High serum neurofilament associates with diffuse white ...appearing white matter (NAWM), which leads to a di ﬀuse neuroaxonal damage not visible in conventional MRI. This dif-fuse

ARTICLE OPEN ACCESS

High serum neurofilament associates withdiffuse white matter damage in MSMaija Saraste PhD Svetlana Bezukladova MSc Markus Matilainen PhD Jouni Tuisku MSc

Eero Rissanen MD PhD Marcus Sucksdorff MD Sini Laaksonen MD Anna Vuorimaa MD

Jens Kuhle MD PhD David Leppert MD PhD and Laura Airas MD PhD

Neurol Neuroimmunol Neuroinflamm 20218e926 doi101212NXI0000000000000926

Correspondence

Dr Saraste

maijasarasteutufi

AbstractObjectiveTo evaluate to which extent serum neurofilament light chain (NfL) increase is related todiffusion tensor imagingndashMRI measurable diffuse normal-appearing white matter (NAWM)damage in MS

MethodsSeventy-nine patients with MS and 10 healthy controls underwent MRI including diffusiontensor sequences and serum NfL determination by single molecule array (Simoa) Fractionalanisotropy and mean axial and radial diffusivities were calculated within the whole and seg-mented (frontal parietal temporal occipital cingulate and deep) NAWM Spearman corre-lations and multiple regression models were used to assess the associations between diffusiontensor imaging volumetric MRI data and NfL

ResultsElevated NfL correlated with decreased fractional anisotropy and increased mean axial andradial diffusivities in the entire and segmented NAWM (for entire NAWM ρ = minus049 p = 0005ρ = 049 p = 0005 ρ = 043 p = 0018 and ρ = 048 p = 0006 respectively) A multipleregression model examining the effect of diffusion tensor indices on NfL showed significantassociations when adjusted for sex age disease type the expanded disability status scaletreatment and presence of relapses In the same model T2 lesion volume was similarlyassociated with NfL

ConclusionsOur findings suggest that elevated serum NfL in MS results from neuroaxonal damage bothwithin the NAWM and focal T2 lesions This pathologic heterogeneity ought to be taken intoaccount when interpreting NfL findings at the individual patient level

From the Turku PET Centre Turku University Hospital and University of Turku (M Saraste SB MM JT ER M Sucksdorff SL AV LA) Division of Clinical Neurosciences (ERM Sucksdorff SL AV LA) Turku University Hospital Finland and Departments of Medicine Biomedicine and Clinical Research Neurologic Clinic and Policlinic (JK DL)University Hospital Basel Switzerland

Go to NeurologyorgNN for full disclosures Funding information is provided at the end of the article

The Article Processing Charge was funded by the authors

This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License 40 (CC BY-NC-ND) which permits downloadingand sharing the work provided it is properly cited The work cannot be changed in any way or used commercially without permission from the journal

Copyright copy 2020 The Author(s) Published by Wolters Kluwer Health Inc on behalf of the American Academy of Neurology 1

MS is an inflammatory autoimmune disease of the CNS inwhich both acute and chronic inflammation lead to de-myelination and neuronal damage1 Neurofilament light chain(NfL) is one of the most promising soluble biomarkers forassessing disease activity in MS2 The cause and underlyingpathology of the increased NfL concentration indicative ofneuroaxonal injury may however vary greatly both inter- andintraindividually Significant CSF or blood NfL elevationshave been shown in association with acute focal inflammationand gadolinium-enhancing lesions in relapsing-remitting MS(RRMS)3ndash8 On the other hand moderate elevations in NfLconcentrations have been measured in patients with chronicprogressive MS568ndash10

We hypothesize that the NfL elevation in the context of moreadvanced MS disease where no signs of acute inflammation arepresent is caused by the diffuse pathologic process in the normal-appearing white matter (NAWM) which leads to a diffuseneuroaxonal damage not visible in conventional MRI This dif-fuse axonal injury in the NAWM can however be sensitivelymeasured using diffusion tensor imaging (DTI)11 Of the DTIscalars fractional anisotropy is highly sensitive for microstructuralchanges overall whereas axial and radial diffusivities are morespecific to axonal and myelin damage respectively12 It is notknown whether DTI scalars associate with NfL in MS

The aim of this study was to measure to which extent serumNfL increase is related to the diffuse damage in the NAWMincluding the type and spatial distribution of these changesTo address this we performed correlation analyses of NAWMDTI and serumNfL levels and showed significant associationsbetween these measures

MethodsStandard protocol approvals registrationsand patient consentsThe study was approved by the Ethical Committee of theHospital District of Southwest Finland Written informedconsent was obtained from all participants according to theDeclaration of Helsinki

Study cohortSeventy-nine patients with MS from the Neurology Out-patient Clinic of the Division of Clinical Neurosciences atthe Turku University Hospital Turku Finland and 10healthy age-matched controls were included in the studyMRI and serum sampling were performed ge30 days after aclinical relapse Clinical disease course disease duration

and patient age were reviewed and the Expanded Dis-ability Status Scale (EDSS)13 score was assessed by theinvestigating neurologist

Serum samplesBlood samples were collected and serum was stored at minus40degCwithin 4 hours of sampling Concentration of serum NfL wasmeasured by single molecule array (Simoa) assay technologyas described previously614

MRI and DTIBrain MRI was performed in Turku PET center with a 3 TMRI Phillips Ingenuity scanner (Philips HealthcareCleveland OH) Conventional MRI (3-dimensional T1-weighted MRI T2 and fluid-attenuated inversion recovery[FLAIR] with spatial resolution of 1 times 1 times 1 mm) and DTIsequences were included in the protocol The details of theimaging protocol have been described previously15 ForDTI sequences the following parameters were used bvalue = 1000 smmminus2 repetition timetime to echo =9500120 milliseconds field of view = 256 times 256 mmspatial resolution 2 times 2 times 2 mm acquisition matrix 128 times128 mm flip angle = 90deg and acceleration factor 2 with 33(n = 15) 64 (n = 48) or 67 gradient directions (n = 16)The number of gradient directions did not have a re-markable impact on fractional anisotropy mean diffusivityradial diffusivity and axial diffusivity15

NAWM region of interest was created by excluding alllesions and the cerebellar white matter from the whitematter region of interest (appendix e-1 linkslwwcomNXIA355) The lesions were identified from FLAIR im-ages using Lesion Segmentation Toolbox16 The NAWMregion of interest was further segmented to 6 subregions(frontal parietal temporal occipital cingulate and deepwhite matter which includes left and right unsegmentedwhite matter and insula) using FreeSurfer software17 Theareas included in subregions are shown in detail in ap-pendix e-2 The FreeSurfer software was also used to definethe volumes of cortical gray matter whole cerebral whitematter NAWM and total T1 and T2 lesion volumesaccording to our previously reported methodology18

The DTI data were preprocessed and analyzed withExploreDTI software19 T1 and raw diffusion-weightedimage files were first flip permuted after which the T1 filewas masked The files were then converted to diffusiontensor maps using robust diffusion tensor estimation afterwhich diffusion tensor maps were corrected for motioneddy current and echo planar imagingsusceptibility

GlossaryDTI = diffusion tensor imaging EDSS = Expanded Disability Status Scale FLAIR = fluid-attenuated inversion recovery IQR =interquartile range NAWM = normal-appearing white matter NfL = neurofilament light chain RRMS = relapsing-remittingMS SPMS = secondary progressive MS

2 Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 1 | January 2021 NeurologyorgNN

induced distortions using the robust estimation of tensorsby outlier rejection tensor estimation method Fractionalanisotropy mean diffusivity radial diffusivity and axialdiffusivity maps were extracted from the corrected DTImaps Coregistering of maps to corresponding T1-weighted images was performed using SPM8 (The Well-come Centre for Human Neuroimaging University Col-lege London) running on MATLAB (The MathWorks

Natick MA) Finally the mean values for fractional an-isotropy mean diffusivity radial diffusivity and axial dif-fusivity within the whole NAWM and in the segmentedregions of NAWM were calculated in MATLAB

Statistical analysisThe statistical analysis was performed using R statistical soft-ware (version 400) The differences between different MS

Table 1 Demographics clinical characteristics volumetric brain MRI data and cingulate DTI-MRI indices of patients withMS and HCs

MS (n = 79) HC (n = 10)pValue RRMS (n = 56) SPMS (n = 23)

pValue

NfL(low)(n = 45)

NfL(high)(n = 34)

pValue

Femalemale 6019 73 07 4511 158 016 378 2311 018

Age 483(43ndash53)

476(44ndash53)

1 474 (42ndash51) 558 (48ndash61) lt0001 462 (42ndash51) 519 (45ndash58) 0003

Durationa 149 (9ndash19) NA mdash 121 (8ndash14) 196 (14ndash25) lt0001 126 (9ndash15) 145 (10ndash21) 009

EDSS score 3 (2ndash375) NA mdash 25 (2ndash3) 4 (35ndash65) lt0001 25 (2ndash3) 35 (3ndash5) 0005

Relapsesb 4 (2ndash6) 3 (2ndash6) 6 (3ndash8) 002 4 (2ndash6) 3 (2ndash625) 10

DMT (yesno) 5425 NA mdash 4511 914 lt0001 3312 2113 033

First linec 28 NA mdash 27 1 mdash 18 10

Second lined 26 NA mdash 18 8 mdash 15 11

NfL (pgmL) 217(15ndash31)

231(21ndash28)

03 188 (14ndash26) 314 (22ndash44) 0001 16 (12ndash19) 319 (29ndash43) lt0001

Volume (cm3)

WM 4521(412ndash485)

4928(463ndash525)

0009 4642 (423ndash501) 4227 (372ndash463) 0002 4521 (415ndash485) 4542 (406ndash481) 06

NAWM 4445(396ndash479)

NA mdash 4578 (407ndash498) 4107 (339ndash446) 0001 4445 (396ndash477) 4345 (391ndash479) 06

GMctx 4288(409ndash465)

473(450ndash512)


T1 lesion 34 (2ndash8) NA mdash 27 (2ndash6) 65 (4ndash10) 0007 31 (2ndash9) 41 (2ndash7) 03

T2 lesion 69 (3ndash18) NA mdash 49 (2ndash12) 15 (13ndash25) lt0001 56 (2ndash15) 96 (4ndash19) 02

Cingulate DTI

FA 046(043ndash048)

049(047ndash050)


MD (mm2s) 091(086ndash096)

083(082ndash087)


AD (mm2s) 141(136ndash146)

135(131ndash139)


RD (mm2s) 065(059ndash071)

058(056ndash062)


Abbreviations AD = axial diffusivity DMT = disease-modifying treatment DTI = diffusion tensor imaging EDSS = Expanded Disability Status Scale FA =fractional anisotropy GMctx = cortical graymatter HC = healthy control MD=meandiffusivity NA = not applicable NAWM=normal-appearingwhitematterNfL = neurofilament light chain RD = radial diffusivity RRMS = relapsing-remitting MS SPMS = secondary progressive MS WM = white matterPatients withMSwere divided into NfL(low) andNfL(high) subgroups based on themedian value of HCs (231 pgmL) Median (interquartile range) values areshown except for sex and DMT where the number of patients is shown The p values are from the Wilcoxon rank-sum test for continuous variables and theFisher exact test for class variables Significant p values are bolded Values of mean axial and radial diffusivities are expressed as e10minus3a Disease duration (years) calculated from the onset of symptomsb Total number of relapses before study onset data of 2 patients are missingc First-line DMT = interferon-beta dimethyl fumarate glatiramer acetate and teriflunomided Second-line DMT = fingolimod natalizumab and rituximab

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 1 | January 2021 3

groups and between patients and healthy controls wereassessed using theWilcoxon rank-sum (Mann-WhitneyU) testHolm multiple comparison adjustment was used in RRMS vssecondary progressive MS (SPMS) vs healthy control multiplecomparisons The division into NfL(low) and NfL(high)subgroups was based on the median NfL value of the healthycontrols

To test our main hypothesis Spearman correlations were calcu-lated to assess the relationships between NfL and DTI indices(fractional anisotropy mean diffusivity radial diffusivity and axialdiffusivity) in the whole NAWM and in NAWM subregionsSpearman correlation was used instead of Pearson correlation toavoid outlier-induced overestimation of correlations The p valuesof correlation analyses were further adjusted with the false dis-covery rate method (Benjamini-Hochberg procedure) for thenumber of investigated parameters (n = 28)20 The NfL valueswere further modeled by the DTI indices and volumetric MRIdata using multiple regression models The logarithm of NfL wasused as the response because nontransformed values led to non-normality of residuals The models were adjusted by sex agedisease type (relapsing-remittingsecondary progressive) theEDSS score disease-modifying treatment (no treatment first line[dimethyl fumarate glatiramer acetate interferon-beta and teri-flunomide] or second line [fingolimod natalizumab and ritux-imab] treatment) and the presence of relapses within 1 yearbefore sampling (yesno) The normality of the residuals waschecked using the Shapiro-Wilk test Variance inflation factorvalues were used to check that independent variables were nothighly correlated with each other Regression coefficients were

standardized tomake themdirectly comparable to each other Forstandardization the regression results weremultiplied by twice theSD of the DTI or volume variables21 The p values of the multipleregression model were adjusted with the false discovery ratemethod for the number of investigated variables (n = 32)

In addition to verify the quality of the data and increase thegeneralizability and comparability of the results we usedSpearman correlation to assess the relationships betweenfollowing parameters NfL with age disease duration theEDSS score parenchymal fractions of NAWM cortical graymatter and T1 and T2 lesion volumes DTI with the EDSSscore and parenchymal fractions of NAWM cortical graymatter and T1 and T2 lesion volumes (results are shown inappendix e-3 linkslwwcomNXIA355) Spearman cor-relation was used instead of Pearson correlation to avoidoutlier-induced overestimation of correlations and becauseSpearman correlation is preferred in case of ordinal vari-ables ie EDSS score All statistical tests were 2 tailed andp = 005 was used as the threshold for statistical significance

Data availabilityThe anonymized raw data will be shared over the next 3 yearson request from a qualified investigator

ResultsThe demographics clinical characteristics and conventionalimaging data of the 79 patients with MS included in the study

Figure 1 Correlation of serum NfL and DTI indices of the whole NAWM in the NfL(high) subgroup

The serum level of NfL is moderately associatedwith fractional anisotropy (A) mean diffusivity(B) axial diffusivity (C) and radial diffusivity (D)The NfL(high) subgroup is comprised of patientswith serum NfL above the median value ofhealthy controls (231 pgmL) Shown areSpearman correlation coefficients (ρ) and p val-ues The black lines indicate the level and di-rection of the relationship AD = axial diffusivityDTI = diffusion tensor imaging FA = fractionalanisotropy MD = mean diffusivity NAWM =normal-appearing white matter NfL = neuro-filament light chain RD = radial diffusivity


Table 2 Spearman correlations between serum NfL concentrations and DTI-MRI indices

All MS NfL(low) NfL(high)

ρ p Value ρ p Value ρ p Value

Entire NAWM

FA minus016 02 minus020 02 minus042 0013

MD 012 03 014 04 047 0005

AD 001 09 005 07 041 0017

RD 013 03 015 03 045 0008

Frontal NAWM


MD 017 01 011 05 047 0005

AD 009 04 004 08 044 0010

RD 015 02 013 04 044 0009

Parietal NAWM


MD 005 07 005 08 043 0011

AD minus004 08 minus003 08 034 0053

RD 007 05 008 06 044 0009

Temporal NAWM


MD 014 02 008 06 043 0012

AD minus003 08 004 08 026 0139

RD 014 02 009 07 038 0026

Occipital NAWM


MD 012 03 017 03 053 0001

AD 004 07 014 04 038 0029

RD 010 04 015 03 048 0004

Cingulate NAWM


MD 008 05 002 09 049 0003

AD 009 04 010 05 046 0007

RD 008 05 004 08 050 0003

Deep NAWM


MD 008 05 010 06 033 0053

AD 003 08 001 09 036 0039

RD 01 04 012 05 036 0036

Abbreviations AD = axial diffusivity DTI = diffusion tensor imaging FA = fractional anisotropy MD = mean diffusivity NAWM = normal-appearing whitematter NfL = neurofilament light chain RD = radial diffusivityPatients with MS were divided into NfL(low) and NfL(high) subgroups based on the median value of healthy controls (231 pgmL) Fractional anisotropy andmean axial and radial diffusivities of the entire NAWMand of 6 parcellated subregions of NAWMwere correlated with serumNfL level in all patients with MSand in NfL(low) and NfL(high) subgroups Shown are Spearman correlation coefficients (ρ) and uncorrected p values Significant p values are boldedSignificance of these correlations was sustained after adjustment using the false discovery ratemethod for the number of DTI parameters (n = 28) except forparietal FA


are shown in table 1 The median (interquartile range [IQR])age of this cohort was 483 (43ndash53) years which is similar tothe age of the healthy individuals in the control group (476[44ndash53] p = 1) Overall the patient cohort was representedwith a quite stable disease as only 13 of the patients (9 RRMSand 1 SPMS) had had a relapse within 1 year before samplingSixty-eight percent of the patients (45 RRMS and 9 SPMS)were on disease-modifying therapy (dimethyl fumarate n = 3fingolimod n = 14 glatiramer acetate n = 5 interferon beta-1a n= 6 natalizumab n = 8 rituximab n = 4 and teriflunomide n =14) In MRI white matter and cortical gray matter volumeswere decreased in patients compared with healthy controlsDTI measures were obtained from both the entire cerebralNAWM and from 6 different NAWM subregions In the cin-gulate area there were significant differences in all 4 DTI pa-rameters in patients with MS compared with healthy controlsfractional anisotropy was decreased whereas mean diffusivityradial diffusivity and axial diffusivity were increased (table 1)

Serum NfL levels in patients with MS andhealthy controlsThe median (IQR) NfL level was higher in patients withSPMS compared with patients with RRMS (314 [22ndash44] vs188 [14ndash26] pgmL p = 0001 table 1) NfL levels in thewhole MS group were not different from healthy controls(217 [15ndash31] vs 231 [21ndash28] pgmL p = 03) regardlesswhether the analysis was performed with age correctionAcute inflammation did not appear to be a factor to affect theNfL level in this cohort with relatively modest acute in-flammatory activity as the median NfL value among thepatients with or without relapse within the previous year wascomparable (223 [16ndash32] vs 186 [15ndash25] pgmL p = 03Wilcoxon rank-sum data not shown)

Characterization of patients with highNfL levelsTo explore associations between increased NfL levels and DTI-measurable diffuse neuroaxonal damage patients with MS weredivided into NfL(high) and NfL(low) subgroups The divisionwas based on the median NfL value measured among healthycontrols (231 [21ndash28] pgmL) In the NfL(high) subgroup theNfL concentration was significantly elevated compared withhealthy controls (p = 0018 table 1) and in the further NfL vsDTI correlation analyses we focused on the NfL(high) subgroupBased on the demographic and clinical data the patients in theNfL(high) subgroupwere at amore advanced stage of the diseaseTheNfL(high) subgroup includedmore patients with SPMS (p =0003) and the patients in the subgroup were also older (p =0003) and had a higher EDSS score (p = 0005 table 1) Inevaluation using conventional MRI the NAWM white matter orcortical gray matter volumes and T1 and T2 lesion loads were notdifferent between NfL(low) and NfL(high) subgroups (table 1)

Associations between NfL and DTIOur results show that DTI metrics of diffuse neuroaxonaldamage within theNAWMassociatewith high serumNfL levelsin MS As a demonstration of this in the NfL(high) subgroup

several (n = 24) significant correlations were found betweenserum NfL levels and NAWM DTI indices both in the entireNAWM and in various brain subregions higher NfL levels wereassociated with lower fractional anisotropy and higher diffusivity(mean axial and radial) in the whole NAWM (figure 1) and inall its subregions except for fractional anisotropy in temporalNAWM axial diffusivity in parietal and temporal NAWM andmean diffusivity in remaining NAWM (table 2) Significance ofthese correlations was sustained after adjustment using the falsediscovery rate method for the number of DTI parameters (n =28) except for parietal fractional anisotropy Results remainedsimilar when the data were analyzed without patients who hadhad a relapse within the previous year before sampling (n = 3data not shown) No correlations between serumNfL levels andDTI metrics were observed in the NfL(low) subgroup or in theoverall MS cohort (table 2)

Multiple regression modeling was performed to further evaluatethe effect of DTI on NfL In the NfL(high) subgroup the DTIindices weremore significantly associated withNfL thanwith theclinical parameters included in the model (sex age disease typeEDSS score treatment and the presence of relapses within 1year before sampling) (table 3) On average 48 of the variancein NfL (SD 44) could be explained by DTI indices and theclinical parameters mentioned above (figure 2) There weremultiple (n = 26) significant associations between NfL and DTIin the whole NAWM and in all its 6 subregions All except 2 ofthe associations remained significant after adjustment with thefalse discovery rate method for the number of investigated var-iables (n = 32) The strongest associations were observed in thecingulate area between higher NfL and higher mean and radialdiffusivity and in deep and frontal NAWM between higher NfLand higher mean diffusivity (figure 2) Analyzing the datawithout the patients with a relapse (n = 3) within the previousyear did not alter the associations (data not shown)

Higher NfL was also associated with lower NAWM andcortical gray matter and higher T2 lesion volumes (figure 2)The volumes and clinical adjustments explained 434494 and 433 of the variance in NfL respectively

DiscussionPresent results demonstrate that the DTI-MRI measures ofNAWM correlate with serumNfL in MS We found that amongpatients with more advanced disease increased serumNfL levelsassociate with DTI measures reflecting diffuse microstructuraldamage ie decreased fractional anisotropy and increasedmeanaxial and radial diffusivities in the NAWM In a multiple re-gression model which was adjusted with sex age disease typeEDSS score treatment and the presence of relapses the DTIindices were more significantly associated with NfL than theabove-mentioned demographic and clinical parameters

The pathophysiology of MS involves acute and chronic mech-anisms that lead to gradual axonal and myelin damage that


Table 3 Multiple regression analyses evaluating separately the effect of DTI-MRI indices or volumetric brain MRI data onNfL in the NfL(high) subgroup of patients with MS

Variable in model DTI Sex Age Type EDSS score DMT Relapses

Entire NAWM

FA 0018 07 08 10 03 04 02

MD 0003 07 09 10 02 04 04

AD 0010 09 10 08 01 07 05

RD 0005 07 10 10 02 04 03

Frontal NAWM

FA 0045 08 10 10 02 07 02

MD 0001 05 07 10 02 06 04

AD 0004 05 09 08 03 07 04

RD 0003 06 09 09 02 06 03

Parietal NAWM

FA 0023 09 09 09 02 05 01

MD 0004 07 07 08 01 05 02

AD 0017 08 10 10 01 06 03

RD 0006 08 08 09 02 05 02

Temporal NAWM

FA 0116 07 07 10 03 05 03

MD 0007 06 10 10 01 05 05

AD 0009 07 08 08 01 05 05

RD 0020 06 09 10 02 05 04

Occipital NAWM

FA 0011 09 06 09 03 03 01

MD 0009 08 08 09 01 06 02

AD 0086 08 07 08 01 08 03

RD 0011 08 08 08 02 05 02

Cingulate NAWM

FA 0004 05 04 08 06 04 03

MD 0001 06 09 08 06 02 07

AD 0003 08 09 08 03 04 07

RD 0001 06 07 08 07 02 06

Deep NAWM

FA 0047 05 06 09 03 05 03

MD 0001 07 09 09 05 02 07

AD 0005 10 08 10 03 04 08

RD 0002 06 08 10 05 02 08

Volume (pf)

NAWM 0019 08 08 08 03 05 05

Continued


eventually manifest by brain atrophy and clinically by steadyworsening of disability in almost all patients over time This diffusepathology which in the majority of cases leads to progressiveworsening of the MS-related symptoms is now the focus of in-terest for patients and physicians as modern disease-modifyingtherapies have led to an almost complete suppression of relapseactivity while their impact on progression is at best modest Ourincomplete understanding of the molecular mechanisms that leadto progression is one of the main reasons for the failure in de-velopment ofmore efficacious therapies for secondary progressiveform of MS Another impediment is the relative insensitivity ofcurrent measurement tools to capture features of progression atsubclinical stages and hence to quantitate the effects of anti-neurodegenerative treatments at a time point where the overallbrain structure is still largely intact Specifically conventional MRIcan only quantitate brain volume loss as an end result of thedisease but does not provide insights into the underlying micro-structural changes DTI is a nonconventional MRI technique thatprovides a measure of such diffuse changes beyond volumetry

NfL is a highly sensitive biomarker to detect neuronal damageand is the first of its kind applicable in blood-derived probes22

However the cause and underlying pathology of the increasedNfL concentration may vary greatly both inter- and intra-individually In clinically isolated syndrome and RRMS acuteNfL elevation is particularly associated with focal in-flammation ie relapses and focal lesion formation4ndash823

However the indication for the contribution of diffuse pa-thology to higher NfL levels has so far been only incidentaland consisted of associations of increased NfL with moreadvanced clinical disability and brain atrophy824 Novelfindings presented here provide more direct evidence for theeffect of diffuse pathologic process in the NAWM on in-creased serum NfL We observed that among a subgroup ofolder and more disabled patients NfL associates both withradial and axial diffusivities In the context of MS these DTIscalars are thought to reflect myelin loss and axonal damagerespectively11 NfL also associated with mean diffusivity thatcan also indicate on axonal andmyelin loss11 The associationswere most pronounced in the cingulate deep and frontalNAWM

NfL differs in 2 important aspects from MRI first as asignal of neurodegeneration it reflects ongoing pathologicprocesses in real time while imaging captures morphologicfeatures that are inherently retrospective Second NfLlevels reflect neuronal damage in the CNS comprehen-sively ie in addition to brain pathology it also capturesspinal cord pathology that is not routinely evaluated byimaging in the workup of MS for individual patients As adownside NfL levels cannot differentiate betweenacute inflammatory and chronic neurodegenerative diseaseactivity Our results suggest that DTI-MRI allows us tocategorize the pathogenic source of NfL at least semi-quantitatively We demonstrate that both diffuse axonaldamage within the NAWM measured using DTI and focalinflammatory white matter lesion load measured usingconventional MRI contributed to the elevated NfL serumlevels This highlights that both focal and diffuse pathologicchanges must be taken into account when levels of NfL inindividual patients are interpreted A somewhat largerproportion of NfL variance was explained by DTI indices inaverage than by T2 lesion load This implies that in non-relapsing patients the ongoing diffuse microstructuraldamage in the NAWM outside lesions might predominateover focal axonal damage that has occurred within lesionsas a source of NfL in serum Accordingly in other neuro-logic disease with brain diffuse pathology such as amyo-trophic lateral sclerosis Alzheimer disease traumatic braininjury and age-related white matter pathology elevatedserum NfL levels have been associated with DTI-measurable diffuse damage within the CNS25ndash28 Thesestudies are further evidence for the concept that braindiffuse chronic neuropathologic mechanisms contribute toNfL release and that nonconventional MRI techniques areable to identify the CNS areas of its morphologic source

Current results demonstrate that the cingulate area is thebrain area with the most prominent white matter tractdamage outside focal lesions Periventricular regions arecharacteristically the brain area with a particularly heavylesion load in MS29 We addressed the question whetherthe cingulum-NAWM DTI abnormalities would arise from

Table 3 Multiple regression analyses evaluating separately the effect of DTI-MRI indices or volumetric brain MRI data onNfL in the NfL(high) subgroup of patients with MS (continued)


GMctx 0004 09 02 04 003 03 06

T2 0019 06 03 07 04 07 03

T1 015 07 04 10 05 07 03

Abbreviations AD = axial diffusivity DMT = disease-modifying treatment DTI = diffusion tensor imaging EDSS = Expanded Disability Status Scale FA =fractional anisotropy GMctx = cortical gray matter MD = mean diffusivity NAWM = normal-appearing white matter NfL = neurofilament light chain pf =parenchymal fraction RD = radial diffusivityPatients withMSwere divided into NfL(low) and NfL(high) subgroups based on themedian NfL value of healthy controls (231 pgmL) Model was adjusted bysex age disease type (relapsing-remittingsecondary progressive) EDSS score treatment (no first- and second-line) and presence of relapses within 1 yearbefore sampling (yesno) Bolded p values indicate that the parameter affected the logarithmofNfL value statistically significantly whenDTI or volume and allthe adjusting variables were in the models


the dirty-appearing white matter due to close proximity to aheavy lesion load but the relative lesion volume in the cingulatearea was not found to be significantly larger compared withlesion load in other regions or in the whole NAWM Thereforeit seems unlikely that differences in the DTI indices would besolely due to dirty-appearing white matter adjacent to lesionswhereas it is more likely that remote lesions lead to DTI changesin the cingulateNAWM throughWallerian degeneration30 Alsoother studies have observed diffusion changes in the cingulatearea of patients with MS31ndash33 Because the cingulum bundle offibers projects from the cingulate gyrus to the entorhinal cortexin the brain allowing for communication between components

of the limbic system the DTI abnormalities may be the mor-phologic substrate for impaired cognitive function and fatiguesymptoms that are typical in progressive MS3435

Present results are in line with previous observations on therelation between NfL concentration and MS disease sub-type with progressive patients having greater NfL levelscompared with relapsing-remitting patients568ndash102324

However the median NfL value of the entire patient cohortwas relatively low compared with healthy controls which isthe main limitation of our study Because of this we werenot able to use the previously defined cutoff values3 for

Figure 2 The effect of DTI-MRI indices or volumetric MRI data on NfL in the NfL(high) subgroup

The NfL(high) subgroup is comprised of patients with serum NfL above the median value of healthy controls (231 pgmL) Logarithmic serum NfL wasmodeled separately by DTI indices of the entire and parcellated NAWM and volumetric brain MRI data using multiple regression analyses Models wereadjusted by sex age disease type (RRMSSPMS) the EDSS score treatment (no first and second line) and presence of relapseswithin 1 year before sampling(yesno) The results are illustrated using dot and whisker plots in which red dots represent standardized regression coefficients and red lines represent theCIs of the estimates Significant p values of the DTI parameters and the percentage of variance in the response that can be explained by the independentvariables (R2) are also shown The results were ordered according to the R2 value All except 2 of the p values (fractional anisotropy of the frontal and deepNAWM) shown in the figure remained significant after adjustment using the false discovery ratemethod for the number of investigated variables (n = 32) AD =axial diffusivity DTI = diffusion tensor imaging EDSS = Expanded Disability Status Scale FA = fractional anisotropy GMctx = cortical gray matter MD =meandiffusivity NAWM = normal-appearing white matter NfL = neurofilament light chain PF = parenchymal fraction RD = radial diffusivity RRMS = relapsing-remitting MS SPMS = secondary progressive MS WM = white matter


NfL(low) and NfL(high) subgroups Instead we used themedian value of healthy controls Although the divider usedin our study was based on a small group of individualssimilar values have been observed in previous largerstudies68 One reason for the low median NfL level couldbe that the patients with RRMS are represented with arelatively benign disease course In addition our patientswith RRMS were sampled during a relapse-free time andhad no signs of ongoing focal inflammatory activity in theMRI Moreover most of the patients (68) were using adisease-modifying treatment at the time of evaluationHence similar as observed in other studies35233637 thisresulted in low serum NfL concentration Following thesimilarity in the NfL levels between healthy controls andpatients with MS the main results of our study are based ona relatively low number of patients In addition the patientnumbers under different immunomodulatory treatmentsvaried substantially However the heterogeneity of thecohort was taken account in the multiple regression modelwhere both EDSS score and disease-modifying treatmentwere used as adjustments

Our findings suggest that elevated serum NfL in MS resultsfrom neuroaxonal damage both within the NAWM and infocal T2 lesions The association between DTI-measurablediffuse microstructural white matter damage and serumNfL isfurther conceptual evidence for the latter being a usefulmonitoring tool in assessing the degree of ongoing neuro-degenerative processes in MS

AcknowledgmentThe authors thank all patients with MS and healthycontrols who participated in this study They also thank thepersonnel of Turku PET Centre for their excellenttechnical assistance

Study fundingThis work was supported by the Finnish Academy the SigridJuselius Foundation

DisclosureThe authors report no disclosures relevant to the manuscriptGo to NeurologyorgNN for full disclosures

Publication historyReceived by Neurology Neuroimmunology amp NeuroinflammationMay 8 2020 Accepted in final form October 21 2020

References1 Reich DS Lucchinetti CF Calabresi PA Multiple sclerosis N Engl J Med 2018378

169ndash1802 Varhaug KN Torkildsen Oslash Myhr KM Vedeler CA Neurofilament light chain as a

biomarker in multiple sclerosis Front Neurol 201910338

Appendix Authors

Author Location Contribution

MaijaSaraste PhD

Turku PET Centre TurkuUniversity Hospital andUniversity of TurkuFinland

Acquisition of dataanalyzed andinterpreted the data anddrafted and revised themanuscript forintellectual content

Appendix (continued)


SvetlanaBezukladovaMSc


Acquisition of dataanalyzed and interpretedthe data and revised themanuscript

MarkusMatilainenPhD


Statistical analysisdrafted the figures andrevised the manuscript

Jouni TuiskuMSc


Major role in theacquisition of data andrevised the manuscript

EeroRissanenMDPhD

Turku PET Centre TurkuUniversity Hospital andUniversity of TurkuDivision of ClinicalNeurosciences TurkuUniversity Hospital TurkuFinland

Designed andconceptualized the studyand acquisition of data

MarcusSucksdorffMD


Major role in theacquisition of data

SiniLaaksonenMD


Acquisition of data

AnnaVuorimaaMD


Acquisition of data

Jens KuhleMD PhD

Neurologic Clinic andPoliclinic Departments ofMedicine Biomedicineand Clinical ResearchUniversity Hospital BaselSwitzerland


DavidLeppert MDPhD


Revised the manuscriptfor intellectual content

Laura AirasMD PhD


Designed andconceptualized the studyand revised themanuscript for intellectualcontent


3 Kuhle J Kropshofer H Haering DA et al Blood neurofilament light chain as abiomarker of MS disease activity and treatment response Neurology 201992e1007ndashe1015

4 Varhaug KN Barro C Bjoslashrnevik K et al Neurofilament light chain predicts diseaseactivity in relapsing-remitting MS Neurol Neuroimmunol Neuroinflamm 20185e422

5 Novakova L Zetterberg H Sundstrom P et al Monitoring disease activity in multiplesclerosis using serum neurofilament light protein Neurology 2017892230ndash2237

6 Disanto G Barro C Benkert P et al Serum neurofilament light a biomarker ofneuronal damage in multiple sclerosis Ann Neurol 201781857ndash870

7 Siller N Kuhle J Muthuraman M et al Serum neurofilament light chain is a bio-marker of acute and chronic neuronal damage in early multiple sclerosis Mult Scler201925678ndash686

8 Barro C Benkert P Disanto G et al Serum neurofilament as a predictor of diseaseworsening and brain and spinal cord atrophy in multiple sclerosis Brain 20181412382ndash2391

9 Ferraro D Guicciardi C De Biasi S et al Plasma neurofilaments correlate withdisability in progressive multiple sclerosis patients Acta Neurol Scand 202014116ndash21

10 Jakimovski D Zivadinov R RamanthanM et al Serum neurofilament light chain levelassociations with clinical and cognitive performance in multiple sclerosis a longitu-dinal retrospective 5-year study Mult Scler 2020261670ndash1681

11 Sbardella E Tona F Petsas N Pantano P DTI measurements in multiple sclerosisevaluation of brain damage and clinical implications Mult Scler Int 20132013671730

12 Tae WS Ham BJ Pyun SB Kang SH Kim BJ Current clinical applications ofdiffusion-tensor imaging in neurological disorders J Clin Neurol 201814129ndash140

13 Kurtzke JF Rating neurologic impairment in multiple sclerosis an expanded disabilitystatus scale (EDSS) Neurology 1983331444ndash1452

14 Kuhle J Barro C Andreasson U et al Comparison of three analytical platforms forquantification of the neurofilament light chain in blood samples ELISA electro-chemiluminescence immunoassay and SimoaClinChemLabMed 2016541655ndash1661

15 Bezukladova S Tuisku J Matilainen M et al Insights into disseminated MS brainpathology with multimodal diffusion tensor and PET imaging Neurol NeuroimmunolNeuroinflamm 20207e691

16 Schmidt P Gaser C Arsic M et al An automated tool for detection of FLAIR-hyperintense white-matter lesions in Multiple Sclerosis Neuroimage 2012593774ndash3783

17 Salat DH Greve DN Pacheco JL et al Regional white matter volume differences innondemented aging and Alzheimerrsquos disease Neuroimage 2009441247ndash1258

18 Rissanen E Tuisku J Vahlberg T et al Microglial activation white matter tractdamage and disability in MS Neurol Neuroimmunol Neuroinflamm 20185e443

19 Leemans A Jeurissen B Sijbers J et al ExploreDTI a graphical toolbox for pro-cessing analysing and visualizing diffusion MR data Presented at the 17th AnnualMeeting of International Society for Magnetic Resonance in Medicine Honolulu HI2009 p 3537

20 Benjamini Y Hochberg Y Controlling the false discovery rate a practical and pow-erful approach to multiple testing J R Stat Soc Ser B 199557289ndash300

21 Gelman A Scaling regression inputs by dividing by two standard deviations Stat Med2008272865ndash2873

22 Khalil M Teunissen CE Otto M et al Neurofilaments as biomarkers in neurologicaldisorders Nat Rev Neurol 201814577ndash589

23 Canto E Barro C Zhao C et al Association between serum neurofilament light chainlevels and long-term disease course among patients with multiple sclerosis followedup for 12 years JAMA Neurol 2019761359ndash1366

24 Hogel H Rissanen E Barro C et al Serum glial fibrillary acidic protein correlates withmultiple sclerosis disease severity Mult Scler 202026210ndash219

25 Menke RA Gray E Lu CH et al CSF neurofilament light chain reflects corticospinaltract degeneration in ALS Ann Clin Transl Neurol 20152748ndash755

26 Moore EE Hohman TJ Badami FS et al Neurofilament relates to white mattermicrostructure in older adults Neurobiol Aging 201870233ndash241

27 Ljungqvist J Zetterberg H Mitsis M Blennow K Skoglund T Serum neurofilamentlight protein as a marker for diffuse axonal injury results from a case series studyJ Neurotrauma 2017341124ndash1127

28 Schultz SA Strain JF Adedokun A et al Serum neurofilament light chain levels areassociated with white matter integrity in autosomal dominant Alzheimerrsquos diseaseNeurobiol Dis 2020142104960

29 Narayanan S Fu L Pioro E et al Imaging of axonal damage in multiple sclerosisspatial distribution of magnetic resonance imaging lesions Ann Neurol 199741385ndash391

30 Ciccarelli O Werring DJ Barker GJ et al A study of the mechanisms of normal-appearing white matter damage in multiple sclerosis using diffusion tensor imagingevidence of Wallerian degeneration J Neurol 2003250287ndash292

31 Pokryszko-Dragan A Banaszek A Nowakowska-Kotas M et al Diffusion tensorimaging findings in the multiple sclerosis patients and their relationships to variousaspects of disability J Neurol Sci 2018391127ndash133

32 Kolasa M Hakulinen U Brander A et al Diffusion tensor imaging and disabilityprogression in multiple sclerosis a 4-year follow-up study Brain Behav 20199e01194

33 Andersen O Hildeman A Longfils M et al Diffusion tensor imaging in multiplesclerosis at different final outcomes Acta Neurol Scand 2018137165ndash173

34 Koenig KA Sakaie KE Lowe MJ et al The relationship between cognitive functionand high-resolution diffusion tensor MRI of the cingulum bundle in multiple sclerosisMult Scler 2015211794ndash1801

35 Pardini M Bonzano L Bergamino M et al Cingulum bundle alterations underliesubjective fatigue in multiple sclerosis Mult Scler 201521442ndash447

36 Sejbaek T Nielsen HH Penner N et al Dimethyl fumarate decreases neurofilamentlight chain in CSF and blood of treatment naıve relapsing MS patients J NeurolNeurosurg Psychiatry 2019901324ndash1330

37 Piehl F Kockum I Khademi M et al Plasma neurofilament light chain levels inpatients with MS switching from injectable therapies to fingolimod Mult Scler 2018241046ndash1054


DOI 101212NXI000000000000092620218 Neurol Neuroimmunol Neuroinflamm

Maija Saraste Svetlana Bezukladova Markus Matilainen et al High serum neurofilament associates with diffuse white matter damage in MS

This information is current as of December 8 2020

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Academy of Neurology All rights reserved Online ISSN 2332-7812Copyright copy 2020 The Author(s) Published by Wolters Kluwer Health Inc on behalf of the AmericanPublished since April 2014 it is an open-access online-only continuous publication journal Copyright

is an official journal of the American Academy of NeurologyNeurol Neuroimmunol Neuroinflamm

MS is an inflammatory autoimmune disease of the CNS inwhich both acute and chronic inflammation lead to de-myelination and neuronal damage1 Neurofilament light chain(NfL) is one of the most promising soluble biomarkers forassessing disease activity in MS2 The cause and underlyingpathology of the increased NfL concentration indicative ofneuroaxonal injury may however vary greatly both inter- andintraindividually Significant CSF or blood NfL elevationshave been shown in association with acute focal inflammationand gadolinium-enhancing lesions in relapsing-remitting MS(RRMS)3ndash8 On the other hand moderate elevations in NfLconcentrations have been measured in patients with chronicprogressive MS568ndash10

We hypothesize that the NfL elevation in the context of moreadvanced MS disease where no signs of acute inflammation arepresent is caused by the diffuse pathologic process in the normal-appearing white matter (NAWM) which leads to a diffuseneuroaxonal damage not visible in conventional MRI This dif-fuse axonal injury in the NAWM can however be sensitivelymeasured using diffusion tensor imaging (DTI)11 Of the DTIscalars fractional anisotropy is highly sensitive for microstructuralchanges overall whereas axial and radial diffusivities are morespecific to axonal and myelin damage respectively12 It is notknown whether DTI scalars associate with NfL in MS

The aim of this study was to measure to which extent serumNfL increase is related to the diffuse damage in the NAWMincluding the type and spatial distribution of these changesTo address this we performed correlation analyses of NAWMDTI and serumNfL levels and showed significant associationsbetween these measures

MethodsStandard protocol approvals registrationsand patient consentsThe study was approved by the Ethical Committee of theHospital District of Southwest Finland Written informedconsent was obtained from all participants according to theDeclaration of Helsinki

Study cohortSeventy-nine patients with MS from the Neurology Out-patient Clinic of the Division of Clinical Neurosciences atthe Turku University Hospital Turku Finland and 10healthy age-matched controls were included in the studyMRI and serum sampling were performed ge30 days after aclinical relapse Clinical disease course disease duration

and patient age were reviewed and the Expanded Dis-ability Status Scale (EDSS)13 score was assessed by theinvestigating neurologist

Serum samplesBlood samples were collected and serum was stored at minus40degCwithin 4 hours of sampling Concentration of serum NfL wasmeasured by single molecule array (Simoa) assay technologyas described previously614

MRI and DTIBrain MRI was performed in Turku PET center with a 3 TMRI Phillips Ingenuity scanner (Philips HealthcareCleveland OH) Conventional MRI (3-dimensional T1-weighted MRI T2 and fluid-attenuated inversion recovery[FLAIR] with spatial resolution of 1 times 1 times 1 mm) and DTIsequences were included in the protocol The details of theimaging protocol have been described previously15 ForDTI sequences the following parameters were used bvalue = 1000 smmminus2 repetition timetime to echo =9500120 milliseconds field of view = 256 times 256 mmspatial resolution 2 times 2 times 2 mm acquisition matrix 128 times128 mm flip angle = 90deg and acceleration factor 2 with 33(n = 15) 64 (n = 48) or 67 gradient directions (n = 16)The number of gradient directions did not have a re-markable impact on fractional anisotropy mean diffusivityradial diffusivity and axial diffusivity15

NAWM region of interest was created by excluding alllesions and the cerebellar white matter from the whitematter region of interest (appendix e-1 linkslwwcomNXIA355) The lesions were identified from FLAIR im-ages using Lesion Segmentation Toolbox16 The NAWMregion of interest was further segmented to 6 subregions(frontal parietal temporal occipital cingulate and deepwhite matter which includes left and right unsegmentedwhite matter and insula) using FreeSurfer software17 Theareas included in subregions are shown in detail in ap-pendix e-2 The FreeSurfer software was also used to definethe volumes of cortical gray matter whole cerebral whitematter NAWM and total T1 and T2 lesion volumesaccording to our previously reported methodology18

The DTI data were preprocessed and analyzed withExploreDTI software19 T1 and raw diffusion-weightedimage files were first flip permuted after which the T1 filewas masked The files were then converted to diffusiontensor maps using robust diffusion tensor estimation afterwhich diffusion tensor maps were corrected for motioneddy current and echo planar imagingsusceptibility

GlossaryDTI = diffusion tensor imaging EDSS = Expanded Disability Status Scale FLAIR = fluid-attenuated inversion recovery IQR =interquartile range NAWM = normal-appearing white matter NfL = neurofilament light chain RRMS = relapsing-remittingMS SPMS = secondary progressive MS







pValue

NfL(low)(n = 45)

NfL(high)(n = 34)

pValue

Femalemale 6019 73 07 4511 158 016 378 2311 018

Age 483(43ndash53)

476(44ndash53)









231(21ndash28)


Volume (cm3)

WM 4521(412ndash485)

4928(463ndash525)





473(450ndash512)




Cingulate DTI

FA 046(043ndash048)

049(047ndash050)


MD (mm2s) 091(086ndash096)

083(082ndash087)


AD (mm2s) 141(136ndash146)

135(131ndash139)


RD (mm2s) 065(059ndash071)

058(056ndash062)
















Entire NAWM


MD 012 03 014 04 047 0005

AD 001 09 005 07 041 0017

RD 013 03 015 03 045 0008

Frontal NAWM


MD 017 01 011 05 047 0005

AD 009 04 004 08 044 0010

RD 015 02 013 04 044 0009

Parietal NAWM


MD 005 07 005 08 043 0011

AD minus004 08 minus003 08 034 0053

RD 007 05 008 06 044 0009

Temporal NAWM


MD 014 02 008 06 043 0012

AD minus003 08 004 08 026 0139

RD 014 02 009 07 038 0026

Occipital NAWM


MD 012 03 017 03 053 0001

AD 004 07 014 04 038 0029

RD 010 04 015 03 048 0004

Cingulate NAWM


MD 008 05 002 09 049 0003

AD 009 04 010 05 046 0007

RD 008 05 004 08 050 0003

Deep NAWM


MD 008 05 010 06 033 0053

AD 003 08 001 09 036 0039

RD 01 04 012 05 036 0036















Entire NAWM

FA 0018 07 08 10 03 04 02

MD 0003 07 09 10 02 04 04

AD 0010 09 10 08 01 07 05

RD 0005 07 10 10 02 04 03

Frontal NAWM

FA 0045 08 10 10 02 07 02

MD 0001 05 07 10 02 06 04

AD 0004 05 09 08 03 07 04

RD 0003 06 09 09 02 06 03

Parietal NAWM

FA 0023 09 09 09 02 05 01

MD 0004 07 07 08 01 05 02

AD 0017 08 10 10 01 06 03

RD 0006 08 08 09 02 05 02

Temporal NAWM

FA 0116 07 07 10 03 05 03

MD 0007 06 10 10 01 05 05

AD 0009 07 08 08 01 05 05

RD 0020 06 09 10 02 05 04

Occipital NAWM

FA 0011 09 06 09 03 03 01

MD 0009 08 08 09 01 06 02

AD 0086 08 07 08 01 08 03

RD 0011 08 08 08 02 05 02

Cingulate NAWM

FA 0004 05 04 08 06 04 03

MD 0001 06 09 08 06 02 07

AD 0003 08 09 08 03 04 07

RD 0001 06 07 08 07 02 06

Deep NAWM

FA 0047 05 06 09 03 05 03

MD 0001 07 09 09 05 02 07

AD 0005 10 08 10 03 04 08

RD 0002 06 08 10 05 02 08

Volume (pf)

NAWM 0019 08 08 08 03 05 05

Continued










GMctx 0004 09 02 04 003 03 06

T2 0019 06 03 07 04 07 03

T1 015 07 04 10 05 07 03



















Appendix Authors


MaijaSaraste PhD








MarkusMatilainenPhD



Jouni TuiskuMSc



EeroRissanenMDPhD



MarcusSucksdorffMD



SiniLaaksonenMD


Acquisition of data

AnnaVuorimaaMD


Acquisition of data

Jens KuhleMD PhD



DavidLeppert MDPhD



Laura AirasMD PhD






















































Reprints









pValue

NfL(low)(n = 45)

NfL(high)(n = 34)

pValue

Femalemale 6019 73 07 4511 158 016 378 2311 018

Age 483(43ndash53)

476(44ndash53)









231(21ndash28)


Volume (cm3)

WM 4521(412ndash485)

4928(463ndash525)





473(450ndash512)




Cingulate DTI

FA 046(043ndash048)

049(047ndash050)


MD (mm2s) 091(086ndash096)

083(082ndash087)


AD (mm2s) 141(136ndash146)

135(131ndash139)


RD (mm2s) 065(059ndash071)

058(056ndash062)
















Entire NAWM


MD 012 03 014 04 047 0005

AD 001 09 005 07 041 0017

RD 013 03 015 03 045 0008

Frontal NAWM


MD 017 01 011 05 047 0005

AD 009 04 004 08 044 0010

RD 015 02 013 04 044 0009

Parietal NAWM


MD 005 07 005 08 043 0011

AD minus004 08 minus003 08 034 0053

RD 007 05 008 06 044 0009

Temporal NAWM


MD 014 02 008 06 043 0012

AD minus003 08 004 08 026 0139

RD 014 02 009 07 038 0026

Occipital NAWM


MD 012 03 017 03 053 0001

AD 004 07 014 04 038 0029

RD 010 04 015 03 048 0004

Cingulate NAWM


MD 008 05 002 09 049 0003

AD 009 04 010 05 046 0007

RD 008 05 004 08 050 0003

Deep NAWM


MD 008 05 010 06 033 0053

AD 003 08 001 09 036 0039

RD 01 04 012 05 036 0036















Entire NAWM

FA 0018 07 08 10 03 04 02

MD 0003 07 09 10 02 04 04

AD 0010 09 10 08 01 07 05

RD 0005 07 10 10 02 04 03

Frontal NAWM

FA 0045 08 10 10 02 07 02

MD 0001 05 07 10 02 06 04

AD 0004 05 09 08 03 07 04

RD 0003 06 09 09 02 06 03

Parietal NAWM

FA 0023 09 09 09 02 05 01

MD 0004 07 07 08 01 05 02

AD 0017 08 10 10 01 06 03

RD 0006 08 08 09 02 05 02

Temporal NAWM

FA 0116 07 07 10 03 05 03

MD 0007 06 10 10 01 05 05

AD 0009 07 08 08 01 05 05

RD 0020 06 09 10 02 05 04

Occipital NAWM

FA 0011 09 06 09 03 03 01

MD 0009 08 08 09 01 06 02

AD 0086 08 07 08 01 08 03

RD 0011 08 08 08 02 05 02

Cingulate NAWM

FA 0004 05 04 08 06 04 03

MD 0001 06 09 08 06 02 07

AD 0003 08 09 08 03 04 07

RD 0001 06 07 08 07 02 06

Deep NAWM

FA 0047 05 06 09 03 05 03

MD 0001 07 09 09 05 02 07

AD 0005 10 08 10 03 04 08

RD 0002 06 08 10 05 02 08

Volume (pf)

NAWM 0019 08 08 08 03 05 05

Continued










GMctx 0004 09 02 04 003 03 06

T2 0019 06 03 07 04 07 03

T1 015 07 04 10 05 07 03



















Appendix Authors


MaijaSaraste PhD








MarkusMatilainenPhD



Jouni TuiskuMSc



EeroRissanenMDPhD



MarcusSucksdorffMD



SiniLaaksonenMD


Acquisition of data

AnnaVuorimaaMD


Acquisition of data

Jens KuhleMD PhD



DavidLeppert MDPhD



Laura AirasMD PhD






















































Reprints
















Entire NAWM


MD 012 03 014 04 047 0005

AD 001 09 005 07 041 0017

RD 013 03 015 03 045 0008

Frontal NAWM


MD 017 01 011 05 047 0005

AD 009 04 004 08 044 0010

RD 015 02 013 04 044 0009

Parietal NAWM


MD 005 07 005 08 043 0011

AD minus004 08 minus003 08 034 0053

RD 007 05 008 06 044 0009

Temporal NAWM


MD 014 02 008 06 043 0012

AD minus003 08 004 08 026 0139

RD 014 02 009 07 038 0026

Occipital NAWM


MD 012 03 017 03 053 0001

AD 004 07 014 04 038 0029

RD 010 04 015 03 048 0004

Cingulate NAWM


MD 008 05 002 09 049 0003

AD 009 04 010 05 046 0007

RD 008 05 004 08 050 0003

Deep NAWM


MD 008 05 010 06 033 0053

AD 003 08 001 09 036 0039

RD 01 04 012 05 036 0036















Entire NAWM

FA 0018 07 08 10 03 04 02

MD 0003 07 09 10 02 04 04

AD 0010 09 10 08 01 07 05

RD 0005 07 10 10 02 04 03

Frontal NAWM

FA 0045 08 10 10 02 07 02

MD 0001 05 07 10 02 06 04

AD 0004 05 09 08 03 07 04

RD 0003 06 09 09 02 06 03

Parietal NAWM

FA 0023 09 09 09 02 05 01

MD 0004 07 07 08 01 05 02

AD 0017 08 10 10 01 06 03

RD 0006 08 08 09 02 05 02

Temporal NAWM

FA 0116 07 07 10 03 05 03

MD 0007 06 10 10 01 05 05

AD 0009 07 08 08 01 05 05

RD 0020 06 09 10 02 05 04

Occipital NAWM

FA 0011 09 06 09 03 03 01

MD 0009 08 08 09 01 06 02

AD 0086 08 07 08 01 08 03

RD 0011 08 08 08 02 05 02

Cingulate NAWM

FA 0004 05 04 08 06 04 03

MD 0001 06 09 08 06 02 07

AD 0003 08 09 08 03 04 07

RD 0001 06 07 08 07 02 06

Deep NAWM

FA 0047 05 06 09 03 05 03

MD 0001 07 09 09 05 02 07

AD 0005 10 08 10 03 04 08

RD 0002 06 08 10 05 02 08

Volume (pf)

NAWM 0019 08 08 08 03 05 05

Continued










GMctx 0004 09 02 04 003 03 06

T2 0019 06 03 07 04 07 03

T1 015 07 04 10 05 07 03



















Appendix Authors


MaijaSaraste PhD








MarkusMatilainenPhD



Jouni TuiskuMSc



EeroRissanenMDPhD



MarcusSucksdorffMD



SiniLaaksonenMD


Acquisition of data

AnnaVuorimaaMD


Acquisition of data

Jens KuhleMD PhD



DavidLeppert MDPhD



Laura AirasMD PhD






















































Reprints







Entire NAWM


MD 012 03 014 04 047 0005

AD 001 09 005 07 041 0017

RD 013 03 015 03 045 0008

Frontal NAWM


MD 017 01 011 05 047 0005

AD 009 04 004 08 044 0010

RD 015 02 013 04 044 0009

Parietal NAWM


MD 005 07 005 08 043 0011

AD minus004 08 minus003 08 034 0053

RD 007 05 008 06 044 0009

Temporal NAWM


MD 014 02 008 06 043 0012

AD minus003 08 004 08 026 0139

RD 014 02 009 07 038 0026

Occipital NAWM


MD 012 03 017 03 053 0001

AD 004 07 014 04 038 0029

RD 010 04 015 03 048 0004

Cingulate NAWM


MD 008 05 002 09 049 0003

AD 009 04 010 05 046 0007

RD 008 05 004 08 050 0003

Deep NAWM


MD 008 05 010 06 033 0053

AD 003 08 001 09 036 0039

RD 01 04 012 05 036 0036















Entire NAWM

FA 0018 07 08 10 03 04 02

MD 0003 07 09 10 02 04 04

AD 0010 09 10 08 01 07 05

RD 0005 07 10 10 02 04 03

Frontal NAWM

FA 0045 08 10 10 02 07 02

MD 0001 05 07 10 02 06 04

AD 0004 05 09 08 03 07 04

RD 0003 06 09 09 02 06 03

Parietal NAWM

FA 0023 09 09 09 02 05 01

MD 0004 07 07 08 01 05 02

AD 0017 08 10 10 01 06 03

RD 0006 08 08 09 02 05 02

Temporal NAWM

FA 0116 07 07 10 03 05 03

MD 0007 06 10 10 01 05 05

AD 0009 07 08 08 01 05 05

RD 0020 06 09 10 02 05 04

Occipital NAWM

FA 0011 09 06 09 03 03 01

MD 0009 08 08 09 01 06 02

AD 0086 08 07 08 01 08 03

RD 0011 08 08 08 02 05 02

Cingulate NAWM

FA 0004 05 04 08 06 04 03

MD 0001 06 09 08 06 02 07

AD 0003 08 09 08 03 04 07

RD 0001 06 07 08 07 02 06

Deep NAWM

FA 0047 05 06 09 03 05 03

MD 0001 07 09 09 05 02 07

AD 0005 10 08 10 03 04 08

RD 0002 06 08 10 05 02 08

Volume (pf)

NAWM 0019 08 08 08 03 05 05

Continued










GMctx 0004 09 02 04 003 03 06

T2 0019 06 03 07 04 07 03

T1 015 07 04 10 05 07 03



















Appendix Authors


MaijaSaraste PhD








MarkusMatilainenPhD



Jouni TuiskuMSc



EeroRissanenMDPhD



MarcusSucksdorffMD



SiniLaaksonenMD


Acquisition of data

AnnaVuorimaaMD


Acquisition of data

Jens KuhleMD PhD



DavidLeppert MDPhD



Laura AirasMD PhD






















































Reprints
















Entire NAWM

FA 0018 07 08 10 03 04 02

MD 0003 07 09 10 02 04 04

AD 0010 09 10 08 01 07 05

RD 0005 07 10 10 02 04 03

Frontal NAWM

FA 0045 08 10 10 02 07 02

MD 0001 05 07 10 02 06 04

AD 0004 05 09 08 03 07 04

RD 0003 06 09 09 02 06 03

Parietal NAWM

FA 0023 09 09 09 02 05 01

MD 0004 07 07 08 01 05 02

AD 0017 08 10 10 01 06 03

RD 0006 08 08 09 02 05 02

Temporal NAWM

FA 0116 07 07 10 03 05 03

MD 0007 06 10 10 01 05 05

AD 0009 07 08 08 01 05 05

RD 0020 06 09 10 02 05 04

Occipital NAWM

FA 0011 09 06 09 03 03 01

MD 0009 08 08 09 01 06 02

AD 0086 08 07 08 01 08 03

RD 0011 08 08 08 02 05 02

Cingulate NAWM

FA 0004 05 04 08 06 04 03

MD 0001 06 09 08 06 02 07

AD 0003 08 09 08 03 04 07

RD 0001 06 07 08 07 02 06

Deep NAWM

FA 0047 05 06 09 03 05 03

MD 0001 07 09 09 05 02 07

AD 0005 10 08 10 03 04 08

RD 0002 06 08 10 05 02 08

Volume (pf)

NAWM 0019 08 08 08 03 05 05

Continued










GMctx 0004 09 02 04 003 03 06

T2 0019 06 03 07 04 07 03

T1 015 07 04 10 05 07 03



















Appendix Authors


MaijaSaraste PhD








MarkusMatilainenPhD



Jouni TuiskuMSc



EeroRissanenMDPhD



MarcusSucksdorffMD



SiniLaaksonenMD


Acquisition of data

AnnaVuorimaaMD


Acquisition of data

Jens KuhleMD PhD



DavidLeppert MDPhD



Laura AirasMD PhD






















































Reprints






Entire NAWM

FA 0018 07 08 10 03 04 02

MD 0003 07 09 10 02 04 04

AD 0010 09 10 08 01 07 05

RD 0005 07 10 10 02 04 03

Frontal NAWM

FA 0045 08 10 10 02 07 02

MD 0001 05 07 10 02 06 04

AD 0004 05 09 08 03 07 04

RD 0003 06 09 09 02 06 03

Parietal NAWM

FA 0023 09 09 09 02 05 01

MD 0004 07 07 08 01 05 02

AD 0017 08 10 10 01 06 03

RD 0006 08 08 09 02 05 02

Temporal NAWM

FA 0116 07 07 10 03 05 03

MD 0007 06 10 10 01 05 05

AD 0009 07 08 08 01 05 05

RD 0020 06 09 10 02 05 04

Occipital NAWM

FA 0011 09 06 09 03 03 01

MD 0009 08 08 09 01 06 02

AD 0086 08 07 08 01 08 03

RD 0011 08 08 08 02 05 02

Cingulate NAWM

FA 0004 05 04 08 06 04 03

MD 0001 06 09 08 06 02 07

AD 0003 08 09 08 03 04 07

RD 0001 06 07 08 07 02 06

Deep NAWM

FA 0047 05 06 09 03 05 03

MD 0001 07 09 09 05 02 07

AD 0005 10 08 10 03 04 08

RD 0002 06 08 10 05 02 08

Volume (pf)

NAWM 0019 08 08 08 03 05 05

Continued










GMctx 0004 09 02 04 003 03 06

T2 0019 06 03 07 04 07 03

T1 015 07 04 10 05 07 03



















Appendix Authors


MaijaSaraste PhD








MarkusMatilainenPhD



Jouni TuiskuMSc



EeroRissanenMDPhD



MarcusSucksdorffMD



SiniLaaksonenMD


Acquisition of data

AnnaVuorimaaMD


Acquisition of data

Jens KuhleMD PhD



DavidLeppert MDPhD



Laura AirasMD PhD






















































Reprints












GMctx 0004 09 02 04 003 03 06

T2 0019 06 03 07 04 07 03

T1 015 07 04 10 05 07 03



















Appendix Authors


MaijaSaraste PhD








MarkusMatilainenPhD



Jouni TuiskuMSc



EeroRissanenMDPhD



MarcusSucksdorffMD



SiniLaaksonenMD


Acquisition of data

AnnaVuorimaaMD


Acquisition of data

Jens KuhleMD PhD



DavidLeppert MDPhD



Laura AirasMD PhD






















































Reprints




















Appendix Authors


MaijaSaraste PhD








MarkusMatilainenPhD



Jouni TuiskuMSc



EeroRissanenMDPhD



MarcusSucksdorffMD



SiniLaaksonenMD


Acquisition of data

AnnaVuorimaaMD


Acquisition of data

Jens KuhleMD PhD



DavidLeppert MDPhD



Laura AirasMD PhD






















































Reprints













Appendix Authors


MaijaSaraste PhD








MarkusMatilainenPhD



Jouni TuiskuMSc



EeroRissanenMDPhD



MarcusSucksdorffMD



SiniLaaksonenMD


Acquisition of data

AnnaVuorimaaMD


Acquisition of data

Jens KuhleMD PhD



DavidLeppert MDPhD



Laura AirasMD PhD






















































Reprints






















































Reprints


















Reprints




Documents

High serum neurofilament associates with diffuse white ...appearing white matter (NAWM), which leads to a di ﬀuse neuroaxonal damage not visible in conventional MRI. This dif-fuse