Myelin deficiencies visualized in vivo: Visually evoked potentials and T2-weighted magnetic...

Myelin Deficiencies Visualized In Vivo:Visually Evoked Potentials andT2-Weighted Magnetic Resonance Imagesof Shiverer Mutant and Wild-Type Mice

Melanie Martin,1* Timothy D. Hiltner,1 John C. Wood,2 Scott E. Fraser,1

Russell E. Jacobs,1 and Carol Readhead1

1Biological Imaging Center, Beckman Institute, Division of Biology, California Institute of Technology,Pasadena, California2Department of Pediatric Cardiology and Radiology, Children’s Hospital of Los Angeles,USC Keck School of Medicine, Los Angeles, California

Visually evoked potentials (VEPs) and micromagneticresonance imaging (lMRI) are widely used as noninva-sive techniques for diagnosis of central nervous system(CNS) diseases, especially myelin diseases, such asmultiple sclerosis. Here we use these techniques in tan-dem to validate the in vivo data in mouse models. Weused the shiverer mutant mouse, which has little or noCNS myelin, as a test model. These data show thatshiverer (MBPshi/MBPshi) has a VEP latency that is 30%longer than that of its wild-type sibling. Surprisingly, theheterozygous (MBPshi/+) mouse, with apparently normalmyelin, nevertheless has a 7% increase in its VEP la-tency vs. wild type. The lMRIs of the same animalsshow that myelinated white matter is hypointense com-pared with gray matter as a result of the shorter T2 inmyelinated regions of the CNS. T2-weighted images ofwild-type and heterozygous shiverer mice show regionsof hypointensity corresponding to the major myelinatedtracts, including the optic nerve and the optic tract ofthe CNS, whereas shiverer mice have no regions of lowintensity and therefore no detectable myelinated areas.In shiverer mice, lMRI can discern hypomyelinationthroughout the brain, including the optic tract, andthese changes correlate with longer VEP latencies. Inaddition, VEPs can also detect changes in the molecu-lar make up of myelin that are not discernible with his-tology or lMR. These data show the potential of usinglMRI in combination with VEPs to follow changes inboth the quality and the quantity of myelin in vivo.These combined methods would be useful for longitudi-nal studies and therapy testing. VVC 2006 Wiley-Liss, Inc.

Key words: dysmyelination; shiverer mutant mouse;MRI; visually evoked potential latency; T2-weightedmagnetic resonance imaging

The central nervous system (CNS) myelin sheath,which is essential for the rapid conduction of nerveimpulses (Huxley and Stamphli, 1949), can be disrupted

for a number of reasons, including myelin gene muta-tions, viral infection, toxins, and autoimmune diseases.Multiple sclerosis (MS), the most common human auto-immune disease, involves recognition of myelin proteinsby T cells, followed by a cascade of destructive actionsby the immune system (Martin et al., 1992). Morerecently, it has been found that MS develops with acomplex predisposing genetic trait and likely requires anenvironmental factor, such as a viral infection, to triggerthe disease (Sospedtra and Martin, 2005).

Magnetic resonance imaging (MRI) and visuallyevoked potentials (VEPs) are used as diagnostic tools forMS. MR can detect clear demyelinating lesions and areasof edema, but correlation of these findings with clinicalsymptoms has been only partially successful (Maaroufet al., 2003; de Andres, 2003; Bagnato and Frank, 2003).Because visual changes are often an early signature ofMS, VEPs are commonly conducted on patients sus-pected of having MS. By recording signals extracraniallyafter a defined visual stimulus, VEP measurements offera highly sensitive assay for detecting even subclinical

Melanie Martin’s current address is Department of Physics, University of

Winnipeg, Winnipeg, Manitoba, Canada R3B 2E9.

*Correspondence to: Melanie Martin, Department of Physics, 515 Por-

tage Avenue, Winnipeg, Manitoba, Canada R3B 2E9.

E-mail: [email protected]

Contract grant sponsor: NIH; Contract grant number: NEI R01-

EY011933; Contract grant sponsor: Human Brain Project; Contract grant

sponsor: National Institute of Biomedical Imaging and Bioengineering;

Contract grant sponsor: NIMH; Contract grant number: P20-DA08944;

Contract grant number: R01-MH61223; Contract grant sponsor:

NCRR; Contract grant number: R01-RR13625; Contract grant num-

ber: P41 RR12642; Contract grant sponsor: Radiological Society of

North America; Contract grant sponsor: Whitaker Foundation.

Received 3 April 2006; Revised 9 August 2006; Accepted 9 August

2006

Published online 16 October 2006 in Wiley InterScience (www.

interscience.wiley.com). DOI: 10.1002/jnr.21086

Journal of Neuroscience Research 84:1716–1726 (2006)

' 2006 Wiley-Liss, Inc.

(silent) lesions in the visual pathway (Halliday et al.,1973; Chiappa, 1983; Mathews, 1985; Hume and Wax-man, 1988). It is thought that a delay in the latency ofthe VEP signal will occur because of damage to themyelin of a nerve axon and that a decrease in intensityof the VEP will occur because of axonal damage (Alten-muller et al., 1990).

We used VEP and micro-MRI (lMRI) in tandemon a dysmyelinating mouse test model to see whetherthese diagnostic tests used in concert would yield bothcorroborative and more detailed information regardingthe myelin status of the CNS and the optic tracts in par-ticular. C57BL/6J 3 DBA/J hybrid mice carrying thedymyelinating shiverer mutation (MBPshi/MBPshi) wereused as test models.

The shiverer mutation is due to the deletion ofmost of the myelin basic protein (MBP) gene (exonsIII–VII; Roach et al., 1985; Takahashi et al., 1985). Thismutation (MBPshi/MBPshi; Biddle et al., 1973) is anautosomal recessive mutation that maps to the distal endof chromosome 18 (Sidman et al., 1985). The shiverermutant mouse CNS is hypomyelinated (Bird et al.,1978). Its CNS myelin is sparse, and, where it does exist,it is uncompacted and lacks a major dense line (Dupoueyet al., 1979; Privat et al., 1979; Kirschner and Ganser,1980). The mutant mouse is characterized by a tremorthat can first be seen at about 14 days of age and contin-ues for the remainder of the mouse’s short life (50–100days). The phenotype can be rescued by transgenesis:clinical symptoms are no longer present in shiverermutants that have integrated the MBP transgene (Read-head et al., 1987). Morphometric analysis of the thick-ness of the myelin sheath is directly proportional to theMBP gene and transgene dosage (Shine et al., 1992).Mice that are heterozygous for the shiverer mutation(MBPshi/+) have myelin that appears normal, althoughthe MBP expression is 50% of normal (Shine et al.,1992). The shiver mutation was maintained on thehybrid (C57BL/6J 3 DBA/J) background by brother-sister mating at Caltech’s animal facility. In this study,we examined wild-type (+/+), heterozygous (MBPshi/+), and shiverer mutant (MBPshi/MBPshi) mice via VEPsand T2-weighted lMRIs.

The VEPs of 2–6-month-old wild-type (+/+),heterozygous (MBPshi/+), and shiverer mutant (MBPshi/MBPshi) mice on a mixed C57BL/6J 3 DBA/J hybridbackground were measured to be between 30 and 40msec. By 2 months, myelination is complete and remainsstable throughout the life of a healthy animal. In parallel,we assessed myelin content in vivo via T2-weightedlMRI.

MATERIALS AND METHODS

Genotyping Wild-Type, Heterozygous, and ShivererMutant Mice

The shiverer mutation was on a mixed C57BL/6J back-ground, and the animals were bred as heterozygotes (MBPshi/+),and the offspring, which consisted of wild-type (+/+), hetero-

zygous (MBPshi/+), and shiverer mutant (MBPshi/MBPshi)mice, were genotyped prior to use in VEP and MRI experi-ments. For genotyping, a small piece of tail was snipped off atweaning, and tail DNA was extracted by using a Purgene(Gentra Systems) DNA extraction kit according to the manu-facturer’s instructions. The DNA was amplified with threeprimers: a 50 primer common to wild-type and shiverer MBPgene complimentary to sequences in the second intron andtwo 30 primers, one of which is found in the second intron ofthe normal gene, and another primer that is specific to theshiverer breakpoint in the second intron. The primers usedwere 50 region of the second normal MBP intron 50-CAGGGG ATG GGG AGT CAG AAG TGA-30; 30, region of thesecond normal MBP intron 50-TGT TTC CCC ACT TGGGAG CCA GTG G-30, 30 region of the second shiverer MBPintron 50-ATG TAT GTG TGT GTG TGC TTA TCTAGT GTA-30. The PCR conditions were 948C for 2 min,followed by 908C for 1 min, 638C for 1 min, 728C 1 min for40 cycles, then 728C for 10 min and a 48C hold. This yieldedthree applicons in heterozygous (MBPshi/+) mice, a 380-bpband representing the shiverer breakpoint, and a 200-bp bandrepresenting the normal MBP gene in the same region. Shiv-erer mice could be distinguished easily form wild-type micebecause of their shiverering phenotype, seizures, and earlydeath but wild-type and heterozygous (MBPshi/+) werebehaviorally identical, although changes could be seen in theirVEPs. Immunohistochemistry of the shiverer optic nerve con-firmed the lack of compact myelin in the shiverer.

VEP Measurements

VEP experiments were performed in a manner similarto that used by Strain and Tedford (1993). Mice were anes-thetized with isoflurane and placed on a water heating padmaintained at 378C. A stereotaxic positioning device, includ-ing ear and tooth bars, was used to orient and immobilize thehead. The whole apparatus was housed in a custom-built Far-aday cage to reduce noise in the electronics. The Faraday cagewas covered with aluminum foil and black felt to preventleakage of light from other sources. The mice were allowedto reach a steady state with the anesthetic and were dark-adapted for 20 min before recording of the VEPs. A flashingvisual stimulus was generated with a home-built strobe light(2 Hz) positioned at a fixed distance (20 cm) directly in frontof the mouse in a darkened room. Subcutaneous platinumneedle recording electrodes were positioned at Fpz (midline,just distal to the interorbital line), Oz (midline, nuchal crest),and a ground placed in the hind limb of the mouse. A DAM50 differential amplifier (World Precision Instruments, NewHaven, CT) with a high-pass filter at 1 Hz and a low-pass fil-ter at 1 kHz was used to amplify the VEP with a gain of10,000. The voltage was digitized and recorded in Labviewfor 250 msec after each strobe flash. The strobe light and Lab-view recordings were triggered by the same external source.The use of subcutaneous electrodes allowed multiple trials ofthe same individuals over many days. C57BL/6J 3 DBA/Jhybrid mice carrying the shiverer mutation (MBPshi/MBPshi)were compared with wild-type (+/+) and heterozygous

Myelin Deficiencies Visualized In Vivo 1717

Journal of Neuroscience Research DOI 10.1002/jnr

(MBPshi/+) littermates The Caltech Internal Animal Care andUse Committee (IACUC) approved all experiments.

VEP Analysis

VEPs from 100 strobe light flashes were averaged toobtain the final waveform. Each waveform was baseline cor-rected by subtracting a line connecting the first and last pointsof the waveform. The largest peak from this waveform wasfitted to a Gaussian (Fig. 1). The center of the Gaussian peakwas taken to be the latency. A minimum of five waveformswas collected from each mouse on a given day, and the la-tency was calculated in this manner for each waveform. Eachmouse was used in one or more sessions over a period of sev-eral weeks. Means and standard deviations of the latencies forall waveforms were calculated for each genotype.

mMRI

Fifteen mice were scanned a least once using lMRI.Six of these mice also had their VEPs measured as describedabove. Three lMRIs studies are reported here. For the firsttwo lMRI studies, mice were initially sedated with a lightdose (0.0025 ml/g body weight) of a ketamine/xylazine mix-ture (0.3 ml 100 mg/ml ketamine plus 0.1 ml 20 mg/ml xyla-zine in 4 ml physiological saline) and placed in a custom-madeholder with ear and tooth bars to maintain the head positionand minimize motion during the experiment. Anesthesia wasmaintained with 1% isoflurane and animal respiration moni-tored during the course of lMR imaging. lMRIs are MRimages with resolutions on the order of 100 lm (Jacksonet al., 1999; Jacobs et al., 2003). lMRI experiments were per-formed with an 11.7-T Bruker DRX Avance spectrometer

with 89 mm vertical bore and maximum gradient strength of25 G/cm with a 38-mm birdcage Bruker (Bruker BioSpin,Billerica, MA) RF coil.

The first study included the 2D spin-echo images of axialslices [thickness 740 lm, 2 cm 3 2 cm field of view (FOV),78 lm in plane resolution] containing the external capsule.Eight echo times (TE ¼ 12, 24, 36, 48, 60, 72, 84, and 96msec) were obtained in a single experiment for the slice. Arecycle time, TR, of 3 sec was used, and two scans wererecorded and averaged for a total imaging time of 26 min.

The density of water, specifically the density of hydrogennuclei in water molecules, M0, and the transverse relaxationtime, T2, of material being imaged affect the MRI signal mea-sured in the form of an exponential decay (M ¼ M0e

�TE/T2

+ C) in the case of simple materials, such as water. For images,the more water in a given voxel element, the larger the M0,the brighter the voxel will be. Strictly speaking, M0 is calledthe equilibrium magnetization and is proportional to the waterdensity, but here we call it the water density. T2 is a measure ofthe time it takes the system to return to equilibrium, when themeasured signal goes down to zero. Thus, the longer the timebetween excitation, when a signal could be recorded, and theactual time when the signal is recorded, called the echo time,TE, the less signal will be measured. Because different tissueshave different T2 values, changing TE in a T2-weighted imageallows for contrast between tissues with different values of T2.To measure the T2 of the mouse brain using these images, thecomplex raw k-space data were reconstructed into real andimaginary components by using custom-written MATLAB rou-tines. Real and imaginary images were wavelet-packet denoizedin Algorithm Design Toolkit software and custom-madethreshold and cost function blocks (Coiffman et al., 1997). Eachreal and imaginary image was decomposed into a completewavelet-packet representation. Shannon entropy was calculatedat each wavelet-packet node from the complex norm, summingacross all echo times. By using a simple parent-child competi-tive algorithm, the basis minimizing global entropy was calcu-lated (Woog, 1996). To achieve noise suppression, this repre-sentation was hard thresholded, using a compression thresholdof 0.65 (Woog, 1996; Wood and Johnson, 1999; Wood et al.,2002). The complex norm was used for thresholding decisions,so real and imaginary coefficients were accepted or rejected intandem, preserving phase consistency. A multipass algorithmwas used, with nine shifts and two iterations similar to previoussingle echo MRI wavelet-packet denoizing (Woog, 1996;Wood and Johnson, 1999). Denoized images were fit to amonoexponential T2 decay function (M ¼ M0e

�TE/T2 + C)by using a Levenberg-Marquadt algorithm (Matlab Optimiza-tion Toolbox).

For the 3D images of the second study, a rapid acquisi-tion with relaxation enhancement (RARE) sequence was usedwith a RARE factor of 4, matrix size 256 3 128 3 128,FOV 2.85 cm 3 1.70 cm 3 1.70 cm TR ¼ 1 sec, echo spac-ing 12 msec, for a total time of 71 min. These images wereacquired to examine the entire brain of the mice.

The third study involved acquiring 3D slab images ofthe optic nerve by using a RARE sequence with a RAREfactor of 8, matrix size of 128 3 128 3 64, FOV 1.5 cm 31.5 cm 3 0.75 cm, TR 1 sec, echo spacing 10.25 sec, eight

Fig. 1. Visually evoked potentials show significant slowing in theshiverer mutant mouse. Typical VEP waveforms from a wild-type,heterozygous, and shiverer mutant (nonmyelinated) mouse areshown. The latency increases with decreasing MBP gene dosage. Thex-axis shows the time in milliseconds after the strobe light flash; they-axis shows the voltage measured across the mouse head beforeamplification.

1718 Martin et al.


acquisitions for a total time of 136 min. These lMRI experi-ments were performed with a 9.4-T Bruker DRX Avancespectrometer with 30 cm horizontal bore, S116 Bruker(Bruker BioSpin, Billerica, MA) gradient set, and a 72-mmbirdcage Bruker (Bruker BioSpin) RF coil for transmission,with a receiver coil for detection. For this study, a C57BL/6Jwild-type mouse was compared with a homozygous shiverermouse on a C57BL/6J 3 DBA/J hybrid background. TheCaltech Internal Animal Care and Use Committee (IACUC)approved all experiments.

Immunohistochemistry of Wild-Type, Heterozygousand Shiverer Mutant Mice

Wild-type (+/+), shiverer mutant (MBPshi/MBPshi), andheterozygous (MBPshi/+) 8-week-old female mice from thesame litter were genotyped by PCR of DNA extracted fromtail biopsies. The mice were deeply anesthetized with 2.5%Avertin and perfused via the right heart ventricle with 30 mlphosphate-buffered saline (PBS), followed by 30 ml 4% para-formaldehyde (PFA) in PBS, pH 7.4. After perfusion, thehead was removed and postfixed in 4% PFA overnight at48C. The eyes, optic nerves, and chiasma were dissected outand fixed for 2 hr in 4% PFA at 48C. The tissue was thenwashed three times in 15% sucrose in PBS for 1 hr each atroom temperature, with gentle rotation. The tissue was thenwashed in 20% sucrose in 103 PBS for 20 min at room tem-perature and immersed in optimal cutting temperature (OCT)compound (Sakura Finetek, Torrance. CA) for 20 min atroom temperature. The tissue was then placed in plasticmolds, covered with OCT, and snap frozen in liquid nitro-gen. Ten-micrometer serial cyrosections were cut alongthe entire length of the optic nerve from the chiasma to theback of the eyeball. The frozen sections were stored overnightat –208C.

The cryosections were stained by using the BrainStainImaging Kit (Molecular Probes, Invitrogen, Eugene, OR)according to the manufacturer’s instructions. Fluoro-Myelin,the green fluorescent myelin stain, was visualized at a wave-length of 480/598 nm on a Zeiss 510 confocal microscopewith a 320 objective in Caltech’s Biological Imaging Center.

RESULTS

This work examines the relationship among myeli-nation, VEPs, and MRI contrast in the CNS. We mea-

sured VEPs and obtained T2-weighted brain images ofthe wild-type (+/+), shiverer mutant (MBPshi/MBPshi),and heterozygous (MBPshi/+) mice, which representednormal, hypomyelinated, and myelinated phenotypes,respectively, but with 50% MBP levels. Some of themice used for MRI studies also had their VEP latenciesmeasured. VEP latency was found to increase withdecreasing myelin content, as shown in Figure 1. Theunexpected latency difference between the wild-typeand heterozygous mice, as shown in Figure 1, was smallbut consistent. Thus the number of mice used for VEPlatency measurements was increased to determine whe-ther the latency difference was significant. In total, 86VEP waveforms were measured for eight wild-typemice, 70 waveforms for 13 heterozygous mice, and 88waveforms for 12 shiverer mutant mice. The mean 6SD latencies for each genotype are shown in Table I.The shiverer mutant mouse mean VEP latency is signifi-cantly different from both the wild-type and the hetero-zygous mouse VEP latency (t-test P < 0.0001). A bor-derline significant difference (P < 0.03) was unexpect-edly found between the wild-type and heterozygousmouse VEP latencies. Figure 2 illustrates the differencesbetween the latencies of the wild-type (n ¼ 8) and shiv-erer mutant (n ¼ 12) mice. The MBPshi/+ mice (n ¼13) have latencies intermediate between those of thewild-type and shiverer mutant mice (Fig. 2). Theincrease in latency confirms the effects of dysmyelinationon nerve transmission speeds. Nerve transmission slowsnoticeably, causing a longer latency, in hypomyelinatednerve tracts. Significant differences can be detected evenbetween the VEP latencies of mice with apparently nor-mal myelin but with only 50% of the normal MBP lev-els (MBPshi/+). These new observations indicate that wecan expect to be able to detect differences in VEP laten-cies of mice with myelin diseases. No difference wasseen between the latency measurements of male andfemale mice for the heterozygous and shiverer mice ge-notypes, as shown in Table I. A small difference wasseen between male and female wild-type mice, whichcould be due to the small number of wild-type micetested. No correlation was found between genotype andeither peak width or height in the VEPs.

As a control experiment, evoked potentials weremeasured in the same manner as VEPs but with thestrobe light covered so that the mouse saw no light. The

TABLE I. Comparison of VEPs of Wild-Type, Heterozygous, and Shiverer Mice{

Genotype No. of mice No. of scans Latency (msec) Width (msec) Intensity (lV)

Wild-type 8 86 30 6 2 (30 6 2 male 31.5 6 0.3 female) 7 6 2 20 6 10

Heterozygous 13 70 32 6 2 P < 0.03* (32 6 1 male 32 6 2 female) 7 6 2 20 6 10

Shiverer 12 88 39 6 1 P < 0.0001* (39 6 1 male 39 6 1 female) 7 6 1 19 6 8

{Summary of the VEP scans on shiverer mutant (n ¼ 12, 2 male and 10 female), heterozygous (n ¼ 13, 6 male and 7 female), and wild-type (n ¼ 8,

6 male and 2 female) mice. The major peak (Fig. 1) represents the latency (msec). The latencies of the shiverer mutant mouse and heterozygous shiv-

erer mutant mice were significantly delayed compared with the wild-type. No difference was seen between the latency measurements of male and

female mice for the heterozygous and shiverer mice genotypes, as shown. A small difference was seen between male and female wild-type mice, which

might be due to the small number of mice tested.

*P vs. wild-type.



main peak at 30–40 msec did not appear in these evokedpotential measurements (data not shown) confirming thatthis peak is a visually evoked potential and not an audi-tory evoked potential from the clicking noise of thestrobe light, as has been suggested by others (Lehmanand Harrison, 2002). We chose to examine the peak at30–40 msec for our studies, because it was the peak withthe largest amplitude. The peak at >85 msec in previousstudies (Lehman and Harrison, 2002) was not above thenoise level in our studies.

Representative T2-weighted images of animalsused in the VEP studies are shown in Figure 3. At a TEof 24 msec, the heavily myelinated external capsules ofthe wild-type and heterozygous mouse brains are clearlyhypointense compared with the rest of the brain. Theexternal capsule in the shiverer mutant mouse brain isisointense to the surrounding tissue because of the lackof myelin and concomitant increase in T2 relative tomyelinated tissue. The hyperintense regions in the heter-ozygous mouse are ventricles that are sometimes en-larged in mice with myelin disorders.

The proton density, M0, and T2 maps resulting fromthe fit to a monoexponential decay (M ¼ M0e

�TE/T2

+ C) are shown in Figure 4 (Wood et al., 2002). Thisfunction works well for homogenous materials such aspure water. The T2 of myelinated white matter is actuallya weighted sum of three exponentials (Webb et al., 2003;

Laule et al., 2004). The long-time component (on theorder of 1 sec) is thought to result from cerebrospinalfluid, the intermediate-time component (on the order of10 msec) from intra- and extracellular water throughoutthe brain, and the short-time component (on the order of1 msec) from water trapped in the layers of the myelinsheath (Webb et al., 2003; Laule et al., 2004). At 11.7 T,with the echo times used in our experiment, the decay isdominated by the intermediate component. We wereunable to see the short component of the T2 exponentialdecay quantitatively, insofar as we were unable to takemeasurements with short enough echo times. Instead,there is an apparent decrease in M0 of heavily myelinatedtissue. The apparent M0 decreases because the signal hasdecayed away so much even at the shortest echo time thatit does not contribute to the measured proton density.

Axial sections from in vivo 3D T2-weightedimages of the entire mouse brain for the wild-type, het-erozygous, and shiverer mutant are shown in Figure 5.These sections contain the optic tract, part of the visualpathway along which the VEPs travel. The optic tract,as with other white matter regions, is clearly not myelin-ated in the shiverer mutant mouse, as evidenced by thelack of contrast.

In a detailed 3D in vivo slab image of the opticnerve of a wild-type mouse, the myelinated bundles ofaxons are seen as a hypointense ribbon running from theback of the eyeball to the optic chiasma (Fig. 6a). Theoptic nerve of the shiverer mouse is more uniformlygray, indicating little to no myelin (Fig. 6b). Theseresults are in agreement with immunohistochemistryresults from the optic nerve (Fig. 7). Optic nerve cyro-sections from wild-type (Fig. 7A) and heterozygous (Fig.7B) and shiverer (Fig. 7C) mice were stained with greenfluorescent myelin stain (Molecular Probes). Whenviewed at 480 nm on a Ziess 510 confocal microscope,the stained cryosections from wild-type and heterozy-gous mice show bright green fluorescent myelin staining,whereas the shiverer mouse shows no detectable stainingat the same settings. This indicates that, although thewild-type and heterozygous shiverer mouse have well-myelinated optic nerves, the shiverer has little detectablecompact myelin.

DISCUSSION

We found that, by using the noninvasive VEP andlMRI methods in tandem on the dysmyelinating mousemodel shiverer, we were able to gather information onthe quality, quantity, and overall distribution of myelinin the CNS of live mice. In addition, we obtained infor-mation regarding the physiological impact of dysmyeli-nation on the optic nerve. There was a significant differ-ence between the latencies of the main peaks in theVEPs for the wild-type and shiverer mutant mouse.Consistently with this difference, we find correspondingchanges in the lMR images. One of the most strikingof these is that there was no contrast in the in vivo T2-

Fig. 2. Significant differences between VEPs from wild-type andshiverer mutant mice. Distribution of latencies calculated from allwaveforms taken on different days from eight wild-type (mean 30 62 msec), 13 heterozygous (mean 32 6 2 msec), and 12 shiverer mu-tant mouse (mean 39 6 1 msec) are shown. The shiverer mutantpopulation is distinct; its mean latency differs significantly (P <0.0001) from the other mice. The difference in mean latenciesbetween the wild-type and heterozygous is of borderline significance(P < 0.03), indicating that MBP has a role in the effectiveness ofpromoting conduction of nerve impulses. Each bar represents thefraction of waveforms for all mice in that group with a latency valueon the x-axis.

1720 Martin et al.


weighted images between regions of gray and whitematter in the CNS of the hypomyelinated shiverer mu-tant (note the somewhat uniform gray of the images inFigs. 3c, 5c). In the lMR images of a wild-type mouse(Figs. 3a, 5a), white matter is of lower intensity com-pared with gray matter, because of the shorter T2 inmyelinated (white matter) regions of the CNS. The re-gional differences in the intensity in the lMR imagesand the loss of this contrast in the homozygous mutantsshow that lMRI can discriminate changes in the myeli-nation of the white matter.

In addition to the wild-type and homozygous shiv-erer mutant mouse, we also studied the heterozygousmutant mouse. Interestingly, there was an unexpectedsmall but significant difference (P < 0.03) between theheterozygous (MBPshi/+) and wild-type (+/+) mouse

VEP latencies. This difference suggests that, although themyelin thickness is normal, as shown by detailed mor-phometric EM studies (Shine et al., 1992), the lowerlevels of MBP (50% of normal; Readhead et al., 1987;Shine et al., 1992) might affect the degree of compactionand thus the quality of myelin and its function. No dif-ferences were detected via lMRI between wild-typeand heterozygous CNS. This is in agreement withdetailed histological and EM studies (Shine et al., 1992).For this genotype, VEPs were more sensitive to subtlephysiological myelin changes than T2-weighted lMRI.Interestingly, VEPs have previously been shown to besuperior to orbital MRI with a triple dose of gadolin-ium, another type of contrast in MRI, in determining theinvolvement of chronic optic nerve in MS in humans(Acar et al., 2004).

Fig. 3. MR brain images show loss of contrast in the dysmyelinatedstate. T2-weighted axial images of wild-type (a), heterozygous (b),and shiverer mutant (c) mouse brains (slice thickness 740 lm, TE 24msec, FOV 2 3 2 cm2, matrix size 256 3 256, TR 3 sec) from an11.7-T scanner are displayed. A schematic of the wild-type mousebrain is shown in d. The myelinated external capsules (e.c.; arrows)and corpus collosums of the wild-type and heterozygous mouse

brains are hypointense compared with the rest of the brain. Con-versely, the external capsule and corpus collosum are not differenti-ated in the shiverer mutant mouse brain because of the absence mye-lin. The hyperintense regions in the heterozygous brain are the ven-tricles. Enlarged ventricles are sometimes seen in mice with myelindisorders.



Fig. 4. Apparent increase in M0 results in lack of contrast in shiverermutant brain. Representative T2 (left) and M0 (right) maps calculatedfrom a voxel-by-voxel monoexponential fit for the wild-type (a), het-erozygous (b), and shiverer mutant (c) mice (slice thickness 740 lm,FOV 2 3 2 cm2, matrix size 256 3 256, TR 2–3 sec, B0 11.7 T) areshown. The shortest echo time used (12 msec) was already of interme-diate length, so the signal from the short component of T2, which has

be shown to correlate with myelin content, had already decayed to 0.The effect of the myelin is thus seen as a decrease in the M0 values, ascan be seen in the hypointense regions of the wild-type and heterozy-gous brains. Shown with arrows are the myelinated external capsule(e.c.) and the optic tract (o.t.). The ventral hyperintense areas, mostnotably in the M0 map of the heterozygous mouse brain, result fromB1 nonhomogeneities. The central hyperintense areas are the ventricles.

1722 Martin et al.


Some studies have used diffusion tensor imaging(DTI) to detect significant differences in DTI parametersresulting from dysmyelination in the shiverer mutantbrain (Song et al., 2002; Tyszka et al., 2006) and mye-lin-deficient rats (Gulani et al., 2001). However, an ear-lier DTI study of shiverer mutant mice (MBPshi/MBPshi)mice found no significant differences in the diffusion an-isotropy or trace of the diffusion tensor between whitematter regions in the spinal chord of wild-type andMBPshi/MBPshi mice (Ahrens et al., 1999), and other stud-ies found that DTI parameters are not significantly influ-enced by myelin in myelinated and unmyelinated garfishnerves (Beaulieu and Allen, 1994; Beaulieu et al., 1998).In addition, late stages of experimental allergic en-cephalomyelitis (EAE), an animal model of MS, showedno significant differences in diffusion tensor parameters indemyelinated regions of the spinal chord (Ahrens et al.,1998). In our study, we focused on T2-weighted imaging,because this is an established technique for imaging ofmyelin. T2 relaxation has been correlated with myelincontent as measured by quantitative histomorphometry(Gareau et al., 2000; Webb et al., 2003; Laule et al.,2004). Here we compare T2 changes that are associatedwith normal and hypomyelination in the brain with VEPlatencies.

VEPs are widely used to assess the functioning ofthe visual pathways in both health and disease (Hallidayet al., 1973; Chiappa, 1983; Mathews, 1985; Hume andWaxman, 1988). In normal animals, the conduction ve-locity in nerve fibers is affected by axonal diameter,myelin thickness, and internode length (Waxman, 1980).For shiverer mutant mice that have little to no myelin,we observed a significant increase in latency, as expected(Altenmuller, 1990; Diem et al., 2003). We also studiedthe amplitude of the VEPs in the different groups. Wefound no consistent difference in the amplitudes of themain peak of the VEPs among these mice. This lack of

correlation might be expected because a decrease inVEP amplitude is generally taken to indicate axonaldamage, which is considered minimal in shiverer mutantmice, although changes in the organization and compo-sition of the axonal skeleton have been found (Bradyet al., 1999). Our observed VEP amplitudes are some-what sensitive to experimental conditions (e.g., electrodelocation and depth), whereas latency measurements werenot. We also found that the body temperature of themouse had a major influence on the VEP measurement,and for this reason the anesthetized mice were alwaysplaced on a 378C circulating water heating pad duringthe data collection. Latency measurements were highlyreproducible on any given day and from day to day (seeFig. 2), whereas amplitude measurements were variable.The latencies varied 0.3–6.1% (standard deviation/mean)during one day and 1.2–5.8% over several weeks for thesame mouse (data not shown). The amplitudes varied2.3–70% (standard deviation/mean) during one day and20–61% over several weeks for the same mouse (datanot shown). Thus, it is possible that our recordings werenot sensitive to small changes in amplitude even thoughthey were sensitive to small changes in latency. Moredetailed analysis can be performed to obtain informationon other peaks in the VEP waveforms, but, because am-plitude is related to axonal damage and latency is relatedto myelin, we focused on the latency of the main peak,a robust parameter of the VEPs, rather than overinter-preting the data to make a simple comparison. In futurestudies, when axonal damage is important, the VEP am-plitude measurements would have to be validated.

Another study (Lehman and Harrison, 2002) com-pared flash VEPs in wild-type C3HeB/FeJ and homozy-gous shiverer C3HeB/FeJ mice with injectable anes-thetic. The authors found significant differences in VEPlatencies of later peaks, which we did not examine, in allbut two of the shiverer mice. As with the Lehman and

Fig. 5. MR brain images show that the optic tract is differentiallysuppressed in the dysmyelinated state. Axial sections from 3D T2-weighted MR images of wild-type (a), heterozygous (b), and shiverermutant (c) mouse brains (111 3 133 3 133 lm resolution, TE 12msec, FOV 2.85 3 1.7 3 1.7 cm3, matrix size 256 3 128 3 128,TR 1 sec) from an 11.7-T scanner are displayed. The myelinatedoptic tract of the wild-type and heterozygous mouse brains (arrows)

are hypointense compared with the rest of the brain. Conversely, theoptic tract is not differentiated in the shiverer mutant mouse brainbecause of the absence of myelin. This is seen in all myelinatedregions of the brain throughout the 3D images. For instance, the cor-pus callosum/subcallosal fasciculus, cerebral peduncle, and subcorticalwhite matter are also differentially suppressed in the shiverer mutantbrain at right.



Harrison study, we also detect no significant differencein the amplitude of the peaks in the VEPs.

In conclusion, the almost complete absence ofcompact myelin in the shiverer mutant mouse caused a

significant increase in VEP latency and a correspondinglack of contrast in T2-weighted lMRIs. An increase ofthe VEP latency, as seen in the heterozygous shiverermutant mouse, was detectable with myelin that appeared

Fig. 6. In vivo T2-weighted MR images show that the optic nerveof a myelinated wild-type mouse can be distinguished from a dys-myelinated shiverer mouse because of a central core of lower inten-sity, indicating myelinated axons. Slices from 3D T2-weighted MRimages of wild-type (a) and shiverer mutant (b) mouse brains (117lm isotropic resolution, TE 42 msec, FOV 1.5 3 1.5 3 0.75 cm3,

matrix size 128 3 128 3 64, TR 1 sec, scan time 136 min) from a9.4-T scanner are displayed. The myelinated optic nerves of thewild-type mouse have a hypointense central core (dark area indicatedby the arrows), indicating myelinated axons. Conversely, the opticnerves of the shiverer mouse (arrows) have a more intense central corebecause of the absence of myelin. Scale bar is given at lower right.

1724 Martin et al.


histologically normal (Shine et al., 1992) but had halfthe wild-type level of MBP. This indicates that VEP la-tency is sensitive enough to detect subtle myelin changesand therefore would be useful in longitudinal studies ofmyelin disorders. T2-weighted MRI was able to distin-guish between shiverer mutant and normal CNS myelinin vivo. The wild-type mouse showed a clear contrastbetween white matter and gray matter, whereas the shiv-erer mutant had little to no contrast, because it has littleto no myelin. These in vivo data were confirmed at theend of the experiments by immunohistochemistry of thefixed optic nerve. Increased VEP latencies in the shiverermutant mouse corresponded to low-contrast MR imagesof the dysmyelinated optic tract. These noninvasive invivo methods will be essential tools for longitudinalstudies of mouse models of myelin diseases and for test-ing therapies for remyelination.

ACKNOWLEDGMENTS

The authors thank John Carpenter, Mary Martin,and Anthony Readhead for help in preparing figures andTai Hooker for initial calibration of the VEP experi-ments.

REFERENCES

Acar G, Ozakbas S, Cakmakci H, Idiman F, Idiman E. 2004. Visual evoked

potential is superior to triple dose magnetic resonance imaging in the di-

agnosis of optic nerve involvement. Int J Neruosci 114:1025–1033.

Ahrens ET, Laidlaw DH, Readhead C, Brosnan CF, Fraser SE, Jacobs RE.

1998. MR microscopy of transgenic mice that spontaneously acquire ex-

perimental allergic encephalomyelitis. Magn Reson Med 40:119–132.

Ahrens ET, Laidlaw DH, Readhead C, Fraser SE, Jacobs RE. 1999. Inves-

tigating white matter diffusion anisotropy using the dysmyelinating shiv-

erer mutant mouse. Proceedings of the International Society for Magnetic

Resonance in Medicine Seventh Scientific Meeting and Exhibition, vol

2. p 966.

Altenmuller E, Ruether K, Dichgans I. 1990. Chapter 3.4 VEP bei

demyelinisierenden Erkrankungen des ZNS of Evozierte Potentiale.

Berlin: Springer.

Bagnato F, Frank JA. 2003. The role of nonconventional magnetic reso-

nance imaging techniques in demyelinating disorders. Curr Neurol

Neurosci Rep 3:238–245.

Beaulieu C, Allen PS. 1994. Determinants of anisotropic water diffusion

in nerves. Magn Reson Med 31:394–400.

Beaulieu C, Fenrich FR, Allen PS. 1998. Multicomponent water proton

transverse relaxation and T-2-discriminated water diffusion in myelin-

ated and nonmyelinated nerve. Magn Reson Imag 16:1201–1210.

Biddle F, March E, Miller JR. 1973. Research news. Mouse Newslett

48:24.

Bird TD, Farrell DF, Sumo SM. 1978. Brain lipid composition of the

shiverer mouse: genetic defect in myelin development. J Neurochem

31:387–391.

Brady ST, Witt AS, Kirkpatrick LL, de Waegh SM, Readhead C, Tu

PH, Lee VMY. 1999. Formation of compact myelin is required for

maturation of the axonal cytoskeleton. J Neuosci 19:7278–7288.

Chiappa KH. 1983. Evoked potentials in clinical medicine. New York:

Raven Press.

Coiffman R, Wickerhauser MV, Woog L. 1997. Adaptive Design Tool-

kit. New Haven, CT: Fast Mathematical Algorithms and Software Cor-

poration.

de Andres C. 2003. The interest of multiple sclerosis attacks physiopa-

thology and therapy. Rev Neurol 36:1058–1064.

Diem R, Tschirne A, Bahr M. 2003. Decreased amplitudes in multiple

sclerosis patients with normal visual acuity: a VEP study. J Clin Neuro-

sci 10:67–70.

Dupouey P, Jacque C, Bourre J, Cesselin F, Privat A, Bauman N. 1979.

Immunochemical studies of myelin basic protein in shiverer mouse

devoid of major dense line of myelin. Neurosci Lett 12:113–118.

Gareau PJ, Rutt BK, Karlik SJ, Mitchell JR. 2000. Magnetization transfer

and multicomponent T2 relaxation measurements in an experimental

model of MS. J Magn Reson Imag 11:586–595.

Gulani V, Webb AG, Duncan ID, Lauterbur PC. 2001. Apparent diffu-

sion tensor measurements in myelin-deficient rat spinal cords. Magn

Reson Med 45:191–195.

Halliday AM, McDonald WI, Mushin J. 1973. Visual evoked-response in

diagnosis of multiple-sclerosis. Br Med J 4:661–664.

Hume AL, Waxman SG. 1988. Evoked potentials in suspected multiple

sclerosis—diagnostic-value and prediction of clinical course. J Neurol

Sci 83:191–210.

Huxley AF, Stampfli R. 1949. Evidence for salatory conduction in pe-

ripheral myelinated nerve fibers. J Physiol 108:315–339.

Fig. 7. Histological sections show that the optic nerve of a wild-type(A) and a heterozygous (B) mouse is myelinated, whereas the opticnerve of a shiverer (C) mouse is dysmyelinated. Cyrosection of thefixed optic nerves from wild-type (A), heterozygous (B), and shiverermutant (C) were stained with Fluoro-Myelin (Molecular Probes) andviewed on a Zeiss 510 confocal microscope at 488 nm with a 320

objective. The optic nerves of the wild-type (A) and heterozygous(B) mouse show bright fluorescent staining, indicating the presenceof myelin, whereas the shiverer mouse (C) shows no staining at thesame settings, indicating severe hypomyelination of the optic nerve.This immunohistochemistry confirms the MRI and VEP data.



Jackson A, Sheppard S, Johnson AC, Annesley D, Laitt RD, Kassner A.

1999. Combined fat- and water-suppressed MR imaging of orbital

tumors. AJNR Am J Neuroradiol 20:1963–1969.

Jacobs RE, Papan C, Ruffins S, Tyszka JM, Fraser SE. 2003. MRI: volu-

metric imaging for vital imaging and atlas construction. Nat Rev Mol

Cell BiolSuppl, SS10–SS16.

Kirschner DA, Ganser AL. 1980. Compact myelin exists in the absence

of basic protein in the shiverer mutant mouse. Nature 283:207–210.

Laule C, Vavasour IM, Moore GRW, Oger J, Li DKB, Paty DW,

MacKay AL. 2004. Water content and myelin water fraction in multi-

ple sclerosis: a T2 relaxation study. J Neurol 251:284–293.

Lehman DM, Harrison JM. 2002. Flash visual evoked potentials in the

hypomyelinated mutant mouse shiverer. Doc Ophthalmol 104:83–95.

Maarouf M, Kuchta J, Miletic H, Ebel H, Hesselmann V, Hilker R,

Sturm V. 2003. Acute demyelination: diagnostic difficulties and the

need for brain biopsy. Acta Neurochir 145:961–969.

Martin R, McFarland HF, McFarlin DE. 1992. Immunological aspects of

demyelinating diseases. Annu Rev Immunol 10:153–187.

Matthews WB. 1985. Clinical aspects. In: Matthews WB, et al., editors.

McAlpines’s multiple sclerosis, 49. Edinburgh: Churchill Livingstone.

Privat A, Jacque C, Bourpe JM, Dupouey P, Bauman N. 1979. Absence

of major dense line in the myelin of the mutant mouse shiverer. Neu-

rosci Lett 12:107–112.

Readhead C, Popko B, Takahashi N, Shine HD, Saavedra RA, Sidman

RL, Hood L. 1987. Expression of a myelin basic-protein gene in trans-

genic shiverer mice—correction of the dysmyelinating phenotype. Cell

48:703–712.

Roach A, Takahashi N, Pravtcheva D, Ruddle F, Hood L. 1985. Chro-

mosomal mapping of mouse myelin basic protein gene and structure

and transcription of the partially deleted gene in shiverer mustant mice.

Cell 42:149–155.

Rosenbluth J. 1980. Central myelin in the mouse mutant shiverer.

J Comp Neurol 194:639–648.

Shine HD, Readhead C, Popko B, Hood L, Sidman RL. 1992. Morphomet-

ric analysis of normal, mutant, and transgenic CNS—correlation of myelin

basic protein expression to myelinogenesis. J Neurochem 58: 342–349.

Sidman RJ, Conover CS, Cardon JH. 1985. Shiverer gene maps near the

distal end of chromosome 18 in the house mouse. Cytogenet Cell

Genet 39:241–245.

Song SK, Sun SW, Ramsbottom MJ, Chang C, Russell J, Cross AH.

2002. Dysmyelination revealed through MRI as increased radial (but

unchanged axial) diffusion of water. Neuroimage 17:1429–1436.

Sospedra M, Martin R. 2005. Immunology of multiple sclerosis. Annu

Rev Immunol 23:683–747.

Strain GM, Tedford BL. 1993. Flash and pattern-reversal visual-evoked

potentials in C57BL/6J and B6CBAF1/J mice. Brain Res Bull 32:57–63.

Takahashi N, Roach A, Teplow DB, Prusiner SB, Hood L. 1985. Clon-

ing and characterization of the myelin basic protein gene from mouse:

one gene can encode both 14 kd and 18.5 kd MBPs by the alternate

use of exons. Cell 42:139–148.

Tyszka JM, Readhead C, Bearer EL, Pautler RG, Jacobs RE. 2006. Sta-

tistical diffusion tensor histology reveals regional dysmyelination effects

in the shiverer mouse mutant. Neuroimage 29:1058–1065.

Waxman SG. 1980. Determinants of conduction velocity in myelinated

nerve fibers. Muscle Nerve 3:141–150.

Webb S, Munro CA, Midha R, Stanisz GJ. 2003. Is multicomponent T2

a good measure of myelin content in the peripheral nerve? Magn

Reson Med 49:638–645.

Wood JC, Johnson KJ. 1999. Wavelet-denoising of magnetic resonance

images: importance of Rician statistics. Magn Reson Med 41:631–635.

Wood JC, Martin M, Readhead C, Jacobs RE. 2002.Improved T2 Maps

using complex wavelet-packet denoising. International Society for Mag-

netic Resonance in Medicine Tenth Scientific Meeting and Exhibition.

p 2291.

Woog L. 1996.Adaptive waveform algorithms for denoising. PhD Thesis,

Department of Computer Science, Yale University.

1726 Martin et al.


Myelin deficiencies visualized in vivo: Visually evoked potentials and T2-weighted magnetic...

Documents

Transcript of Myelin deficiencies visualized in vivo: Visually evoked potentials and T2-weighted magnetic...