ARTICLE OPEN ACCESS

In vivo evidence of remote neural degeneration inthe lumbar enlargement after cervical injuryGergely David MSc Maryam Seif PhD Eveline Huber PhD Markus Hupp MD Jan Rosner MD

Volker Dietz MD Nikolaus Weiskopf PhD Siawoosh Mohammadi PhD and Patrick Freund MD PhD

Neurologyreg 2019921-11 doi101212WNL0000000000007137

Correspondence

Prof Freund

PatrickFreundBalgristch

AbstractObjectiveTo characterize remote secondary neurodegeneration of spinal tracts and neurons belowa cervical spinal cord injury (SCI) and its relation to the severity of injury the integrity ofefferent and afferent pathways and clinical impairment

MethodsA comprehensive high-resolution MRI protocol was acquired in 17 traumatic cervical SCIpatients and 14 controls at 3T At the cervical lesion a sagittal T2-weighted scan providedinformation on the width of preserved midsagittal tissue bridges In the lumbar enlargementhigh-resolution T2-weighted and diffusion-weighted scans were used to calculate tissue-specific cross-sectional areas and diffusion indices respectively Regression analyses determinedassociations between MRI readouts and the electrophysiologic and clinical measures

ResultsAt the cervical injury level preserved midsagittal tissue bridges were present in the majority ofpatients In the lumbar enlargement neurodegenerationmdashin terms of macrostructural andmicrostructural MRI changesmdashwas evident in the white matter and ventral and dorsal hornsPatients with thinner midsagittal tissue bridges had smaller ventral horn area higher radialdiffusivity in the gray matter smaller motor evoked potential amplitude from the lower ex-tremities and lower motor score In addition smaller width of midsagittal tissue bridges wasalso associated with smaller tibialis sensory evoked potential amplitude and lower light-touchscore

ConclusionsThis study shows extensive tissue-specific cord pathology in infralesional spinal networksfollowing cervical SCI its magnitude relating to lesion severity electrophysiologic integrity andclinical impairment of the lower extremity The clinical eloquence of remote neurodegenerativechanges speaks to the application of neuroimaging biomarkers in diagnostic workup andplanning of clinical trials

These authors contributed equally to this work

From the Spinal Cord Injury Center Balgrist (GD MS EH MH JR VD PF) University Hospital Zurich University of Zurich Switzerland Wellcome Trust Centre forNeuroimaging (NW SM PF) UCL Institute of Neurology London UK Department of Neurophysics (MS NW PF) Max Planck Institute for Human Cognitive and Brain SciencesLeipzig and Department of Systems Neuroscience (SM) University Medical Center Hamburg-Eppendorf Hamburg Germany

Go to NeurologyorgN for full disclosures Funding information and disclosures deemed relevant by the authors if any are provided at the end of the article

The Article Processing Charge was funded by Wellcome Trust

This is an open access article distributed under the terms of the Creative Commons Attribution License 40 (CC BY) which permits unrestricted use distribution and reproduction in anymedium provided the original work is properly cited

Copyright copy 2019 The Author(s) Published by Wolters Kluwer Health Inc on behalf of the American Academy of Neurology 1

Published Ahead of Print on February 15 2019 as 101212WNL0000000000007137

Besides the primary damage to the lesion site traumatic spinalcord injury (SCI) triggers a cascade of pathologic processesremote from injury12 These are reflected by neuronal dys-function such as premature exhaustion of motor neurons andimpaired sensorimotor function below the level of thelesion3ndash9 Moreover remote dynamic neurodegenerative andreorganizational processes of the neural circuits are believedto play a critical role in the patientsrsquo long-term recovery1 andmight determine the success of future regenerative therapies

To better understand the interaction between degenerativeprocesses at and caudal to a cervical lesion and their relation toelectrophysiologic and clinical measures of the lower extremity3 questions were investigated (1) Are the degenerative pro-cesses in the lumbar enlargement similar to those demon-strated within the high cervical cord above the injury10 (2) Isthere a relationship between lesion severity and the magnitudeof neurodegeneration in the lumbar enlargement (3) Is therea relationship between structural changes at and below the levelof injury and electrophysiologic and clinical measures

We applied high-resolution multimodal MRI to the lumbarenlargement1112 and quantified electrophysiologic and clini-cal measures of lower limb to investigate tissue-specific cordpathology in chronic cervical SCI patients We hypothesizedthat (1) remote tissue-specific neurodegeneration (reflectedby macrostructural and microstructural MRI changes) occursin the lumbar enlargement (2) preserved midsagittal tissuebridges at the lesion site are related to the magnitude of tissue-specific cord pathology in the lumbar enlargement and(3) preserved midsagittal tissue bridges and remote tissue-specific neurodegeneration correlate with electrophysiologicand clinical measures of lower limb function

MethodsStandard protocol approvals registrationsand patient consentsThe study protocol was designed in accordance with theDeclaration of Helsinki and was approved by the local ethicscommittee (EK-2010-0271) All participants provided writteninformed consent prior to study enrollment

ParticipantsSeventeen SCI patients (4 female age 427 plusmn 140 years[mean plusmn SD]) were recruited and admitted to the out-patient clinic at Balgrist University Hospital Zurich Swit-zerland between August 19 2015 and December 6 2016In addition 14 healthy volunteers (4 female age 424 plusmn 172years) formed the control dataset SCI patients fulfilled thefollowing inclusion criteria (1) traumatic cervical SCI (2)no other neurologic or psychiatric disorders (3) no MRIcontraindications and (4) no pregnancy

Clinical examinationIn patients neurologic impairment was assessed by means ofthe International Standards for Neurologic Classification ofSpinal Cord Injury (ISNCSCI) protocol13 Single motor andsensory scores in the ISNCSCI scoring sheet were summedup between L2 and S4-5 neurologic levels and are referred toas lower extremity motor (LEMS) light touch (LELT) andpinprick scores (LEPP) throughout the article Daily life in-dependence (ie self-care respiration sphincter managementand mobility) was assessed by the Spinal Cord IndependenceMeasure (SCIM)14

Electrophysiologic measurementsThe electrophysiologic examinations were conductedaccording to the standard protocol of the European Multi-center Study about Spinal Cord Injury (emsciorg) Abduc-tor hallucis (AH) and tibialis anterior (TA) motor evokedpotentials (MEP) were acquired simultaneously by single-pulse transcranial magnetic stimulation placing the coil at 4cm rostral of Cz provoking a response in the AH and TAmuscles A sample frequency of 2000 Hz biphasic stimulusduration of 200 μs and a band-pass filter of 30 Hzndash1 kHz wereused The time from the stimulation to the muscle responseonset determined the MEP latency and the amplitude wasmeasured from baseline to the highest negative peak of thepotential

To obtain tibial sensory evoked potential (SEP) posteriortibial nerves were stimulated bilaterally at the ankle Thestimulation was performed until a motor response was in-duced Cortical responses were recorded with active electrodeat Czrsquo (2 cm posterior to Cz) and referenced to Fz according

GlossaryAD = axial diffusivityAH = abductor hallucisAIS =American Spinal Injury Association Impairment Scale dGMA = dorsal graymatter area DTI = diffusion tensor imaging FA = fractional anisotropy FOV = field of view GM = gray matter GMA = graymatter area ISNCSCI = International Standards for Neurologic Classification of Spinal Cord Injury LELT = InternationalStandards for Neurologic Classification of Spinal Cord Injury lower extremity light touch score LEMS = InternationalStandards for Neurologic Classification of Spinal Cord Injury lower extremity motor score LEPP = International Standards forNeurologic Classification of Spinal Cord Injury lower extremity pinprick scoreMD = mean diffusivityMEP = motor evokedpotential RD = radial diffusivity ROI = region of interest SC = spinal cord SCA = spinal cord area SCI = spinal cord injurySCIM = Spinal Cord Independence Measure SEP = sensory evoked potential SNR = signal-to-noise ratio TA = tibialisanterior TE = echo time TR = repetition time tSEP = tibial sensory evoked potential vGMA = ventral horn gray matter areaWM = white matter WMA = white matter area

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to the 10ndash20 EEG system The impedance was maintainedunder 5 kΩ Two sets of 150 responses were averaged andsuperimposed The SEP P40 latency wasmeasured as the timefrom the stimulation to the first positive peak of the primarycomplex and the amplitude as the difference between the P40and N50 (first negative) peaks

Means of both sides (left and right) of the electrophysiologicmeasures were used for analysis as MRI readouts wereextracted from the whole cross-section of the spinal cord(SC) Evoked potential latencies were normalized for heightFor MEP and SEP patients without any recordable potentialwere given an amplitude of 0 mVμV Only patients withbilateral responses were included into the latency analysisDue to the small number of participants with bilateral MEPresponses MEP latency analysis was not performed

Image acquisitionAll MRI measurements were performed on a clinical 3TSiemens (Erlangen Germany) SkyraFit system using a stan-dard radiofrequency body coil for transmission and thecombination of 16-channel radiofrequency head and neck coiland standard spine matrix coil for reception Foam positionerswere placed under the knees to reduce the normal cord lor-dosis and maximize the contact between the spine coil and thelower SC In addition an MRI-compatible cervical collar(Laerdal Medical Stavanger Norway) was used to reducemotion in the cervical cord15

At the lesion level a standard clinical sagittal T2-weightedimage was acquired to assess the extent of the lesion Thefollowing settings were used repetition time (TR) 3430 msecho time (TE) 90 ms flip angle 150deg field of view (FOV)218 times 218 times 55 mm3 resolution 034 times 034 times 275 mm3

In the lumbar enlargement high-resolution structural data wereacquired using a T2-weighed 3D multi-echo gradient-echosequence (multi-echo data image reconstruction sequence)The 20 axialndashoblique slices were centered at the widest point ofthe enlargement as appearing in a localizing sagittal T2-weighted image following the procedure described in Yian-nakas et al11 Depending on the participant the widest pointwas located between the T10 and L1 vertebral levels Fourmeasurements were acquired using the following parametersslice thickness 25 mm in-plane resolution 05 times 05 mm3 in-plane FOV192 times 162mm2 TE 19ms TR 44ms flip angle 11degreadout bandwidth 260 Hzpixel 4 measurements total ac-quisition time 832 minutes Zero filling interpolation was usedto double the apparent in-plane resolution (025 times 025 mm2)

A diffusion MRI dataset consisting of 60 diffusion-weighted(b = 500 secondsmm2) and 7 T2-weighted (b = 0 secondsmm2) volumes was also acquired using a reduced-FOV single-shot spin-echo echo-planar imaging sequence with identicalslice prescription as the T2-weighed images Acquisitionmeasures were as follows slice thickness 5 mm (10 gap) in-plane resolution 076 times 076 mm2 in-plane FOV 133 times

30 mm2 TE 71 ms TR 350 ms 58 partial Fourier in phase-encoding direction (anteriorndashposterior direction) The ac-quisition time varied with the participantrsquos heart rate witha nominal acquisition time of 52 minutes The acquisition wascardiac gated (minimal duration between 2 successive trig-gers 1800 ms) to reduce artifacts related to CSF pulsationThe in-plane apparent resolution was doubled by zero fillinginterpolation (038 times 038 mm2)

Image analysis

Lesion segmentationIn SCI patients the lesion appearing as a hyperintense area inthe T2-weighted image was segmented as previously de-scribed16 On the midsagittal slice we quantified the width ofmidsagittal tissue bridges16 which was defined as the width ofthe normal-appearing tissue bundles ventral and dorsal to thevisible lesion The lesion characteristics could not be assessedin 4 out of 17 patients due to artifacts caused by the ortho-pedic fixations

Processing of structural MRI dataAn average of the 4 T2-weighted volumes was created usingserial longitudinal registration (SPM12 MATLAB 2013) toaccount for between-scan motion The averaged image wasresliced to 5 mm slice thickness to increase signal to noiseratio Cross-sectional SC area (SCA) was obtained in all slicesusing the semi-automatic 3D active surface cord segmentationmethod as implemented in JIM 7017 Three slices around theslice having the largest SCA were considered for furthersegmentation and analysis to ensure comparable and re-producible anatomical coverage of the lumbar enlargement11

Gray matter (GM) was segmented manually (using sub-voxelsegmentation in JIM 70) to determine cross-sectional GMarea (GMA) measures White matter area (WMA) was cal-culated as the difference between SCA and GMA Using thesame manual segmentation tools GMA was further sub-divided into bilateral dorsal (dGMA) and ventral horn(vGMA) areas as previously described1018 For the statisticalanalysis the slice-averaged tissue areas including SCA WMAGMA dGMA and vGMA were used

Processing of diffusion MRI dataWe used the SPM-based ACID toolbox for processing thediffusion tensor imaging (DTI) data following the proceduredescribed in our previous studies1920 In short we performedslice-wise registration between all 67 volumes to correct formotion and eddy-current artifacts Then we fitted the diffu-sion tensor model using a robust tensor fitting algorithmwhich has been shown to effectively reduce physiologic arti-facts residual motion artifacts and misregistrations20 Tensorfitting generated the following DTI index maps fractionalanisotropy (FA) mean diffusivity (MD) axial diffusivity(AD) and radial diffusivity (RD) After tensor fitting all DTImaps were registered to the corresponding T2-weighedimage using a nonlinear transformation (BSplineSyn algo-rithm21) implemented in the Spinal Cord Toolbox22 The SC

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GM and white matter (WM) masks generated on the T2-weighted structural image were then applied to the DTI mapsand were manually adjusted if necessary to account for slightregistration errors All binary masks were one-voxel eroded toreduce partial volume effects Finally mean FA MD AD andRD values were extracted from the SC GM and WM binarymasks which were used for subsequent analyses

Statistical analysisAll statistical analyses were performed in Stata 14 (StataCorpLP TX) Age and sex differences between SCI patients andcontrols were assessed usingMann-WhitneyU test and Fisherexact test respectively We excluded 4 SCI patients and 1healthy control from the cross-sectional area and DTI meas-urements due to extensive motion artifacts or signal dropoutFirst to assess remote tissue-specific neurodegenerationcross-sectional tissue areas (SCA WMA GMA vGMA anddGMA) and DTI measures (FA MD AD RD) were com-pared between SCI patients and controls using a 2-sample ttest (1-tailed unequal variances α = 005) Second correla-tion analysis was used to investigate linear associations be-tween these remote MRI readouts and lesion measures (α =

005) Finally the relationships between the MRI readouts(lesion-level and in the lumbar enlargement) electrophysio-logic assessments and clinical scores were assessed usingcorrelation analysis (α = 005)

Data availabilityThe authors certify they have documented all data methodsand materials used to conduct the research presented Ano-nymized data pertaining to the research presented will bemade available by request from qualified investigators

ResultsDemographic clinical electrophysiologic andradiologic characteristicsThere was no significant difference between the SCI patientsand controls in terms of sex (Fisher exact test p = 0370) andage (Mann-Whitney U test z = minus0437 p = 066) Of the17 patients 3 were classified as American Spinal Injury As-sociation Impairment Scale (AIS) A 4 as AIS C and 10 as AISD (table 1) Patients were scanned on average 745 plusmn 600

Table 1 Demographic clinical lesion and neurophysiologic information of the spinal cord injury patients

PatientID Sex

Agey

Timesinceinjurymo

Levelofinjury AIS

LEMS(max50)

LELT(max32)

LEPP(max32)

TBmm

MEPampl(AH)mV

MEPampl(TA)mV

SEPamplμV

MEPlat(AH)ms

MEPlat(TA)ms

SEPlatms

1 M 29 12 C5 A 0 0 0 042 0 0 0 ND ND ND

2 M 23 13 C7 A 0 0 0 0 0 0 0 ND ND ND

3 M 33 32 C5 A 0 0 0 036 0 0 0 ND ND ND

4 F 32 23 C5 C 18 0 0 NA 039 008 0 487 ND ND

5 M 69 4 C2 C 26 9 0 NA 015 0 0 436 ND ND

6 F 40 84 C4 C 1 16 0 124 021 0 009 474 ND 485

7 M 30 135 C7 C 14 16 6 123 010 006 027 ND ND 374

8 M 41 146 C6 D 50 32 32 349 NA NA 193 NA NA 402

9 M 49 181 C8 D 50 16 24 146 NA NA 082 NA NA 373

10 M 27 59 C7 D 23 16 0 141 037 009 380 458 366 385

11 M 41 55 C6 D 44 26 22 NA NA NA NA NA NA NA

12 M 50 91 C4 D 47 16 16 271 095 054 0 441 354 ND

13 M 48 22 C5 D 50 32 29 689 NA NA NA NA NA NA

14 M 64 49 C2 D 45 17 14 366 085 043 123 414 320 541

15 M 68 158 C7 D 50 32 30 265 NA NA 122 NA NA 410

16 M 39 40 T1 D 44 32 32 NA 059 030 131 432 331 438

17 M 43 163 C5 D 29 7 8 240 155 025 212 411 332 563

Abbreviations AH = abductor hallucis AIS = American Spinal Injury Association Impairment Scale amp = amplitude lat = latency LELT = InternationalStandards for Neurologic Classification of Spinal Cord Injury lower extremity light touch score LEMS = International Standards for Neurologic Classification ofSpinal Cord Injury lower extremity motor score LEPP = International Standards for Neurologic Classification of Spinal Cord Injury lower extremity pinprickscore MEP = motor evoked potential NA = not available ND = not defined SEP = sensory evoked potential TA = tibialis anterior TB = width of preservedmidsagittal tissue bridges

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months following the injury Midsagittal tissue bridges werepresent in all incomplete patients (AIS B-D) and in 23complete patients (AIS A) The amplitudes of the recordedsignals are shown in table 1 No bilateral AH and TA MEPsignal was detected in motor complete patients while 2 out of8 motor incomplete patients did not have bilateral TA MEPsignal Tibial SEP (tSEP) signal was not recordable in com-plete and 3 out of 12 incomplete patients The mean (plusmnSD)amplitudes for AH and TAMEP as well as tSEP were 043 plusmn048 mV 015 plusmn 019 mV and 085 plusmn 111 μV respectivelyThe mean latencies for AH MEP TA MEP and tSEP were4441 plusmn 271 3405 plusmn 188 and 4413 plusmn 718 ms respectively

Remote tissue-specific neurodegeneration inthe lumbar enlargementIn the lumbar enlargement SCI patients had lower WMA(minus108 p = 0011) and GMA (minus130 p = 0005) comparedto controls with both ventral GMA (minus93 p = 0046) anddorsal GMA (minus191 p lt 0001) being affected (figure 1 and

table 2) In the atrophied lumbar enlargement patients hadlower FA and AD values in both GM and WM compared tocontrols (figure 2 and table 2) In addition higher RD of WMand lower MD of GM were observed in SCI patients

Relation of tissue bridges to cord pathologyPatients with larger width of the midsagittal tissue bridges hadlarger vGMA (r = 062 p = 0041) (figure 3A) and lower MD(WM r = minus076 p = 0006 GM r = minus086 p lt 0001) and RD(WM r = minus071 p = 0015 GM r = minus087 p lt 0001)(figure 3B)

Relationship between tissue bridges cordpathology and electrophysiologic measuresPatients with larger width of midsagittal tissue bridges hadlarger MEP amplitudes (AH r = 081 p = 0008 TA r =0908 p lt 0001) (figure 3G) but not SEP amplitudes (r =041 p = 0192) In addition smaller MEP amplitudes wereassociated with smaller GMA (AH r = 068 p = 0043 TA r =075 p = 0020) and higher RD in the GM (AH r = minus083 p =0006 TA r = minus089 p = 0001) (figure 3 C and D) Patientswith longer SEP latencies had lower FA in both WM and GM(WM r = minus085 p = 0030 GM r = minus081 p = 0049) (figure3 E and F)

Relation of lesion measures and cordpathology to clinical outcomeAt the lesion level the width of midsagittal tissue bridgescorrelated positively with LEMS (r = 075 p = 0003) LELT(r = 076 p = 0003) LEPP (r = 073 p = 0046) (figure 4AndashC) and SCIM (r = 070 p = 0017) In the lumbar en-largement RD of both GM and WM were negatively corre-lated with LEMS (WM r = minus060 p = 0029 GM r = minus063 p= 0021) LELT (WM r = minus058 p = 0039 GM r = minus062 p= 0025) LEPP (WM r = minus059 p = 0033 GM r = minus063 p =0020) and SCIM (WM r = minus061 p = 0045 GM r = minus070p = 0017 figure 4 DndashF) In addition MD of the WM cor-related negatively with LEMS (r = minus072 p = 0006) LELT(r = minus061 p = 0028) LEPP (r = minus069 p = 0009) and SCIM(r = minus065 p = 0032)

DiscussionWhile the extent of secondary remote changes in the cervicalcord after traumatic SCI has been described in vivo this studyshows in vivo evidence of secondary remote neurodegenera-tive changes affecting infralesional spinal networks We ob-served marked macrostructural (reflected by cross-sectionalarea measurements) and microstructural (reflected by diffu-sion MRI) signs of degeneration in both the GM and WMwithin the lumbar enlargement The magnitude of (trans-synaptic) neurodegeneration was associated with changes inpreserved electrophysiologic information flow of afferent andefferent pathways and lower limb function Thus next totissue-specific supralesional cord pathology102324 the lumbarenlargement after a cervical SCI also undergoes neurode-generative changes both in WM and GM tissue

Figure 1Box plots of tissue-specific cross-sectional areas inthe lumbar enlargement

Spinal cord (SC) gray matter (GM) and white matter (WM) areas are illus-trated in (A) while dorsal GM and ventral GM areas resulting from GMsubsegmentation are plotted in (B) In case of significant group-level dif-ference (p lt 005) the percent group difference is also indicated Both WMand GM were significantly smaller in patients (minus108 and minus130 re-spectively) where dorsal GM contributed proportionally more to the GMatrophy Dots represent outliers that fall belowQ1 minus 15 times interquartile range(IQR) or above Q3 + 15 times IQR (Q1 Q3 = first and third quartiles respectivelyIQR = Q3 minus Q1)

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Remote tissue-specific neurodegeneration inthe lumbar enlargementIn the WM of the lumbar enlargement AD was decreased andRD increased which led to the overall reduction in FA Fur-thermore impaired microstructure also translated to tissueatrophy in the WM These observations are consistent withprevious studies that showed that axonal degeneration includingdisintegration of the axonal skeleton and membrane as well asaccompanying demyelination leads to increased RD and de-creased AD after experimental or human SCI25ndash27 This suggeststhat the dominant histopathologic substrates of the observed invivo human WM changes are likely to be anterograde and ret-rograde degeneration of descending motor pathways and as-cending afferent spinal projections (for an overview of WMdegeneration processes see figure 5)Moreover accumulation ofcellular debris in the extracellular space could further reorganizethe microstructural architecture and lead to reduced anisotropyand diffusivity in the WM28 Interestingly the magnitude ofneurodegeneration in the lumbar enlargement was similar to theabove-level neurodegeneration in the upper cervical cord10

In the GM of the lumbar enlargement we found decreasedAD which led to reduction in MD and FA In the acute and

subacute phases animal SCI models have demonstratedmorphologic changes in the GM including decreased numberbut increased length of the remaining dendrites and enlargedsoma size2930 In humans however remote structural changesin the cordsrsquo GM after SCI are understudied The atrophy ofventral horns presumably reflects transsynaptic degenerationof flexor motor neuron pool due to deprivation from supra-spinal input (figure 5)73132 In contrast the extensor motorneuron pool in the lumbar cord continues to receive pro-prioceptive input to become activated It has been reportedthat the extensor neurons are likely to survive after the injurycompared to flexor neurons5 Similarly dorsal horn neuronsinterneurons and propriospinal networks33 are also prone toundergo a partially transsynaptic degeneration (figure 5)Other mechanisms including vascular remodeling andchanges in the amount of glial cells34 could also play a role inGM pathology however their degree of contribution is notclear

Relation of tissue bridges to cord pathologyFollowing the initial injury a post-traumatic cyst develops inthe majority of patients within the first month16 It has beenshown that the magnitude of such neuronal tissue loss

Table 2 List of tissue-specific cross-sectional areas and diffusion tensor imaging scalar values in the lumbosacralenlargement in both spinal cord injury patients and controls

MRI readout ROI

Lumbar enlargement

Controls Patients Difference p Value

Cross-sectional area mm2 SC 687 plusmn 61 608 plusmn 65 minus116 0002

GM 215 plusmn 28 187 plusmn 23 minus130 0005

WM 472 plusmn 49 421 plusmn 57 minus108 0011

dGM 63 plusmn 07 51 plusmn 09 minus191 lt0001

vGM 108 plusmn 16 98 plusmn 13 minus93 0046

FA SC 046 plusmn 005 042 plusmn 004 minus105 0004

GM 032 plusmn 004 028 plusmn 004 minus102 0023

WM 058 plusmn 004 052 plusmn 003 minus103 lt0001

MD μm2ms SC 104 plusmn 006 100 plusmn 005 minus36 0064

GM 094 plusmn 006 090 plusmn 005 minus42 0044

WM 111 plusmn 007 111 plusmn 006 minus03 0454

AD μm2ms SC 164 plusmn 011 150 plusmn 009 minus82 0002

GM 127 plusmn 009 117 plusmn 008 minus76 0005

WM 193 plusmn 010 182 plusmn 010 minus60 0005

RD μm2ms SC 074 plusmn 006 075 plusmn 006 16 0319

GM 078 plusmn 006 077 plusmn 004 minus15 0302

WM 070 plusmn 007 076 plusmn 005 75 0026

Abbreviations AD= axial diffusivity dGM=dorsal horn graymatter FA = fractional anisotropy GM=graymatterMD=meandiffusivity RD= radial diffusivityROI = region of interest SC = spinal cord vGM = ventral horn gray matter WM = white matterResults are given as group-level mean plusmn SD

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(ie lesion severity) is associated with reduced cross-sectionalGM andWM area above the level of lesion10 In this study weshow relationships between lesion severity (ie preservedmidsaggital tissue bridges) and remote cord pathology belowthe level of injury We found associations between the widthof the midsagittal tissue bridges and the magnitude of ventralhorn atrophy as well as microstructural alterations within thelumbar enlargement Thus the relation between the severityof lesion and below-level neurodegeneration indicates that theinitial damage to the cervical cord primarily drives (initiates)remote neurodegenerative processes3536

Relationship between tissue bridges cordpathology and electrophysiologic measuresElectrophysiologic measures obtained after SCI are predictive offunctional recovery37 We previously demonstrated that pre-served tissue bridges underwrite electrophysiologic communi-cation16 This finding has been confirmed in our data (1) noMEP and SEP signals were present in the patient without tissuebridges and (2) patients with larger width of tissue bridges alsohad larger MEP amplitudes However here we show that notonly does the severity of lesion correlate with the cervical im-pairment of conductivity but it is also associated with the tissue-specific pathology in the lumbar enlargement That is patientswith GM atrophy and altered microstructure (reflected by RDand MD) within the GM had lower MEP amplitudes of theabductor halluces (extensor) and tibialis anterior (flexor)

muscles These associations are thought to reflect transsynapticchanges within both extensor and flexor motor neuron poolsBased on the literature we suggest that the leg extensor moto-neurons are less affected as they continue to receive pro-prioceptive input even after the injury while a loss of flexormotoneurons occurs after deprivation from supraspinal input531

Thus a great part of the neurodegenerative changes observedwithin the GM might directly relate to the loss of supraspinaldrive onto the flexor motor neuron pools

Relation of lesion measures and cordpathology to clinical outcomeThe width of midsagittal tissue bridges measured at 1 monthpost-SCI predicts clinical recovery at 1 year post-SCI16 Inaddition chronic SCI patients show an association betweencervical macrostructural and microstructural changes andclinical impairment10 Our findings are in agreement with thisobservation patients with smaller width of midsagittal tissuebridges had greater functional impairment below the lesionWhile several DTI measures in the WM (MD RD) and GM(MD RD) were related significantly to the motor impair-ment no correlation was found between the ventral horn areain the lumbar enlargement and the ISNCSCI motor scoreThe reason for this might be that motor scores merely reflectmuscle strength quantitatively on a coarse 5-grade scaleHence macrostructural changes within the ventral horn canhardly be translated into measurable functional loss

Figure 2 Box plots of tissue-specific diffusion tensor imaging scalar values (fractional anisotropy [FA] mean diffusivity[MD] axial diffusivity [AD] radial diffusivity [RD]) comparing controls and patients

In case of significant group-level difference (p lt 005) the percent group difference is also reported Spinal cord injury (SCI) patients had lower FA and AD andhigher RD in thewhitematter (WM) In the graymatter (GM) patients had lower FAMD and AD Dots represent outliers that fall belowQ1 minus 15 times interquartilerange (IQR) or above Q3 + 15 times IQR (Q1 Q3 = first and third quartiles respectively IQR = Q3 minus Q1)

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Figure 3 Significant associations between the severity of the lesion (width of midsaggital tissue bridges) remote structuralchanges in the lumbar enlargement and electrophysiologic measures

(A B) Width of midsaggital tissue bridges is associated with cross-sectional gray matter (GM) area and radial diffusivity in the GM of the lumbosacralenlargement (C D) GMarea and radial diffusivity of GMare associatedwithmotor evoked potential (MEP) amplitudes (E F) Fractional anisotropy in thewhitematter (WM) and GM is associated with sensory evoked potential (SEP) latencies (G) Width of tissue bridges is associated with the MEP amplitudes AH =abductor hallucis

8 Neurology | Volume 92 Number 12 | March 19 2019 NeurologyorgN

LimitationsThe study had several limitations First this cross-sectionalstudy included a heterogeneous patient cohort in terms ofinjury severity time since injury and lesion level In additionMRI readouts in the spinal cord have been shown to alter withage and sex38 which might have affected the groupwisecomparisons between DTI measures To address this werecruited sex- and age-matched controls Furthermore theexpected sex- and age-related effects were smaller than theSCI-induced ones

Another limitation is related to the technical feasibility oflumbar cord imaging such as the low signal-to-noise ratio(SNR) susceptibility motion and other physiologic artifacts

We have addressed these issues at the acquisition and imageprocessing stage To compensate for the relatively low SNR ofthe lumbar images due to the application of a spine matrix coil(instead of head and neck surface coil in the cervical coil) wehave (1) placed foam positioners under the knee to increasethe contact between the spine and the coil (2) acquired 4averages for the T2-weighted and relatively many volumes(67) for the DTI dataset and (3) acquired thick slices(5 mm) To compensate for the susceptibility artifacts af-fecting mainly the DTI dataset we have coregistered thedistorted DTI dataset to the nondistorted T2-weightedimages Motion artifacts were probably the biggest issuewhich resulted in the exclusion of 4 patients and 1 healthycontrol due to extensive blurring In addition we used cardiac

Figure 4 Significant associations between the severity of the lesion (width of midsaggital tissue bridges) remote structuralchanges in the lumbosacral enlargement and clinical outcome

(AndashC) Width of midsaggital tissue bridges is associated with ISNCSCI extremity motor light touch and pinprick scores (DndashF) Radial diffusivity in the whitematter (WM) correlates with lower extremity motor light touch and pinprick scores

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gating for the DTI dataset to minimize the effect of CSFpulsation In all participants between-volume motion wascorrected by realigning the T2-weighted and DTI imagesusing serial longitudinal registration and slice-wise linearregistration respectively Finally the application of robusttensor fitting further reduced residual motion artifacts in theDTI dataset as previously demonstrated20

At present automatic segmentation algorithms have beenvalidated only for the cervical GM39 Although the segmen-tation of the cross-sectional area of the lumbar cord was semi-automatic (only the midpoint of the cord had to be setmanually) the GM was segmented manually To be as ac-curate and reliable as possible in the region of interest (ROI)analysis (1) GM was segmented on the T2-weighted imagedue to the better contrast and applied on the DTI dataset aftercoregistration (2) all the segmentation was performed by thesame experienced user (3) all SC GM and WM masks were1-voxel eroded to reduce partial volume effects and (4) allfinal masks were visually inspected and corrected if necessary

However despite the careful approach applied in the ROIselection remaining partial volume effects and imperfectregistration could have affected the ROI-based analysis butwe considered their extent to be minor

DiscussionTracking trauma-induced tissue-specific neurodegenerativeand reorganizational changes in infralesional spinal networksdemonstrates the far-reaching consequences of a focal CNSinjury Our findings suggest that the macrostructural andmicrostructural changes reflect signs of transsynaptic de-generation in sensorimotor pathways These are for examplereflected in the premature exhaustion of motoneurons and animpaired sensorimotor function below the lesion level457

The clinical consequence of remote neurodegenerativechangesmdashincluding axonal and transsynaptic changesmdashfavors the application of multimodal MRI approaches inroutine clinical decision-making and planning of clinical trialsNeuroimaging biomarkers of remote cord pathology offerefficient targeting of therapeutic agents and monitoring inclinical trials

Author contributionsGergely David study concept and design data acquisitionanalysis and interpretation of data statistical analysis writingthe manuscript Maryam Seif analysis of data critical revisionof manuscript for intellectual content Eveline Huber studyconcept and design data acquisition interpretation of datacritical revision of manuscript for intellectual content MarkusHupp data collection interpretation of data critical revisionof manuscript for intellectual content Jan Rosner in-terpretation of data critical revision of manuscript for in-tellectual content Volker Dietz interpretation of data criticalrevision of manuscript for intellectual content NikolausWeiskopf study concept and design critical revision ofmanuscript for intellectual content Siawoosh Mohammadistudy concept and design critical revision of manuscript forintellectual content Patrick Freund study concept and de-sign interpretation of data writing the manuscript studysupervision

AcknowledgmentThe authors thank all the patients and healthy volunteers whoparticipated in this study and the staff of the Department ofRadiology and Neurology at the University Hospital Balgrist

Study fundingPF is funded by a SNF Eccellenza Professorial Fellowshipgrant (PCEFP3_1813621) The study was supported by theInternational Foundation for Research in Paraplegia (IRP-158) MS was funded by Wings for Life Austria (WFL-CH-00714) and the EU project (Horizon2020 ldquoNISCIrdquo grantagreement n_681094) PF NW and SM received fundingfrom the ERANET NEURON (hMRIofSCI) and the BMBF

Figure 5 Secondary degenerative processes occurring re-motely above and below the primary injury site

Sensory and motor tracts affected by the injury undergo anterograde orretrograde (depending on the direction) axonal degeneration and accom-panying demyelination In the lumbar cord the lower motor neurons lo-cated in the ventral horn may undergo transsynaptic degeneration due tothe loss of input from the injured corticospinal tracts Similarly second-order sensory neurons of the spinothalamic and dorsal column mediallemniscus systems can also be affected by transsynaptic degeneration MEP= motor evoked potential SEP = sensory evoked potential

10 Neurology | Volume 92 Number 12 | March 19 2019 NeurologyorgN

(01EW1711A and B) SM was supported by the MarieSklodowska-Curie Individual Fellowship MSCA-IF-2015 (EUHorizon 2020) and the Deutsche Forschungsgemeinschaft(grant MO 23974-1) NW was supported by the EuropeanResearch Council under the European Unionrsquos SeventhFramework Programme (FP72007-2013)ERC grant agree-ment 616905 The Wellcome Trust Centre for Neuroimagingis supported by core funding from the Wellcome Trust 0915Z10Z TheWellcomeTrust Centre for Neuroimaging has aninstitutional research agreement with and receives supportfrom Siemens Healthcare The Article Processing Charge wasfunded by Wellcome Trust grant 091593Z10Z

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

Publication historyReceived by Neurology June 25 2018 Accepted in final form November7 2018

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10 Huber E David G Thompson AJ et al Dorsal and ventral horn atrophy is associatedwith clinical outcome after spinal cord injury Neurology 2018901510ndash1522

11 Yiannakas MC Kakar P Hoy LR Miller DH Wheeler-Kingshott CAM The use ofthe lumbosacral enlargement as an intrinsic imaging biomarker feasibility of greymatter and white matter cross-sectional area measurements using MRI at 3T PLoSOne 20149e105544

12 Yiannakas MC Grussu F Louka P et al Reduced field-of-view diffusion-weightedimaging of the lumbosacral enlargement a pilot in vivo study of the healthy spinalcord at 3T PLoS One 2016111ndash15

13 Kirshblum SCWaringW Biering-Sorensen F et al Reference for the 2011 revision ofthe international standards for neurological classification of spinal cord injury J SpinalCord Med 201134547ndash554

14 Catz A Itzkovich M Steinberg F et al The Catz-Itzkovich SCIM a revised version ofthe spinal cord independence measure Disabil Rehabil 200123263ndash268

15 Yiannakas MC Kearney H Samson RS et al Feasibility of grey matter and whitematter segmentation of the upper cervical cord in vivo a pilot study with applicationto magnetisation transfer measurements Neuroimage 2012631054ndash1059

16 Huber E Lachappelle P Sutter R Curt A Freund P Are midsagittal tissue bridgespredictive of outcome after cervical spinal cord injury Ann Neurol 201781740ndash748

17 Horsfield MA Sala S Neema M et al Rapid semi-automatic segmentation of thespinal cord from magnetic resonance images application in multiple sclerosis Neu-roimage 201050446ndash455

18 Grabher P Mohammadi S David G Freund P Neurodegeneration in the spinalventral horn prior to motor impairment in cervical spondylotic myelopathyJ Neurotrauma 2017342329ndash2334

19 David G Freund P Mohammadi S The efficiency of retrospective artifact correctionmethods in improving the statistical power of between-group differences in spinalcord DTI Neuroimage 2017158296ndash307

20 Mohammadi S Freund P Feiweier T Curt A Weiskopf N The impact of post-processing on spinal cord diffusion tensor imaging Neuroimage 201370377ndash385

21 Tustison NJ Avants BB Explicit B-spline regularization in diffeomorphic imageregistration Front Neuroinform 201371ndash13

22 De Leener B Levy S Dupont SM et al SCT spinal Cord Toolbox an open-sourcesoftware for processing spinal cord MRI data Neuroimage 201714524ndash43

23 Cohen-Adad J El Mendili MM Lehericy S et al Demyelination and degeneration inthe injured human spinal cord detected with diffusion and magnetization transferMRI Neuroimage 2011551024ndash1033

24 Freund P Schneider T Nagy Z et al Degeneration of the injured cervical cord isassociated with remote changes in corticospinal tract integrity and upper limb im-pairment PLoS One 20127e51729

25 Song SK Sun SW Ju WK Lin SJ Cross AH Neufeld AH Diffusion tensor imagingdetects and differentiates axon and myelin degeneration in mouse optic nerve afterretinal ischemia Neuroimage 2003201714ndash1722

26 Zhang J Jones M Deboy CA et al Diffusion tensor magnetic resonance imaging ofWallerian degeneration in rat spinal cord after dorsal root axotomy J Neurosci 2009293160ndash3171

27 Brennan FH Cowin GJ Kurniawan ND Ruitenberg MJ Longitudinal assessment ofwhite matter pathology in the injured mouse spinal cord through ultra-high field(164T) in vivo diffusion tensor imaging Neuroimage 201382574ndash585

28 Beaulieu C Does MD Snyder RE Allen PS Changes in water diffusion due toWallerian degeneration in peripheral nerve Magn Reson Med 2005361ndash5

29 Kitzman P Alteration in axial motoneuronal morphology in the spinal cord injuredspastic rat Exp Neurol 2005192100ndash108

30 Uchida K Baba H Maezawa Y Furukawa S Furusawa N Imura S Histologicalinvestigation of spinal cord lesions in the spinal hyperostotic mouse (twytwyl)morphological changes in anterior horn cells and immunoreactivity to neurotropicfactors J Neurol 1998245781ndash793

31 Lin CS Macefield VG Elam M Wallin BG Engel S Kiernan MC Axonal changes inspinal cord injured patients distal to the site of injury Brain 2007130985ndash994

32 Beauparlant J Van Den Brand R Barraud Q et al Undirected compensatory plasticitycontributes to neuronal dysfunction after severe spinal cord injury Brain 20131363347ndash3361

33 Filli L Schwab ME Structural and functional reorganization of propriospinal con-nections promotes functional recovery after spinal cord injury Neural Regen Res201510509ndash513

34 Schwab ME Bartholdi D Degeneration and regeneration of axons in the lesionedspinal cord Physiol Rev 199676319ndash370

35 Konomi T Fujiyoshi K Hikishima K et al Conditions for quantitative evaluation ofinjured spinal cord by in vivo diffusion tensor imaging and tractography preclinicallongitudinal study in common marmosets Neuroimage 2012631841ndash1853

36 Kelley BJ Harel NY Kim CY et al Diffusion tensor imaging as a predictor oflocomotor function after experimental spinal cord injury and recovery J Neurotrauma2014311362ndash1373

37 Petersen JA Spiess M Curt A et al Upper limb recovery in spinal cord injuryinvolvement of central and peripheral motor pathways Neurorehabil Neural Repair201731432ndash441

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39 Prados F Ashburner J Blaiotta C et al Spinal cord grey matter segmentation chal-lenge Neuroimage 2017152312ndash329

NeurologyorgN Neurology | Volume 92 Number 12 | March 19 2019 11

DOI 101212WNL0000000000007137 published online February 15 2019Neurology

Gergely David Maryam Seif Eveline Huber et al cervical injury

In vivo evidence of remote neural degeneration in the lumbar enlargement after

This information is current as of February 15 2019

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