specifications: 254

206 (immediately after completing the 10-session course (TR/TE) = (2000 ms/25 ms), FOV = 240 × 240 mm2 , 255

207 of treatment, post-intervention), and at a two-month FOV phase = 100%, voxel size = 3.8 × 3.8 × 50.0 256

follow-up (8 weeks after the therapeutic session fin- mm3 , slice thickness = 5 mm, and 32 sagittal slices.
d

208 257

209 ished). Specifically, each patient completed global The functional run lasted for 316 s with 155 volumes 258
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210 cognitive function evaluation through Mini-Mental acquired. During the scanning, subjects passively 259

211 State Exam (MMSE) and Addenbrooke’s Cognitive viewed a fixation cross, not focused on anything in 260

212 Examination (ACE-III). Tests used to assess individ- particular and were kept awake during the resting- 261

213 ual domains of cognition included the logic memory state data acquisition. 262

214 test (short story test immediate recall), Auditory Ver- Data prepossessing in this study was per- 263
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215 bal Learning Test (AVLT) – immediate free recall/ formed using Data Processing Assistant for 264

216 (5 min/20 min) delayed free recall/recognition con- Resting State fMRI (DPARSF) V4.3, which is 265

217 taining 12 items, Trail Making Test A/B (TMTA/B), based on Statistical Parametric Mapping (SPM12; 266

218 digital span (forward/backward), as well as Geriatric http://www.Fil.ion.ucl.ac.uk/spm) and the toolbox 267
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219 Depression Scale (GDS). Different summary scores of Data Processing & Analysis of Brain Imaging 268

220 derived from raw AVLT results were used in this (DPABI, http://rfmri.org/DPABI) [39, 40] including 269

221 study as the primary endpoint to evaluate the base- slice timing, head motion correction (nuisance 270

222 line and intervention effect of rTMS treatment both covariates including white matter signal, cere- 271
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223 in rTMS and sham groups [37, 38]. Demographics brospinal fluid signal, linear trend, and Friston 272

224 and baseline neuropsychological tests were analyzed 24-parameter model of head-motion parameter 273

225 using Fisher’s exact test for categorical variables and model were regressed out), normalization, spatial 274

226 two-sample two-tail Student’s t-test/non-parametric smoothing (FWHM = 6 mm) and band-pass filtering 275

227 (Mann-Whitney) test for continuous variables. The (0.01 Hz<f<0.08 Hz). These processed images were 276

228 intervention effect was investigated by conduct- used for seed-based connectivity analysis and the 277

229 ing mixed-design analysis of variance (ANOVA) subsequent correlation study. We did not use global 278

230 with Group (rTMS/sham) as the between-subjects signal regression because of possible distortion of 279

231 factor and time (pre/post-intervention, follow-up) group differences in functional connectivity [41, 42]. 280
 H. Cui et al. / Hypoconnected DMN Improves Cognition in aMCI 5

281 An additional quality control measure for the wise connectivity maps generated through PCG. The 331

282 motion artifacts was performed using mean frame- overall correlations for each group before and after 332

283 wise displacement (FD) according to Power et al. the rTMS intervention were then forwarded to a 333

284 [43] in order to reflect the temporal derivative of mixed-design analysis of variance (ANOVA), with 334

285 movement parameters [44]. No participant was group (rTMS group/sham group) as the between- 335

286 classified as an outlier nor excluded from the main subjects factor, and time (pre/post-rTMS) as the 336

287 analysis. Images from the two groups did not differ in within-subjects factor. Mean time courses of each 337

288 mean FD (rTMS group/pre-intervention M = 0.104, region of significance were extracted and redefined as 338

f
289 SD = 0.055, range = 0.161, minimum = 0.051, max- specified regions of interest (ROI) for further analy- 339

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290 imum = 0.213; rTMS/post-intervention M = 0.101, sis. The functional connectivity between PCG and the 340

291 SD = 0.061, range = 0.173, minimum = 0.036, max- newly defined ROIs were reexamined using mixed- 341

292 imum = 0.209; sham/pre-intervention M = 0.094, design ANOVA through SPSS software for stricter 342

293 SD = 0.050, range = 0.131, minimum = 0.027, max- comparison (p < 0.05, Bonferroni corrected). Subse- 343

294 imum = 0.158; sham/post-intervention M = 0.095, quent contrasts were performed in order to examine 344

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295 SD = 0.307, range = 0.098, minimum = 0.042, maxi- the connectivity differences between each group 345

296 mum = 0.139). No significant group difference was (post-intervention scan: rTMS/sham) as well as dif- 346

297 found in motion parameter (F (3, 18.342) = 0.085, ferences between each scanning time point (pre/post 347

298 p = 0.967). intervention). Correlations between their alterations 348

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and AVLT score changes (AVLT-D5, AVLT-D20, 349

299 Functional connectivity analysis AVLT-I, and AVLT-R) were also calculated. In all 350

analysis, familywise error (FWE) multiple compari- 351

300 The function of rTMS on regional connectivity son corrections for the group maps were performed 352

301 could be a large-scale modulation affecting a mul- by estimating the smoothness of the data after pre- 353
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302 tiplicity of regions within different networks, thus possessing procedure (3dFWHMx) ahead of 5000 354

303 we first performed a seed-based functional connec- Monte Carlo simulations using AlphaSim correction 355

304 tivity within the DMN by placing a 6-mm seed in the to determine statistical thresholds for voxel cluster 356

305 PCG at the given coordinates at the Montreal Neu- size needed for the achievement of a family-wise 357

306 rological Institute template coordinates of (0, –51, ␣ < 0.05 at a voxel-wise p < 0.005. 358

29) in order to calculate areas across the whole brain To investigate the possible causes for poten-
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307 359

308 closely associated with it [45]. PCG is commonly tial discrepancies of cognitive improvement among 360
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309 identified as a critical node in the traditional DMN the patients receiving therapeutic rTMS, correlation 361

310 [17, 46, 47]. fMRI and PET studies have proven analysis was carried out to investigate the relationship 362

311 the essential role of PCG within DMN, both for its between the improved neuropsychological exams and 363

312 wide connection among other regions, such as the the initial status of brain represented by the base- 364

313 medial prefrontal cortex, anterior cingulate cortex, line functional connectivity within DMN. Regions of 365
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314 inferior parietal lobule and medial temporal regions DMN proved to have changed through the course of 366

315 (e.g., hippocampal/parahippocampal gyri), as well rTMS therapy and adjacent regions overlapping with 367

316 as its vulnerability of being impaired during neu- one another observed through a close look into the 368

317 ropathological process like AD [16, 48]. The average clusters with significant differences were extracted 369
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318 blood oxygen level–dependent (BOLD) signal time as ROIs and the correlation within the mean time 370

319 course within the ROI of PCG was then correlated to course of each seed ROI was calculated. The bilat- 371

320 every voxel in the brain for each subject using Pear- eral PCG, the adjacent precuneus (PCUN), inferior 372

321 son correlation coefficients so as to identify specific parietal lobule (IPL), and ventral medial prefrontal 373
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322 regions within DMN that might have been changed. cortex (vMPFC) were defined as ROIs, and the base- 374

323 The correlation coefficients were then converted to line functional connectivity strength of each pair of 375

324 z scores using the Fisher r-to-z transformation in ROIs were then forwarded into correlation analysis 376

325 order to generate the resting-state functional connec- with the alterations in AVLT-I and AVLT-R (pre- 377

326 tivity z-map of DMN. We performed a one-sample post/pre). Statistics for the value were performed 378

327 t test showing the general activation of the baseline through GraphPad Prism software and validated by 379

328 FC within DMN in both groups (rTMS/sham). An SPSS. The FDR correction was implemented for mul- 380

329 independent-sample t test was used to exclude the tiple comparison using the Benjamini and Hochberg 381

330 possibility of baseline group differences in voxel- (1995) procedure. 382

 6 H. Cui et al. / Hypoconnected DMN Improves Cognition in aMCI

383 RESULTS benefit from rTMS therapy. Significant correlation 431

between percent change in AVLT-R and gender 432

384 Subjects characterization (r = 0.321, p = 0.336), age (r = 0.126, p = 0.712), edu- 433

cation (r = 0.163, p = 0.632), and RMT (r = 0.177, 434

385 A total of 25 aMCI patients were initially recruited p = 0.602) was not found, but baseline AVLT-R corre- 435

386 for receiving rTMS/sham stimulation, and 21 of them lated significantly with AVLT-R change (r = –0.698, 436

387 finished the whole session and rsfMRI scanning p = 0.017). 437

388 with no adverse effect reported (two excluded for

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389 incomplete course of rTMS treatment, another two Functional imaging results 438

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390 excluded for abnormal structural findings unqualified
391 for inclusion criteria (Supplementary Figure 1). No Changes in functional connectivity induced by 439

392 significant differences were found between the two rTMS 440

393 groups regarding demographic and neuropsycholog- At baseline, the DMN map generated through 441

394 ical characteristics (Supplementary Table 1). the seed of PCG showed no significant difference 442

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between the two groups (rTMS/sham) (Supplemen- 443

395 rTMS improves short-term memory tary Figure 2). The functional connectivity pattern 444

was in line with known areas, roughly includ- 445

396 Analysis of the effects of rTMS on neu- ing the ventral/dorsal medial prefrontal cortex, 446

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397 ropsychological performance improvements revealed PCG/retrosplenial cortex (Rsp) and adjacent pre- 447

398 significant Group × Time interactions (p < 0.05) on cuneus with the addition of lateral parietal cortex [21, 448

399 the memory domain. At the end of intervention 37, 38]. As for the alterations in functional connec- 449

400 (post-rTMS), significant improvement on AVLT- tivity between the PCG node and whole brain area, 450

401 Immediate free recall (AVLT-I) was observed in the clusters revealed significant Group × Time interac- 451
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402 rTMS group (p = 0.002) rather than the sham group tion (p < 0.05, FWE corrected), modeled as the effect 452

403 (p = 0.215) comparing to baseline level (Fig. 1C); of interest. Information regarding the identified clus- 453

404 Scores on AVLT-5 min delayed free recall (AVLT- ters are visualized in Supplementary Table 2. Clusters 454

405 D5) (Fig. 1A) and AVLT-20 min delayed free recall of size with volume greater than 540 mm3 were saved 455

406 (AVLT-D20) (Fig. 1B) were significantly increased as a single map (ROI) and were literally defined on 456

in the rTMS group (p < 0.001 and p = 0.004, respec- the voxels that showed peak activation compared with
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407 457

408 tively), but unchanged in the sham group (p = 0.443 other voxels in the same cluster, namely under this 458
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409 and 0.504, respectively). A better performance trend condition, and they were left anterior cingulate gyrus 459

410 was observed in AVLT-Recognition (AVLT-R) com- (BA32, ACG.L), left superior frontal gyrus (BA8, 460

411 pared to the sham group (p = 0.086) (Fig. 1D) after SFG.L), right fusiform gyrus (BA37, FFG.R), and 461

412 the intervention. As for the follow-up reassessment left supplementary motor area (BA4, SMA.L). We 462

413 (8 weeks after the therapeutic regimen finished), extracted the mean time series of each newly defined 463
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414 significant improvements still existed in the rTMS ROI and calculated its correlation with PCG. Results 464

415 group concerning AVLT-I (p = 0.004), AVLT-D5 were analyzed by mixed-design analysis of variance 465

416 (p = 0.002), AVLT-D20 (p = 0.006), and AVLT-R (ANOVA) again for further validation and correction 466

417 (p = 0.011). Improvements with time were found in (Bonferroni corrected for four comparisons, thresh- 467
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418 ACE-III, TMTA without group differences. No other old at 0.05/4 = 0.0125). With the exception of ACG.L 468

419 significant results appeared in the rest of test scores. (F(1,19) = 3.908, p = 0.063), FFG.R (F(1,19) = 17.154, 469

420 Moreover, no significant correlation in the rTMS p = 0.001), SFG.L (F(1,19) = 11.230, p = 0.003), and 470

421 group was found between percent change in AVLT- SMA.L (F(1,19) = 8.634, p = 0.008) showed signifi- 471
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422 I (where symptomatic improvement was defined as cant Group × Time interaction. Simple main effects 472

423 a positive change in test score: (post-pre)/pre) and analysis was carried out within these connectiv- 473

424 gender (r = 0.067, p = 0.844), education (r = 0.521, ity pairs (PCG-FFG.R/SFG.L/SMA.L) to determine 474

425 p = 0.101), nor baseline AVLT-I score (r = 0.011, the difference between each group (rTMS versus 475

426 p = 0.975), although age (r = –0.797, p = 0.003) and sham) at each time point (pre and post). As shown 476

427 resting motor threshold (RMT) (r = 0.603, p = 0.050) in Fig. 2A, the connectivity pair PCG-ACG.L was 477

428 did correlate significantly with AVLT-I, suggesting approaching significance (p = 0.063). A paired t-test 478

429 that younger patients with less activated corti- showed a tendency toward decrease after interven- 479

430 cal excitability (higher RMT) would gain more tion in the rTMS group (p = 0.094) and the two 480
 H. Cui et al. / Hypoconnected DMN Improves Cognition in aMCI 7

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Fig. 1. Differences in AVLT scores at each time point (post-rTMS, two-month follow-up) from baseline measurements. A) AVLT-5 min
delayed free recall (AVLT-D5), (B) AVLT-20 min delayed free recall (AVLT-D20), (C) AVLT-immediate free recall (AVLT-I) and (D) AVLT-
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Recognition (AVLT-R) changes; bars colored black for rTMS group while red for sham group. *p < 0.05, **p < 0.01. Error bars indicate
SEM.

481 groups were widely divergent in functional connec- functional connectivity (from z(r) = –0.121 to 0.166, 495
co

482 tivity after the therapy (z(r) = 0.179 (rTMS group) p = 0.024) without a baseline difference (p = 0.108) 496

483 to 0.540 (sham group), p = 0.028) without baseline (Fig. 3). The analysis of intervention effect on PCG- 497

484 difference (p = 0.897). Moreover, there was a slight SFG.L portrayed no significant changes in the rTMS 498

485 non-significance (r = –0.600, p = 0.051) for the corre- group while the same analysis of the sham group 499
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486 lation of changes in PCG-ACG.L connectivity with revealed a dramatic increase in functional correlation 500

487 improvements in AVLT-D5 rather than AVLT-D20 (z(r) = –0.142 to 0.353, p = 0.002) (Supplementary 501

488 (r = –0.452, p = 0.163) and other tests (Fig. 2B). The Figure 3). PCG has also been found to be corre- 502

489 connectivity between PCG and FFG.R varied through lated with SMA.L outside the DMN district with a 503

490 the course of intervention, where the rTMS group significant interaction effect. After the intervention, 504

491 showed a dramatic decrease in functional connectiv- the rTMS group had a significantly higher functional 505

492 ity after rTMS therapy (from z(r) = 0.151 to –0.234, connectivity (z(r) = 0.361 to –0.0198, p = 0.038) 506

493 p = 0.003), while in the sham group, an opposite trend compared to the sham group without baseline dif- 507

494 could be observed with a significant increment in ference (p = 0.452) (Supplementary Figure 4). No 508
 8 H. Cui et al. / Hypoconnected DMN Improves Cognition in aMCI

f
roo
rP
tho
Fig. 2. Changes of the functional connectivity generated through PCG demonstrated significant Group × Time interactions for ACG.L with
deactivation effect (p < 0.05, Bonferroni corrected). Clusters of significance were demonstrated in the sagittal, coronal and axial views
(p < 0.05, family-wise error corrected) with mean correlation z(r) of each connectivity pair. A) The functional connectivity between posterior
Au
cingulate gyrus (PCG) and left anterior cingulate gyrus (ACG.L) showed a post-regimen difference of significance (p < 0.05) with a trend for
deactivation in the rTMS group (p = 0.094). Color bars are presented with t values. Bars show means and error bars represent SEM *p < 0.05,
**p < 0.01. B) Marginally significant correlation between changes in PCG-ACG.L connectivity and improvements in AVLT-D5, but not for
AVLT-D20, were seen. Dashed line indicates 95% confidence interval.

509 significant correlation of changes in connectiv- and other previous studies (Fig. 4A). Significant 532

ity between PCG and FFG.R/SFG.L/SMA.L with correlation between baseline functional connectivity
d

510 533

511 rTMS-induced alterations in AVLT scores was found. and neuropsychological test improvements in rTMS 534

In conclusion, all ROIs within the DMN area showed groups after FDR correction was found. Specifi-
cte

512 535

513 intervention-related deactivation (i.e., they presented cally, the baseline functional connectivity between 536

514 with reduction in functional connectivity) while the PCG.R and PCUN.R (r = –0.848, p < 0.05), IPL.L 537

515 only outlier ROI (SMA.L) exhibited a hyperactiva- (r = –0.817, p < 0.05), and PCUN.L (r = –0.850, 538

516 tion tendency after rTMS intervention. p < 0.05), was significantly negatively correlated 539
rre

with AVLT-R alterations in the rTMS group but 540

517 Less activated baseline functional connectivity not the sham group (Fig. 4B-D). However, the 541

518 within DMN indicates a better response toward functional connectivity between PCG.R-PCG.L 542

519 rTMS therapy was not significantly correlated with AVLT-R 543
co

improvement (r = –0.624, p = 0.161) (Fig. 4G). The 544

520 In order to investigate the potential mechanisms functional connectivity between IPL.L and PCG.L, 545

521 for a difference in response regarding cognitive PCUN.L, and vMPFC.L were also negatively 546

522 improvement among the patients receiving thera- correlated with AVLT-R with an expected trend 547
Un

523 peutic rTMS, we further analyzed the relationship (respectively for PCG.L, r = –0.753, p = 0.053; for 548

524 between the improving neuropsychological assess- PCUN.L, r = –0.617, p = 0.081; and for vMPFC.L, 549

525 ments (AVLT-I/R) and the initial status of brain r = –0.624, p = 0.189) (Fig. 4E, F, H). No signif- 550

526 represented by the baseline connectivity within icant correlations were found between AVLT-I 551

527 DMN. We mainly focused on four pairs of ROIs improvement and ROIs, although a negative corre- 552

528 including bilateral precuneus (PCUN.L/R), inferior lation with vMPFC.R-IPL.L (r = –0.765, p = 0.107, 553

529 parietal lobe (IPL.L/R), ventral medial prefrontal FDR corrected) approached significance (Supple- 554

530 cortex (vMPFC.L/R) as well as posterior cingulate mentary Figure 5A). Among the ROIs showing 555

531 gyrus (PCG.L/R) based on the present rTMS results significant Group × Time interaction changing 556
 H. Cui et al. / Hypoconnected DMN Improves Cognition in aMCI 9

f
roo
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tho
Fig. 3. Changes of the functional connectivity generated through PCG demonstrated significant Group × Time interactions for FFG.R with
Au
deactivation effect (p < 0.05, Bonferroni corrected). Clusters of significance were demonstrated in the sagittal, coronal and axial views
(p < 0.05, FWE corrected) with mean correlation z(r). The functional connectivity between PCG and right fusiform gyrus (FFG.R) showed
significant within-group (p < 0.01) as well as between-group (p < 0.01) differences of functional connectivity reduction. Color bars are
presented with t values. Bars show means and error bars represent SEM. *p < 0.05, **p < 0.01.

after rTMS therapy, only PCG-SMA.L showed a rTMS effects on resting-state DMN
d

557 578

558 significant positive correlation with AVLT-R

improvement (r = 0.718, p < 0.05) (Supplementary After rTMS therapy, three regions located within
cte

559 579

560 Figure 5B). the DMN have decreased FC with PCG, and the 580

FC changes between PCG and ACG.L are corre- 581

lated with AVLR improvement. As part of the medial 582

561 DISCUSSION prefrontal cortex that constitutes the anterior DMN 583
rre

(BA32, ACG.L), this result indicated that decreased 584

562 As is well-appreciated, to date, there has been lim- FC within DMN during resting state is responsible 585

563 ited research focusing on the longitudinal changes for the cognitive function improvement. This find- 586

564 of DMN network activity in aMCI and the resting- ing might have far-reaching significance and could 587

help explain a series of previously discovered phe-

co

565 state network modification caused by invasive or 588

566 non-invasive methods of therapeutic intervention. nomena which had indicated the susceptibility to 589

567 Here, in the present study, our results not only AD-related pathology due to excessive metabolic 590

568 provide evidence for the effectiveness of rTMS ther- demand by neural activity and leads to further deduc- 591
Un

569 apy to ameliorate memory deficits of aMCI patients tions regarding the abnormalities of the DMN in 592

570 through improved neuropsychological performance, aMCI patients [16, 49, 50]. During the process of 593

571 but also reveal a potential mechanism associated with aging, as well as the pathological development of cog- 594

572 connectivity-based DMN alterations via rsfMRI. nitive impairment, the DMN is deeply involved, but 595

573 Moreover, our results show for the first time that changes over time have been seen to reflect altered 596

574 less activated baseline functional connectivity (FC) (increased/decreased) internal connections revealed 597

575 within the DMN indicates a better likelihood of by FC, varying according to different stages [21, 598

576 response to rTMS therapy, which itself also generally 51–57]. While a decreased FC in DMN is generally 599

577 leads to DMN deactivation. regarded as a biomarker of functional deterioration 600

 10 H. Cui et al. / Hypoconnected DMN Improves Cognition in aMCI

f
roo
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tho
d Au
Fig. 4. Correlations between symptoms improvements and baseline functional connectivity between pairs of ROIs within DMN (p < 0.05,
FDR corrected). Symptoms improvement is percent change in AVLT-R (post-pre/pre). The black dots are individual data from subjects of
rTMS group, while the red triangle mark represented subjects of sham group. Black and red lines are the lines of best fit for the two group
cte

along with r and p values, respectively. A) Anatomical location of each ROI selected within DMN: posterior cingulate gyrus (PCG.L/R),
inferior parietal lobe (IPL.L/R), precuneus (PCUN.L/R) and ventral medial prefrontal cortex (vMPFC.L/R). B-D) The baseline functional
connectivity between PCG.R and PCUN.R (r = –0.848, p < 0.05), IPL.L (r = –0.817, P < 0.05), PCUN.L (r = –0.850, p < 0.05) revealed negative
correlation of significance with AVLT-R improvement in rTMS group, but not in sham group. G) The functional pair between PCG.L-R
and AVLT-R change (r = –0.624, p = 0.161). E, F, H) IPL.L-PCG.L/PCUN.L/vMPFC.L negatively correlated with AVLT-R with an expected
trend (r = –0.753, p = 0.053; r = –0.617, p = 0.081; and r = –0.624, p = 0.189, respectively).
rre

601 during the stage of AD, explanations vary for the probably already exists and persists from the pro- 616

602 bidirectional changes of connectivity seen in different dromal stage of AD, and remains as the disease 617
co

603 studies with aMCI patients, especially an enhanced progresses to aMCI. Thus, the so-called compen- 618

604 interaction with PCG. Some hypothesize that the satory increase in FC within certain parts of the 619

605 increased FC of certain components within DMN DMN is unlikely to make up for degraded cog- 620

606 could be explained as a “compensatory process” of nitive performance presenting with memory loss 621
Un

607 the brain aiming to make up for the less activated and executive dysfunction. A recent study focus- 622

608 brain regions engaged in the episodic memory pro- ing on asymptomatic individuals with AD family 623

609 cess [51, 58, 59]. Still, other studies questioned the history illustrated a similar scenario [61]. When 624

610 permanence of the response as well as the abil- divided into two separate groups based on subjec- 625

611 ity of enhanced DMN FC to facilitate memory tive cognitive decline (SCD) complaints, SCD [–] 626

612 function [60]. Our finding suggests that this compen- subjects showed lower posterior DMN-medial tem- 627

613 sation (hyperconnectivity within DMN) may not be poral memory system (MTMS) connectivity, and 628

614 effective as expected, and is possibly counterproduc- posterior DMN-MTMS connectivity was negatively 629

615 tive. As aforementioned, the functional abnormality correlated with immediate memory over time [61]. 630
 H. Cui et al. / Hypoconnected DMN Improves Cognition in aMCI 11

631 Another longitudinal study also demonstrated the Baseline functional connectivity predicts 659

632 close relationship between the increasingly overac- treatment response 660

633 tivated DMN and the worsening status of cognition

634 through the course of MCI [62]. Our results fur- It is of vital importance to be able to predict the 661

635 ther indicate that deactivation of the overactivated possible response to rTMS through a resting-state 662

636 DMN in aMCI patients can efficiently reverse mem- fMRI scan because rsfMRI is the most applica- 663

637 ory deficits and is potentially beneficial for the ble, easily-controlled means of imaging that can be 664

638 maintenance and normalization of a normally acti- widely implemented regardless of varying clinical 665

f
639 vated DMN. Meanwhile, there is a growing body of situations. In the present study, the subjects that pre- 666

roo
640 evidence underscoring the unwanted consequences sented with better therapeutic effects were those with 667

641 associated with chronic hyperconnectivity. In the relatively low resting-state functional connectivity at 668

642 context of the growing awareness of the dispro- baseline (no significant correlation was seen in the 669

643 portionately higher A␤ deposition within highly sham group). All baseline connectivity pairs of ROIs 670

644 connected networks (e.g., posterior DMN) due to chosen, including among bilateral PCG, PCUN, IPL, 671

rP
645 elevated metabolism [63, 64], it is possible that the and vMPFC demonstrated obvious negative correla- 672

646 increased brain metabolism caused by local hyper- tion with the improvement in cognitive performance 673

647 connectivity would lead to sites of neurodegeneration seen for the rTMS group, indicating the importance 674

648 resulting in a cascade of network failure [21, 65], of a relative deactivation of DMN preceding a bet- 675

ter treatment response. It can be further deduced that

tho
649 although the relationship between elevated activity 676

650 and amyloid pathology could be reciprocal [66, 67]. patients with less activated DMN during the resting 677

651 Therefore, the risk of progressive cognitive decline state are more likely to be at an earlier stage of disease 678

652 in AD patients with A␤/tau-associated neuronal with relatively favorable prognosis, while those with 679

653 toxicity in hub regions of DMN could be accen- overactivated DMN are more likely to be approach- 680
Au
654 tuated through chronic hyperconnectivity [21]. The ing a later stage of MCI and consequent advanced 681

655 hypothetical developmental sequence of dementia deterioration. Based on the results obtained through 682

656 concerning cognitive decline and the corresponding rsfMRI in the present study, we surmise that the deac- 683

657 changes in activation of certain components of the tivated pattern within certain regions of the DMN 684

658 DMN is illustrated in Fig. 5. during resting state might help ease the difficulty in 685

normalizing the overactivated DMN to reduce its con-

d

686

nectivity back to a normal activation status. Thus, 687

reduced activation in the DMN facilitates the pro-

cte

688

cess of reallocating resources towards task-positive 689

networks under active use, perhaps even when the 690

normal circuits are insufficient or compromised. In 691

turn, deviant hyperactivation within the DMN at rest 692

rre

would hamper the process of deactivation modulation 693

(timely inhibitive response) in response to increasing 694

task load. As a consequence, aMCI patients with a 695

less activated DMN respond better to rTMS treat- 696

co

ment. 697

One limitation of the present study is the absence 698

Fig. 5. Hypothetical model of the clinical trajectory of cognitive of task-related fMRI that could directly monitor the 699
impairment from preclinical stage to AD. Patients in the stage
of mild cognitive impairment (MCI), which follows the preclin-
alteration of functional network during cognitive 700
Un

ical stage of AD, undergo specific symptoms such as memory activities requiring initiative, such as memorizing and 701

loss and simultaneously present with hyperactivated DMN within calculating. Although we discovered a hyperactivated 702
certain circuit components. Based on work in this study, the mech- connection between PCG and SMA.L (a key node of 703
anism of rTMS ameliorating memory deficits is to deactivate the
abnormally overactivated DMN toward its normal activation sta- the sensory-motor network, SMN) (Supplementary 704

tus. Note that this diagram represents a hypothetical model for the Figure 4) and a positive correlation between base- 705

neuropsychological-neurofunctional continuum of AD but does line FC and neuropsychological score improvement 706
not affirm the exact time point of transition from overactivation (Supplementary Figure 5B), this potentially inspiring 707
to deactivation, as well as its relative extent of change. In clinical
practice, situations of different individuals can vary considerably result, indicating the possibility of a rTMS-induced 708

due to variable clinical backgrounds and other confounding factors. useful compensatory circuit beyond DMN (SMN) 709
 12 H. Cui et al. / Hypoconnected DMN Improves Cognition in aMCI

710 cannot be validated without further evidence from ACKNOWLEDGMENTS 759

711 task-induced hyperactivation. However, a factor of

712 unpredictability associated with task-related fMRI This study was supported by the National Natu- 760

713 includes variable degrees of collaboration and com- ral Science Foundation of China (No. 81671043), 761

714 prehension by patients that lead to uncertain impact the “Shuguang Program (16SG15)” supported by 762

715 on the coherence of the assessment. Cognitive fluc- Shanghai Education Development Foundation, the 763

716 tuation is common in aMCI and a reliable outcome Shanghai Municipal Commission of Health and 764

717 obtained through carefully devised task-related fMRI Family Planning (20174Y0021), and the Shanghai 765

f
718 cannot be guaranteed. One of the most important Municipal Education Commission - Gaofeng Clinical 766

roo
719 advantages of rsfMRI imaging, on the other hand, Medicine Grant (20172001). 767

720 is the ability to scan patients who are impaired in The authors would like to thank all the patients 768

721 cognitive function and unable to actively respond to for their participation in this study and Dr. Eric B. 769

722 any task-based scanning paradigm [56]. Meanwhile, Dammer, School of Medicine, Emory University, 770

723 although the changes in DMN we saw have been Atlanta, USA, for critical reading and comments 771

rP
724 consistently reported and replicated across diverse on this manuscript. We thank the three anonymous 772

725 clinical cohorts via different analytical methods, an reviewers for their constructive comments and sug- 773

726 exclusive focus on single network connectivity might gestions. 774

727 have ignored co-morbid disease status or aging pro- Authors’ disclosures available online (https:// 775

tho
728 cesses that beset patients with cognitive decline, www.j-alz.com/manuscript-disclosures/18-1296r2). 776

729 ranging from psychological disturbances such as

730 depression and hypochondriasis to somatic discom- SUPPLEMENTARY MATERIAL 777

731 forts like headache and chronic pain [68–70]. These

732 confounding factors, inescapable as they seem to The supplementary material is available in the 778
Au
733 be, could be carefully controlled through a rigor- electronic version of this article: http://dx.doi.org/ 779

734 ous pre-intervention assessment before enrollment 10.3233/JAD-181296. 780

735 for research. In order to minimize the potential impact

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