in vivo diffusion tensor imaging of the rat spinal cord at 9.4t

Technical Note

In Vivo Diffusion Tensor Imaging of the Rat SpinalCord at 9.4T

Benjamin M. Ellingson, MS,1 Shekar N. Kurpad, MD, PhD,2 Shi-Jiang Li, PhD,3 andBrian D. Schmit, PhD1*

Purpose: To determine differences in diffusion measure-ments in white matter (WM) and gray matter (GM) regionsof the rat cervical, thoracic, and cauda equina spinal cordusing in vivo diffusion tensor imaging (DTI) with a 9.4T MRscanner.

Materials and Methods: DTI was performed on seven ratsin three slices at the cervical, thoracic, and cauda equinaregions of the spinal cord using a 9.4T magnet. Axial diffu-sion weighted images (DWIs) were collected at a b-value of1000 seconds/mm2 in six directions. Regions of interestwere identified via T2-weighted images for the lateral, dor-sal, and ventral funiculi, along with GM regions.

Results: Analysis of variance (ANOVA) results indicated sig-nificant differences between every WM funiculus compared toGM for longitudinal apparent diffusion coefficient (lADC),transverse apparent diffusion coefficient (tADC), fractionalanisotropy (FA), measured longitudinal anisotropy (MA1), andanisotropy index (AI). A significant difference in mean diffu-sivity (MD) between regions of the spinal cord was not found.Diffusion measurements were significantly different at eachspinal level. In general, GM regions were significantly differentthan WM regions; however, there were few significant differ-ences between individual WM regions.

Conclusion: In vivo DTI of the rat spinal cord at 9.4Tappears sensitive to the architecture of neural structures inthe rat spinal cord and may be a useful tool in studyingtrauma and pathologies in the spinal cord.

Key Words: spinal cord; MRI; DTI; rat; diffusion tensorimagingJ. Magn. Reson. Imaging 2008;27:634–642.© 2008 Wiley-Liss, Inc.

THE PURPOSE OF THIS STUDY was to test a high-field (9.4T) magnetic resonance (MR) scanner for invivo diffusion tensor imaging (DTI) of the rat spinalcord. DTI has shown great promise for detectingchanges in the spinal cord structure through mea-surements of the diffusion anisotropy, or preferentialdiffusion orientation of water (1). DTI exploits thediffusion properties of water in different tissue typesto delineate anatomical boundaries. Specifically,anisotropic diffusion in the central nervous system isprimarily caused by barriers to diffusion such ascell membranes, and previous studies have shownthat anisotropy increases with an increase in ax-onal fiber density, a decrease in axonal fiber diame-ter, or a decrease in membrane permeability (2). Asa result, changes in diffusion can reflect underly-ing tissue changes associated with injury and heal-ing processes. The development of high-field scan-ners for small animals may make it possible tononinvasively monitor structural changes in the ro-dent spinal cord in experimental models of injury andduring testing of new treatment paradigms using invivo DTI.

In the current study, in vivo diffusion measure-ments of seven rat spinal cords were made using a9.4T scanner. Most measurements of diffusion in therat spinal cord have been limited to ex vivo studies(1,3–5) and in vivo studies using implantable coils(6–12). Recently, Gullapalli et al (13) were able toacquire in vivo diffusion properties of various spinaltract regions at the thoracic level in the rat using a4.7T MR scanner. We hypothesized that better im-ages, specifically images with higher signal-to-noiseratio (SNR), could be acquired using higher fieldstrength while still detecting the anisotropy charac-teristics of spinal cord regions. Consequently, wemeasured the diffusion coefficients for the rat spinalcord with a 9.4T magnet using a standard spin-echodiffusion-weighted (DW) pulse sequence at the cervi-cal, thoracic, and cauda equina levels. In addition,the diffusion characteristics of gray matter (GM) andregional white matter (WM) tracts in the cervical, tho-racic, and cauda equina levels were compared to eachother and to published measurements using otherapproaches and different field strength scanners.

1Department of Biomedical Engineering, Marquette University, Mil-waukee, Wisconsin.2Department of Neurosurgery, Medical College of Wisconsin, Milwau-kee, Wisconsin.3Department of Biophysics, Medical College of Wisconsin, Milwaukee,Wisconsin.Contract grant sponsor: National Institutes of Health; Contract grantnumber: R24-HD039627.*Address reprint requests to: B.D.S., Marquette University, Departmentof Biomedical Engineering, P.O. Box 1881, Milwaukee, WI 53201-1881.E-mail: [email protected] October 6, 2006; Accepted October 2, 2007.DOI 10.1002/jmri.21249Published online in Wiley InterScience (www.interscience.wiley.com).

JOURNAL OF MAGNETIC RESONANCE IMAGING 27:634–642 (2008)

© 2008 Wiley-Liss, Inc. 634

MATERIALS AND METHODS

MRI and Animal Preparation

A total of seven adult female Sprague-Dawley rats(250–300 g) were used in this study. A volume coil(Model T10325; Bruker BioSpin, Ettlingen, Germany)with an inside diameter of 72 mm was used for radiofrequency (RF) transmission and a 2-cm diametersingle-loop surface coil (Model T9208; Bruker Bio-Spin), retrofitted to a custom G10 fiberglass housing,was used for receiving the resulting RF signal. Anes-thetized animals (ketamine and medetomidine at 75mg/kg body weight and 0.5 mg/kg body weight, re-spectively) were placed supine on the surface coilwith the spinal cord centered on the receiving coil.Using tape, animals were firmly fastened to the fiber-glass housing in the region directly above the receivecoil. A respiratory sensor pillow (SA Instruments,Stony Brook, NY, USA) was placed on the abdomen ofthe animals to gate each RF spin-echo (i.e., each lineof k-space) to the same position in the respiratorycycle and to monitor the status of the anesthesia.

The in vivo DTI images were acquired using a 9.4T,31-cm horizontal bore MR scanner (9.4T Bruker Bio-Spec 94/30 USR In vivo Spectroscopy Imaging Sys-tem; Bruker BioSpin), which provides a homogeneousfield to within 0.1 ppm over 70 mm with 12 user-adjustable shim coils and gradient coils capable ofproducing 100 mT/m. Fast low-angle shot (FLASH)images were used as scouts for the subsequent diffu-sion images. A single T2-weighted image (b � 0 sec-onds/mm2) and DW images were acquired at TE/TR � 31 msec/500 msec, slice thickness � 2 mm,field-of-view (FOV) � 3.84 cm, number of excitation(NEX) � 1, and an acquisition matrix � 128 � 128using a standard spin-echo DW sequence. Addition-ally, DW images had a diffusion gradient pulse width(�) of 3 msec, a duration between diffusion gradientpulses (�) of 15 msec, and resulting effective b-valuesranging from 995–1011 seconds/mm2 depending onorientation with respect to imaging gradients (targetb-value � 1000 seconds/mm2) with images acquiredin a total of six DW directions ([� 0.33, 0.67, 0.67],[0.67, � 0.33, 0.67], [0.67, 0.67, � 0.33]). In order tolimit cardiac related aliasing artifacts, we chose thephase-encode direction in the left-right (L-R) orienta-tion and the frequency-encode direction in the ante-rior-posterior (A-P) orientation.

Three images were collected at the midcervical region(1 cm caudal to the base of the skull), T8 thoracic level(center of surface coil placed on T8 vertebral body), andthe cauda equina region (approximately 2 cm rostral toiliac crest) for a total of nine images per specimen.Images were oriented oblique to the main field gradientsso the image plane was perpendicular to the orientationof the spinal cord. Image acquisition in each of the threeregions (cervical, thoracic, cauda) took an average of 23minutes to complete. Total image acquisition and spec-imen preparation took an average of 1.8 hours per spec-imen. The position of the animal was centered on thereceive coil before each new scan location was mea-sured. The SNR was calculated for the T2 images at

each spinal level by using the technique described byKaufman et al (14).

DTI Processing and Region of Interest Selection

Images were imported into AFNI (http://afni.nimh.nih-.gov/afni) for analysis. The diffusion weighted images(DWIs) were coregistered with the T2-weighted imagesin AFNI to correct for eddy-current and susceptibilitydistortions, and then exported to Matlab using the AFNIMatlab Library functions. The effective b-value and ac-tual gradient directions calculated by the Bruker™ sys-tem were imported into Matlab to accurately calculatethe tensor. Longitudinal and transverse apparent diffu-sion coefficients (tADCs) (defined as largest eigenvalue,�1, and average of smaller eigenvalues, (�2 � �3)/2,respectively), mean diffusivity (MD) (defined as the av-erage of the three eigenvalues, or (�1 � �2 � �3)/3),fractional anisotropy (FA) maps, measured longitudinalanisotropy (MA1) (defined as the difference between thelargest eigenvalue and MD, or �1 � MD) and anisotropyindex (AI) (defined as tADC/longitudinal apparent dif-fusion coefficient [lADC]) were calculated in Matlab andused for further analysis.

Regions of interest (ROIs) were selected in the dor-sal funiculus (DF), ventral funiculus (VF), left lateralfuniculi (LLF), right lateral funiculi (RLF), and GMregions in the three slices from the cervical and tho-racic regions of the spinal cord for each animal (Fig.1a and b) using the T2-weighted images (Fig. 1d ande and Fig. 1g and h). Right and left GM regions weregrouped together into a single ROI for statistical anal-ysis. A single region was selected from each of thethree slices in the cauda equina region. ROI maskswere retained from those placed on the T2-weightedimages and used to extract precisely the same ROI foreach DTI parameter map (lADC, tADC, MD, FA, MA1,and AI). ROIs selected from the three consecutiveslices from each ROI were averaged for each respec-tive spinal level and then averaged across all animals.In addition, we explored the FA values in the specificWM tracts defined by Gullapalli et al (13), which in-cluded the rubrospinal (RubSp), vestibulospinal(VSp), dorsal corticospinal (dCSp), and reticulospinal(RetSp) tracts, as well as the fasciculus gracilis (FG)within the thoracic spinal cord.

Phantom Study

Using the same pulse sequence and data processingmethods, a water phantom was scanned on two dif-ferent days to verify the consistency of our technique.The phantom was not moved within the scanner be-tween sessions, resulting in identical alignment of theimages. An identical region of low anisotropy wasselected and compared between the two scan ses-sions (named Phantom 1 and Phantom 2) on the samewater phantom.

Statistical Analysis

A total of five statistical tests were conducted in thisstudy, aimed at testing five different hypotheses. First,diffusion measurements in a targeted ROI from the wa-

In Vivo DTI of Rat Spinal Cord at 9.4T 635

ter phantom were compared using a paired t-test toverify system stability and consistency of the pulse se-quence. Second, to test whether the diffusion measure-ments of WM regions were similar across level (fixedfactor: cervical, thoracic, and cauda levels) and acrossspecimens (random factor), a repeated-measures anal-ysis of variance (ANOVA) was conducted for each of thediffusion measurements. If the difference was signifi-cant, post hoc analysis was performed using a pairedt-test with Bonferroni adjustment for multiple compar-isons. Third, to test whether the diffusion measure-ments varied across various regions in the spinal cord(fixed factor: dorsal funiculi, ventral funiculi, left lateralfuniculus, right lateral funiculus, and GM) and withinspecimens (random factor), a repeated-measuresANOVA was conducted for each of the diffusion mea-surements, with level included as a fixed factor. If thedifference was significant, post hoc analysis was per-formed using a paired t-test with Bonferroni adjust-ment for multiple comparisons. Next, to test whetherthere is a difference in diffusion characteristics betweenlevels and regions within a single animal, a two-wayANOVA (level � ROI) was conducted for each of the

diffusion measurements. Tukey’s test was used formultiple comparisons. Lastly, a one-way repeated-mea-sures ANOVA was used to examine the FA in specificWM tracts (fixed factor: RubSp, RetSp, FG, dCSp, VSp)across specimens (random factor). If the difference wassignificant, post hoc analysis was performed using apaired t-test with Bonferroni adjustment for multiplecomparisons. Statistical analyses were conducted us-ing Minitab statistical software (Minitab� for Windows;Minitab Inc., State College, PA, USA; http://www.minitab.com). The level of significance was set at �0.05 for all statistical tests.

RESULTS

Typical images of the lADC, tADC, MD, and FA for thecervical, thoracic, and cauda equina regions are shownin Fig. 2. Contrast between regions of white and GM inthe lADC, tADC and FA was observed, with little con-trast observed between these tissue types in the MDimages. The central region in the cauda equina showedlow values of diffusion, corresponding anatomically

Figure 1. Schematic of the rat cervical spinal cord (a), thoracic spinal cord (b), and cauda equina region (c). Selected regions ofinterest in the cervical and thoracic region are illustrated for GM, dorsal funiculi, lateral funiculi, and ventral funiculi. Selectedregions of interest in the cauda equina are illustrated for cauda WM. T2-weighted images of the cervical spinal cord (d), thoracicspinal cord (e), and cauda equina (f) are illustrated alongside T2-weighted images with selected ROIs (g–i). Note that images wereevaluated in standard radiological orientation.

636 Ellingson et al.

with the filum terminale, and high diffusion in the re-gions surrounding the central portion, correspondinganatomically to areas containing lumbar and sacralspinal nerves. Diffusion characteristics of the cervical,thoracic, and cauda levels are shown in Table 1. TheSNR for the T2 images was 31.25 � 5.91 (range �23.45–39.02) in the cervical region, 27.80 � 2.40(range � 24.81–32.33) in the thoracic region, and25.92 � 7.61 (range � 16.23–37.63) in the region of thecauda equina.

In general, diffusion parameters were significantlydifferent between each of the spinal levels. Evaluationof WM regions across the cervical, thoracic, and caudaspinal levels using a repeated-measures ANOVA indi-cated significant differences when examining all diffu-sion measurements; specifically, significant differencesbetween levels were apparent in the lADC (P 0.001),tADC (P 0.001), MD (P 0.001), FA (P 0.001), MA1(P 0.001), and AI (P 0.001). Post hoc analysis indi-cated significant differences in all diffusion measure-ments (lADC, tADC, MD, FA, MA1, and AI) between alllevels (cervical, thoracic, and cauda).

Repeated-measures ANOVAs indicated significantdifferences in diffusion anisotropy between the variouswhite and GM regions within the spinal cord. Generally,differences between the WM regions in the same levelwere not significant, although a few exceptions werenoted. A summary of the differences is shown in theright column in Table 1. ANOVA results indicated therewere significant differences in lADC, tADC, FA, MA1,and AI between ROIs (P 0.001) and levels (P 0.05).Further, post hoc analyses indicated significant differ-ences between GM and the WM tracts (DF, VF, LLF,RLF) for lADC (P 0.0001), FA (P 0.0001), MA1 (P

0.0001), and AI (P 0.0001) within the same level.Conversely, there were no significant differences be-tween the various WM tracts for lADC, FA, MA1, or AI(see Table 1 for P values) within the same level. The posthoc analysis for tADC was unique in that there weresignificant differences between GM and DF, LLF, RLF (P 0.0001), but not between GM and VF (P � 0.068).Further investigation indicated significant differencesin tADC between the lateral funiculi compared with theventral funiculi, specifically LLF vs. VF (P � 0.048) andRLF vs. VF (P � 0.0345). ANOVA results indicated MDwas not significantly different between ROIs (P � 0.095)or levels (P � 0.076).

The results from the two-way ANOVA used to exam-ine differences in diffusion characteristics within a sin-gle specimen showed similar results to differences seenacross specimens (Table 2). Specifically, lADC, tADC,and MA1 showed a significant difference between thecervical and thoracic levels (Tukey test, P 0.001) andthe cervical spinal cord showed a significant differencebetween WM regions compared to GM (DF vs. GM, P 0.001; VF vs. GM, P 0.001; LLF vs. GM, P 0.001;RLF vs. GM, P 0.001). There were no significant dif-ferences between regions in the thoracic spinal cord fortADC, lADC, or MA1. Similar to trends observed intADC and lADC, mean diffusivity (MD) showed signifi-cant differences between WM regions and GM in thecervical spinal cord (DF vs. GM, P 0.001; RLF vs. GM,P � 0.020; LLF vs. GM, P � 0.018; VF vs. GM, P �0.047). Significant differences in FA between most WMand GM regions were observed in both the cervical andthoracic levels; however the VF was not significantlydifferent from GM (VF vs. GM, P � 0.058). The AI wassignificant different between the cervical and thoracic

Figure 2. Representative images for lADC (first column from left), tADC (second column from left), MD (third column from left),and FA (right column) for the cervical spinal cord (first row from top), thoracic spinal cord (second row from top), and caudaequina (bottom row).


Tab

le1

Diff

usio

nC

hara

cter

istic

sin

Whi

tean

dG

ray

Mat

ter

ofR

atC

ervi

calS

pina

lCor

d,T

hora

cic

Spi

nalC

ord,

and

Cau

daE

quin

aE

valu

ated

Acr

oss

Spe

cim

ens

(N�

7)

DT

Ipa

ram

eter

sS

pina

lle

vel

(1)

Whi

tem

atte

r(2

)G

ray

mat

ter

(3)

Dor

sal

funi

culu

s(4

)V

entr

alfu

nicu

lus

(5)

Left

late

ral

funi

culu

s

(6)

Rig

htla

tera

lfu

nicu

lus

Diff

eren

ces

Bet

wee

nW

Mtr

acts

N(v

oxel

s/sp

ecim

en)

Cer

vica

l78

–120

62–1

0620

–31

18–3

818

–27

21–3

1T

hora

cic

49–8

428

–75

10–1

811

–26

10–1

88–

20C

auda

37–6

2A

cros

ssp

ecim

enco

mpa

rison

s(N

�7)

Long

itudi

nala

ppar

ent

diffu

sion

coef

ficie

nt(lA

DC

)(�

10�

3

mm

2/s

econ

d)

Cer

vica

l0.

851

�0.

008

0.72

4�

0.00

80.

857

�0.

015

0.85

4�

0.01

80.

840

�0.

017

0.85

2�

0.01

73,

4,5,

6vs

.2

Tho

raci

c0.

719

�0.

013

0.70

6�

0.00

20.

731

�0.

026

0.72

4�

0.02

20.

699

�0.

027

0.72

1�

0.02

6C

auda

0.96

8�

0.01

7T

rans

vers

eap

pare

ntdi

ffusi

onco

effic

ient

(tA

DC

)(�

10�

3

mm

2/s

econ

d)

Cer

vica

l0.

230

�0.

003

0.26

2�

0.00

40.

227

�0.

006

0.24

9�

0.00

80.

219

�0.

007

0.22

6�

0.00

63,

5,6

vs.

2T

hora

cic

0.24

6�

0.00

40.

278

�0.

006

0.24

1�

0.00

90.

259

�0.

009

0.24

1�

0.00

80.

240

�0.

008

5,6

vs.

3C

auda

0.38

4�

0.00

8M

ean

diffu

sivi

ty(M

D)

(�10

�3

mm

2/s

econ

d)C

ervi

cal

0.43

7�

0.00

40.

416

�0.

004

0.43

7�

0.00

60.

450

�0.

009

0.42

6�

0.00

70.

435

�0.

008

Tho

raci

c0.

403

�0.

006

0.42

1�

0.00

60.

404

�0.

012

0.41

4�

0.01

10.

394

�0.

012

0.40

0�

0.01

1C

auda

0.57

9�

0.00

9F

ract

iona

lani

sotr

opy

(FA

)C

ervi

cal

0.69

6�

0.00

50.

589

�0.

008

0.70

0�

0.01

00.

679

�0.

011

0.70

2�

0.01

20.

703

�0.

009

3,4,

5,6

vs.

2T

hora

cic

0.62

4�

0.00

70.

573

�0.

010

0.63

4�

0.01

50.

604

�0.

015

0.62

4�

0.01

30.

636

�0.

013

Cau

da0.

558

�0.

009

Mea

sure

dlo

ngitu

dina

lan

isot

ropy

(MA

1)(�

10�

3

mm

2/s

econ

d)

Cer

vica

l0.

414

�0.

006

0.30

8�

0.00

60.

420

�0.

011

0.40

3�

0.01

20.

414

�0.

013

0.41

7�

0.01

13,

4,5,

6vs

.2

Tho

raci

c0.

315

�0.

008

0.28

6�

0.00

90.

327

�0.

017

0.31

0�

0.01

40.

305

�0.

017

0.32

1�

0.01

7C

auda

0.38

9�

0.01

2A

niso

trop

yin

dex

(AI)

(tA

DC

/lA

DC

)C

ervi

cal

0.28

8�

0.00

50.

393

�0.

007

0.28

1�

0.01

00.

308

�0.

011

0.28

0�

0.01

10.

284

�0.

009

3,4,

5,6

vs.

2T

hora

cic

0.36

6�

0.00

70.

417

�0.

010

0.35

2�

0.01

40.

378

�0.

013

0.37

2�

0.01

30.

361

�0.

013

Cau

da0.

429

�0.

009


levels (P 0.001) and WM regions were significantlylower than GM regions in both the cervical and thoraciclevels (GM vs. LLF, P 0.001; GM vs. RLF, P 0.001;GM vs. DF, P 0.001; GM vs. VF, P � 0.009). Table 2shows the diffusion characteristics for the cervical andthoracic levels and individual ROIs within the singlespecimen along with significant comparisons.

Using the specific WM tracts, we examined the diffu-sion characteristics of the RubSp, VSp, dCSp, andRetSp tracts as well as the FG within the thoracic spinalcord. Results indicated a higher FA in regions of the FG(FA � 0.856 � 0.011, reported as standard error ofmean across specimens, N � 7), RubSp tract (FA �0.828 � 0.013), and VSp tract (FA � 0.752 � 0.019)compared to the dCSp tract (FA � 0.661 � 0.026) andRetSp tracts (FA � 0.656 � 0.011). A one-way repeated-measures ANOVA indicated significantly different FAvalues between spinal tracts (ANOVA; P 0.001).Tukey’s test for multiple comparisons indicated the FGand the RubSp tracts had a significantly higher FAcompared to the RetSp tracts (FG vs. RetSp, P 0.001;RubSp vs. RetSp, P 0.001), dCSp tracts (FG vs. dCSp,P 0.001; RubSp vs. dCSp, P 0.001), and VSp tracts(FG vs. VSp, P 0.001; RubSp vs. VSp, P 0.001).Also, both the dCSp tracts and RetSp tracts had a lowerFA than the VSp tracts (VSp vs. dCSp, P 0.001; VSpvs. RetSp, P 0.001). There were no significant differ-ences between the dCSp tracts and the RetSp tracts(dCSp vs. RetSp, P � 0.995) or between the FG andRubSp tracts (FG vs. RubSp, P � 0.296).

Diffusion characteristics for the low anisotropy re-gions within the water phantom are shown in Table 3. Apaired t-test indicated no significant differences be-tween the two scan sessions when measuring tADC (P �0.258), FA (P � 0.206), MA1 (P � 0.152), or AI (P �0.274). However, small but significant differences inmeasurements between the water phantoms in the two

scan sessions were evident when measuring lADC (P �0.012) and MD (P 0.001). The magnitude of the dif-ference between these measurements was small (lADCdifference � 0.033 � 10�3 mm2/second; MD differ-ence � 0.016 � 10�3 mm2/second) and was most likelyinfluenced by slight differences in sample temperatureat the time of scanning. Although the internal temper-ature of the phantom was not measured in this exper-iment, the outside of the phantom sample maintained aconsistent temperature of approximately 21.3 � 0.4°Cthroughout the experiment, as measured with an MR-compatible temperature probe. We anticipate that lo-calized heating will occur from the small diameter sur-

Table 2Diffusion Characteristics in White and Gray Matter of Rat Cervical Spinal Cord, Thoracic Spinal Cord, and Cauda Equina Evaluated in aSingle Specimen

DTI parameters Spinal level(2) Graymatter

(3) Dorsalfuniculus

(4) Ventralfuniculus

(5) Left lateralfuniculus

(6) Rightlateral

funiculus

Differencesbetween WM

tracts

N (voxels) Cervical 94 30 35 24 22Thoracic 37 18 11 18 18

Longitudinal apparentdiffusion coefficient(lADC) (�10�3 mm2/second)

Cervical 0.686 � 0.017 0.905 � 0.025 0.842 � 0.032 0.908 � 0.028 0.833 � 0.031 3, 4, 5, 6 vs. 2

Thoracic 0.729 � 0.029 0.767 � 0.040 0.693 � 0.050 0.684 � 0.032 0.660 � 0.042

Transverse apparentdiffusion coefficient(tADC) (�10�3 mm2/second)

Cervical 0.225 � 0.006 0.197 � 0.015 0.178 � 0.016 0.127 � 0.018 0.129 � 0.014 3, 4, 5, 6 vs. 2

Thoracic 0.251 � 0.012 0.204 � 0.017 0.187 � 0.015 0.215 � 0.021 0.216 � 0.012

Mean diffusivity (MD)(�10�3 mm2/second)

Cervical 0.365 � 0.015 0.433 � 0.013 0.399 � 0.011 0.412 � 0.009 0.410 � 0.011 3, 4, 5, 6 vs. 2Thoracic 0.410 � 0.012 0.391 � 0.017 0.375 � 0.025 0.371 � 0.014 0.345 � 0.020 6 vs. 2

Fractional anisotropy (FA) Cervical 0.673 � 0.015 0.766 � 0.020 0.756 � 0.028 0.848 � 0.013 0.793 � 0.023 3, 5, 6 vs. 2Thoracic 0.626 � 0.023 0.706 � 0.031 0.682 � 0.033 0.669 � 0.022 0.706 � 0.021 3, 5, 6 vs. 2

Measured longitudinalanisotropy (MA1)(�10�3 mm2/second)

Cervical 0.321 � 0.014 0.472 � 0.020 0.442 � 0.028 0.521 � 0.021 0.423 � 0.026 3, 4, 5, 6 vs. 2

Thoracic 0.319 � 0.021 0.375 � 0.029 0.318 � 0.032 0.312 � 0.022 0.315 � 0.026

Anisotropy index (AI)(tADC/lADC)

Cervical 0.326 � 0.056 0.224 � 0.020 0.239 � 0.029 0.144 � 0.012 0.154 � 0.023 3, 4, 5, 6 vs. 2Thoracic 0.364 � 0.022 0.280 � 0.029 0.320 � 0.031 0.334 � 0.023 0.297 � 0.026 3, 4, 5, 6 vs. 2

Table 3Diffusion Characteristics of Water Phantom on Two Different Days

DTI parameters Phantom Values

N (voxels) Phantom 1 469Phantom 2 469

Longitudinal apparentdiffusion coefficient(lADC) (�10�3 mm2/second)

Phantom 1 2.846 � 0.009Phantom 2 2.879 � 0.010

Transverse apparentdiffusion coefficient(tADC) (�10�3 mm2/second)

Phantom 1 2.254 � 0.004Phantom 2 2.261 � 0.005

Mean diffusivity (MD)(�10�3 mm2/second)

Phantom 1 2.451 � 0.003Phantom 2 2.467 � 0.003

Fractional anisotropy (FA) Phantom 1 0.162 � 0.003Phantom 2 0.168 � 0.003

Measured longitudinalanisotropy (MA1)(�10�3 mm2/second)

Phantom 1 0.395 � 0.008Phantom 2 0.412 � 0.009

Anisotropy index (AI)(tADC/lADC)

Phantom 1 0.796 � 0.004Phantom 2 0.790 � 0.004


face coil; however, the effect of this heating on thediffusion characteristics of internal structures in a livespecimen was expected to be negligible.

DISCUSSION

In the present study we found significant differencesin diffusion properties between spinal cord WM andGM using a 9.4T scanner. As expected, WM regionshad higher anisotropy compared to GM. This phe-nomenon has been well documented in the centralnervous system (5,10,15) and most likely reflects dif-ferences in cellular architecture between WM and GM(15).

Our study demonstrated significant differences be-tween WM located within the spinal cord and WMlocated in the cauda equina. Specifically, WM in thecauda equina had significantly larger lADC comparedwith WM located in the spinal cord (cervical and tho-racic levels), which may correspond to larger axondiameter and/or a smaller number of axon counts peraxial cross sectional area (16), as well as a greaterproportion of cerebrospinal fluid (CSF) present withinthe measured voxels. Further, WM in the caudaequina demonstrated significantly larger tADC com-pared with WM located in the spinal cord, which maycorrespond to an increase in axon spacing, increasein extracellular volume fraction, and/or decrease inaxon counts per axial cross sectional area (16). Thisis consistent with differences in the correspondinganatomy between WM in the spinal cord and WM inthe cauda equina. The cauda equina, which is lesstightly packed and contains relatively fewer axons,would be expected to exhibit fewer barriers to diffu-sion and thus have higher apparent diffusion coeffi-cients. In addition to the anatomical differencesbetween these regions, motion-related signal attenu-ation in the cauda equina region may have also arti-ficially inflated the diffusion measurements.

Diffusion characteristics between the cervical andthoracic regions were expected to be similar, but in-stead showed a statistically significant difference. Ana-tomical differences between WM tracts of the cervicaland thoracic spinal cord most likely attributed to thesedifferences. The higher lADC and lower tADC observedin the cervical spinal cord may be due to a greaterpercentage of large diameter, tightly packed axonaltracts which innervate the upper extremities. Simula-tions by Ford et al (2) and experiments by Schwartz et al(16) show that tightly packed, highly myelinated axonshave a lower tADC compared to unmyelinated axonsthat are loosely packed. Further, Schwartz et al (16)demonstrated that axon diameter is positively corre-lated with lADC, suggesting the higher percentage oflarge diameter axons in the cervical WM pathways mayhave contributed to this effect.

Contrary to a recent study using a 4.7T scanner (13),we observed no significant difference between individ-ual WM regions in either the cervical or thoracic spinalcord. Our study had comparable image resolution tothe previous study (300 �m square axial voxel size inour study compared to 234 �m square axial voxel size),but may have had at least twice the SNR because of the

higher field strength used in the current setup. Thenumber of averages, temperature, differences betweenthe pulse sequence parameters (b-value, diffusion time,diffusion sensitizing gradient strength, TR, TE, etc.),and specimen motion may also account for differencesin the results.

Differences in diffusion parameters of individual WMtracts within each funiculi were observed in the currentstudy; however, possible partial volume effects and im-age artifacts may confound any conclusions aboutthese differences. Specifically, we found a higher FA inthe regions of the FG, RubSp tract, and VSp tract,which are located at the edge of the spinal cord, com-pared to the dorsal cortical spinal tract and RetSptracts, which are located more medial, consistent withthe results reported previously (13). Further, we foundsignificantly higher tADC in the VF compared to bothlateral funiculi, which is also consistent with the re-sults reported by Gullapalli et al (13). The measure-ments from specific tracts, particularly on the edge ofthe cord may be influenced by partial volume effects orartifacts. For example, the RetSp tract and the VSptract covered only about 2–4 voxels per slice at a reso-lution of 300 �m; thus, partial volume effects werelikely to have significant effects on the diffusion mea-surements. Further, the effects of Gibb’s ringing at theedge of the spinal cord may have contributed to signif-icant differences observed between WM tracts at theedge of the cord, compared to medial tracts.

Motion artifact, particularly due to CSF pulsations,could have caused a greater degree of partial volumecontamination in voxels at the edge of the cord andresulted in inflated apparent diffusion coefficients fromincreased signal attenuation. Many imaging sequenceshave been developed to overcome these challenges in-cluding fast imaging techniques such as single- or mul-tiple-shot diffusion-weighted echoplanar imaging (DW-EPI), although EPI of the spinal cord may suffer fromsusceptibility-related phase distortions due to theharsh magnetic environment surrounding the spinalcord. Other sequence designs have focused on compro-mising between pulsed gradient spin-echo DWI and tra-ditional EPI, including line scan diffusion imaging (17),navigator-echo based EPI techniques (18–20), and par-allel imaging DTI techniques (21,22).

The effect of image artifacts (such as eddy-current,susceptibility, and motion induced distortions) on thediffusion measurements is a substantial concern wheninvestigating the use of high magnetic fields for diffu-sion imaging techniques. However, since the resultswere relatively consistent with similar studies aftercoregistration of DWIs and T2-weighted images in AFNI,we were confident that significant artifacts were notpresent. To illustrate the similarity between our resultsand similar studies, lADC and tADC were plotted andlinear trend lines were fit to both the datasets for WMand GM (Fig. 3). When the results of this study werecompared to the results of similar studies (including exvivo and in vivo studies such as Franconi et al (23)using a volume coil at 7-T, Elshafiey et al (7) using animplanted coil at 7-T, and Gullapalli et al (13) using asurface coil at 4.7T), the trends indicated that althoughthe magnitude of apparent diffusion coefficients dif-


fered between studies, the AI was similar for each tissuetype. Specifically, the AI for WM was found to be ap-proximately 0.18 (r2 � 0.72) and the AI for GM wasfound to be approximately 0.43 (r2 � 0.66). Differencesin the magnitudes of tADC and lADC were most likelythe result of variations in pulse sequence parameters,especially the diffusion time (�). For example, Gullapalliet al (13) reported lADCs around twice what we ob-tained, which is likely due to the diffusion times differ-ing by a factor of two (� � 15 msec in our study vs. � �30 msec for Gullapalli et al (13)).

Our results were particularly close to published val-ues by Elshafiey et al (7), who observed a lADC of 0.79 �10�3 mm2/second for WM (compared to 0.72 � 10�3

mm2/second and 0.85 � 10�3 mm2/second found inthe thoracic and cervical regions in our study, respec-tively) and 0.71 � 10�3 mm2/second for GM (comparedto 0.71 � 10�3 mm2/second and 0.72 � 10�3 mm2/second found in the thoracic and cervical regions in ourstudy, respectively). Similarly, tADC values were alsosimilar, with 0.11 � 10�3 mm2/second reported for WM(compared to 0.23 � 10�3 mm2/second and 0.25 �10�3 mm2/second found in the cervical and thoracicregions in our study, respectively) and 0.23 � 10�3

mm2/second for GM (compared to 0.26 � 10�3 mm2/second and 0.28 � 10�3 mm2/second found in thecervical and thoracic regions in our study, respectively).The similarity of these results can be attributed, at leastin part, to similarities in the pulse sequences sinceElshafiey et al (7) used a diffusion time of 18.9 mseccompared with 15 msec in our study. The similaritiesbetween the current results and previous studiesshows promise for the use of high-field, noninvasivetechniques for in vivo DTI quantification of the rat spi-nal cord, although more quality control testing (repeat-

ability, reproducibility, and consistency with waterphantoms) would be useful in order to further validatethe use of DTI in the spinal cord at 9.4T.

FA values obtained in this study for WM were gener-ally similar to previous studies. In our study, WM FAwas approximately 0.70 for the cervical spinal cord and0.62 for the thoracic spinal cord, which is similar to theresults of Fenyes and Narayana (9,10) who reported FAvalues of 0.59–0.67 for WM. Elshafiey et al (7), reportedsimilar apparent diffusion coefficients to the currentstudy; however, they found FA values of 0.95, which islarger than the results of our study. Further, in vivostudies by Bilgen et al (12), Franconi et al (23), andMadi et al (11) also reported values of FA for WM thatwere greater than 0.89, which is higher than the FAmeasurements in the current study.

The use of in vivo DTI at 9.4T produced contrastingvalues of diffusion between GM and WM in the spinalcord similar to those observed in studies at lower fieldstrengths. In addition, significant differences in dif-fusion characteristics were observed between spinalcord WM and cauda equina WM, which reflect ana-tomical differences between these regions and possi-ble motion-related signal attenuation. Few significantdifferences were observed between individual WM fu-niculi in the spinal cord, although the DF had slightlyhigher FA and lower MD compared with the VF inboth the cervical and thoracic spinal cord. This trendis consistent with previous reports from various invivo rat studies (6,11,13), ex vivo rat studies (24), exvivo MD trends in the cat spinal cord (25) and theanisotropy characteristics of live, excised lampreyspinal cord (26).

The results of this study demonstrate promise forthe use of high-field DTI in monitoring the progres-

Figure 3. tADC vs. lADC for WM (open diamonds) and GM (open boxes) in various studies illustrating the feasibility of in vivoDTI of the rat spinal cord at high field strength. The results from this study are shown as filled triangles.


sion and treatment of spinal trauma. Previous animalstudies involving DTI in spinal cord injury haveshown tADC approximately doubles and lADC is de-creased by approximately half in the acute stage afterinjury (6,27). Given the variance in apparent diffu-sion coefficients in this study were relatively high, wepredict more than seven specimens will be needed toshow statistical significance after spinal trauma us-ing the current experimental setup. Additional mod-ifications to the pulse sequence design including ed-dy-current and susceptibility related corrections,such as those performed by Gullapalli et al (13),would likely decrease the variance of measured ADCsand make the technique more amenable to spinalcord injury research. In conclusion, to the best of ourknowledge this is the first study to document thediffusion properties of the cervical, thoracic, andcauda equina levels in vivo, and noninvasively, at afield strength of 9.4T.

REFERENCES1. Schwartz ED, Duda JT, Shumsky JS, Cooper ET, Gee JC. Spinal

cord diffusion tensor imaging and fiber tracking can identify whitematter tract disruption and glial scar orientation following lateralfuniculotomy. J Neurotrauma 2005;22:1388–1398.

2. Ford JC, Hackney DB, Lavi E, Phillips M, Patel U. Dependence ofapparent diffusion coefficients on axonal spacing, membrane per-meability, and diffusion time in spinal cord white matter. J MagnReson Imaging 1998;8:775–782.

3. Schwartz ED, Chin CL, Shumsky JS, et al. Apparent diffusioncoefficients in spinal cord transplants and surrounding white mat-ter correlate with degree of axonal dieback after injury in rats. AJNRAm J Neuroradiol 2005;26:7–18.

4. Schwartz ED, Cooper ET, Chin CL, Wehrli S, Tessler A, HackneyDB. Ex vivo evaluation of ADC values within spinal cord whitematter tracts. AJNR Am J Neuroradiol 2005;26:390–397.

5. Schwartz ED, Hackney DB. Diffusion-weighted MRI and the eval-uation of spinal cord axonal integrity following injury and treat-ment. Exp Neurol 2003;184:570–589.

6. Deo AA, Grill RJ, Hasan K, Narayana PA. In vivo serial diffusiontensor imaging of experimental spinal cord injury. J Neurosci Res2006;83:801–810.

7. Elshafiey I, Bilgen M, He R, Narayana PA. In vivo diffusion tensorimaging of rat spinal cord at 7 T. Magn Reson Imaging 2002;20:243–247.

8. Fenyes DA, Narayana PA. In vivo echo-planar imaging of rat spinalcord. Magn Reson Imaging 1998;16:1249–1255.

9. Fenyes DA, Narayana PA. In vivo diffusion tensor imaging of ratspinal cord with echo planar imaging. Magn Reson Med 1999;42:300–306.

10. Fenyes DA, Narayana PA. In vivo diffusion characteristics of ratspinal cord. Magn Reson Imaging 1999;17:717–722.

11. Madi S, Hasan KM, Narayana PA. Diffusion tensor imaging of invivo and excised rat spinal cord at 7T with an icosahedral encodingscheme. Magn Reson Med 2005;53:118–125.

12. Bilgen M, Elshafiey I, Narayana PA. In vivo magnetic resonancemicroscopy of rat spinal cord at 7T using implantable RF coils.Magn Reson Med 2001;46:1250–1253.

13. Gullapalli J, Krejza J, Schwartz ED. In vivo DTI evaluation of whitematter tracts in rat spinal cord. J Magn Reson Imaging 2006;24:231–234.

14. Kaufman L, Kramer DM, Crooks LE, Ortendahl DA. Measuringsignal-to-noise ratios in MR imaging. Radiology 1989;173:265–267.

15. Moseley ME, Cohen Y, Kucharczyk J, et al. Diffusion-weighted MRimaging of anisotropic water diffusion in cat central nervous sys-tem. Radiology 1990;176:439–445.

16. Schwartz ED, Cooper ET, Fan Y, et al. MRI diffusion coefficients inspinal cord correlate with axon morphometry. Neuroreport 2005;16:73–76.

17. Gudbjartsson H, Haier SE, Mulkern RV, Moocz IA, Patz S, JoleszFA. Line scan diffusion imaging. Magn Reson Med 1996;36:509–519.

18. Holder CA, Muthupillai R, Mukundan S Jr, Eastwood JD, HudginPA. Diffusion-weighted MR imaging of the normal human spinalcord in vivo. AJNR Am J Neuroradiol 2000;21:1799–1806.

19. Demir A, Ries M, Moonen CT, et al. Diffusion-weighted MR imagingwith apparent diffusion coefficient and apparent diffusion tensormaps in cervical spondylotic myelopathy. Radiology 2003;229:37–43.

20. Bammer R, Fazekas F, Augustin M, et al. Diffusion-weighted MRimaging of the spinal cord. AJNR Am J Neuroradiol 2000;21:587–591.

21. Cercignani M, Horsfield MA, Agosta F, Filippi M. Sensitivity-en-coded diffusion tensor MR imaging of the cervical cord. AJNR Am JNeuroradiol 2003;24:1254–1256.

22. Tsuchiya K, Fujikawa A, Suzuki Y. Diffusion tractography of thecervical spinal cord by using parallel imaging. Am J Neuroradiol2005;26:398–400.

23. Franconi F, Lemaire L, Marescaux L, Jallet P, Le Jeune JJ. In vivoquantitative microimaging of rat spinal cord at 7T. Magn ResonMed 2000;44:893–898.

24. Inglis BA, Yang L, Wirth ED, 3rd, Plant D, Mareci TH. Diffusionanisotropy in excised normal rat spinal cord measured by NMRmicroscopy. Magn Reson Imaging 1997;15:441–450.

25. Pattany PM, Puckett WR, Klose KJ, et al. High-resolution diffusion-weighted MR of fresh and fixed cat spinal cords: evaluation ofdiffusion coefficients and anisotropy. AJNR Am J Neuroradiol1997;18:1049–1056.

26. Takahashi M, Hackney DB, Zhang G, et al. Magnetic resonancemicroimaging of intraaxonal water diffusion in live excised lampreyspinal cord. Proc Nat Acad Sci USA 2002;99:16192–16196.

27. Ford JC, Hackney DB, Alsop DC, et al. MRI characterization ofdiffusion coefficients in a rat spinal cord injury model. Magn ResonMed 1994;31:488–494.


in vivo diffusion tensor imaging of the rat spinal cord at 9.4t

Documents