Motor neuron pathology in experimental autoimmune - Brain

doi:10.1093/brain/awh550 Brain (2005), 128, 1877–1886

Motor neuron pathology in experimentalautoimmune encephalomyelitis: studies inTHY1-YFP transgenic mice

P. G. Bannerman,1 A. Hahn,1 S. Ramirez,1 M. Morley,1 C. Bonnemann,1 S. Yu,2 G.-X. Zhang,2

A. Rostami2 and D. Pleasure1

1Neurology Research, Abramson Pediatric Research Center, Children’s Hospital of Philadelphia and 2Department ofNeurology, Thomas Jefferson Hospital, Philadelphia, Pennsylvannia, USA

Correspondence to: David Pleasure MD, Room 516H Abramson Research Building, The Children’s Hospital of Philadelphia,34th and Civic Center Blvd, Philadelphia, PA, USAE-mail: [email protected]

Using adult male C57BL/6 mice that express a yellow fluorescent protein transgene in their motor neurons,we induced experimental autoimmune encephalomyelitis (EAE) by immunization withmyelin oligodendrocyteglycoprotein peptide 35–55 (MOG peptide) in complete Freund’s adjuvant (CFA). Control mice of the sametransgenic strain received CFAwithout MOG peptide. Early in the course of their illness, the EAEmice showedlumbosacral spinal cord inflammation, demyelination and axonal fragmentation. By 14 weeks post-MOGpeptide, these abnormalities were much less prominent, but the mice remained weak and, as in patientswith progressive multiple sclerosis, spinal cord atrophy had developed. There was no significant loss of lumbarspinal cord motor neurons in the MOG peptide-EAE mice. However, early in the course of the illness, motorneuron dendrites were disrupted and motor neuron expression of hypophosphorylated neurofilament-H(hypoP-NF-H) immunoreactivity was diminished. By 14 weeks post-MOG peptide, hypoP-NF-H expressionhad returned to normal, but motor neuron dendritic abnormalities persisted and motor neuron perikaryalatrophy had appeared. We hypothesize that these motor neuron abnormalities contribute to weakness in thisform of EAE and speculate that similar motor neuron abnormalities are present in patients with progressivemultiple sclerosis.

Keywords: motor neuron; experimental autoimmune encephalomyelitis (EAE); dendrite; axonal degeneration;multiple sclerosis

Abbreviations: CFA = complete Freund’s adjuvant; EAE = experimental autoimmune encephalomyelitis; hypoP-NF-H =

hypophosphorylated neurofilament heavy; MAP2a = microtubule-associated protein 2a; MBP = myelin basic protein;MOG = myelin oligodendrocyte glycoprotein; PBS = phosphate-buffered saline; YFP = yellow fluorescent protein

Received February 11, 2005. Revised April 4, 2005. Accepted April 21, 2005. Advance Access publication May 18, 2005

IntroductionMultiple sclerosis is an immune-mediated disease character-

ized clinically by relapsing-remitting or progressive neurolo-

gical deficits, and pathologically by multiple plaques of CNS

inflammation and demyelination. While multiple sclerosis

was for many years considered to be primarily a myelin sheath

disorder, it is now clear that, as Charcot (1877) first observed,

axonal fragmentation is an important component of the dis-

ease (Trapp et al., 1998; Perry and Anthony, 1999; Bjartmar

et al., 2000, 2003). In vivo neuroimaging supports the concept

that loss of CNS axons contributes substantially to progressive

multiple sclerosis disability (De Stefano et al., 1998, 2001;

Edwards et al., 1999; Miller et al., 2002; Lin et al., 2003,

2004). Occasionally, neuronal perikarya are also lost in

multiple sclerosis (Peterson et al., 2001).

Experimental autoimmune encephalomyelitis (EAE) is

elicited in susceptible animal strains by immunization with

various myelin antigens. As in multiple sclerosis, pathological

features of EAE include foci of CNS inflammation, demy-

elination, and axonal blebbing, tortuosity and fragmenta-

tion (Slavin et al., 1998; Pitt et al., 2000; Kornek et al., 2001;

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Onuki et al., 2001). Reversible motor neuron dendritic

beading has also been observed during the acute phase of

myelin basic protein (MBP)-induced EAE in Lewis rats

(Zhu et al., 2003). Death of neurons has been documented

in greater detail in EAE than in multiple sclerosis. For

example, approximately one quarter of spinal cord ventral

horn neurons are lost in Lewis rats with MBP-induced

EAE (Smith et al., 2000) and neuronal apoptosis occurs in

the CNS of mice and rats with MBP- or myelin oligodendro-

cyte glycoprotein (MOG) peptide-induced EAE (Meyer et al.,

2001; Ahmed et al., 2002; Diem et al., 2003; Hobom et al.,

2004; Kanwar et al., 2004).

Our goal was to characterize motor neuron pathology in

EAE. To this end, we induced EAE in a transgenic line of

C57BL/6 mice expressing yellow fluorescent protein (YFP) in

the perikarya and processes of a variety of neurons, including

all motor neurons (Feng et al., 2000), by immunizing them

with a peptide homologous to residues 35–55 of rodent MOG

(MOG peptide) (Slavin et al., 1998; Onuki et al., 2001; Gran

et al., 2002; Zhang et al., 2003).

The intense perikaryal YFP fluorescence of spinal cord

motor neurons in these mice was helpful for motor neuron

counting and cell body size measurements, and facilitated

motor neuron localization of proteins of interest by dual

label fluorescence microscopy. Our results indicate that,

while these chronically weak MOG peptide-EAE mice do

not lose motor neurons, they do develop persistent motor

neuron dendritic abnormalities and late motor neuron

perikaryal atrophy.

MethodsThy1-YFP transgenic miceFounders for our colony of C57BL/6mice expressing a YFP transgene

driven by thy1 promoter elements (Feng et al., 2000) (thy1-YFP

mice) were obtained from Jackson Labs Bar Harbor, Maine, USA.

(B6.Cg-TgN(Thy1-YFPH)2Jrs).

MOG peptide-EAEWhile female mice are more susceptible than male mice to some

forms of EAE, preliminary observations demonstrated an approx-

imately equal clinical severity of MOG peptide-induced EAE in

female and male C57BL/6 thy1-YFP transgenic mice. Because it

was more efficient to generate males from our colony of these

mice, all our subsequent studies employed male thy1-YFP transgenic

mice. MOG peptide in complete Freund’s adjuvant (CFA) or CFA

withoutMOGpeptidewasadministered to thesemice at age3months.

Rodent MOG peptide (300 mg, amino acids 35–55, synthetic, reverse

phase chromatographically purified) in CFA containing 4 mg/ml of

killed Mycobacterium tuberculosis (Becton Dickinson Frankin Lakes,

NJ, USA) was administered by subcutaneous flank injection on

day 0, followed by tail vein administration of 75 ng of pertussis

toxin (List Biological Labs, Campbell, CA, USA) on days 0 and 2

post-immunization (Gran et al., 2002; Zhang et al., 2003). Control

mice received CFA and pertussis toxin, but no MOG peptide.

The mice were weighed and examined daily. Neurological deficits

were graded on a five-point scale (limp tail or waddling gait = 1; limp

tail and ataxia = 2; single limb paresis and ataxia = 2.5; single limb

paralysis = 3; single limb paralysis and paresis of second limb = 3.5;

full paralysis of two limbs = 4; moribund = 4.5; and death = 5) (Gran

et al., 2002; Zhang et al., 2003). MOG-EAE and control thy1-YFP

transgenic mice were sacrificed shortly after the onset of deficits

(14 days post-immunization), at peak mean clinical severity

(21 days post-immunization) and late in the course of their illness

(98 days post-immunization).

Spinal cord tissue fixation and processingMOG peptide-EAE and control thy1-YFP mice were anaesthetized

with ketamine/xylazine, then perfused via the heart with saline fol-

lowed by phosphate-buffered 4% (w/v) paraformaldehyde. Spinal

cords were removed, post-fixed at 25�C with phosphate-buffered

4% paraformaldehyde for 1 h, then transected in the mid-lumbar

region and processed for paraffin and cryostat sectioning. The par-

affin sections corresponded to spinal cord levels L5 and L6, whereas

the cryostat sections corresponded to spinal cord levels L5 and L4.

To prepare paraffin sections for fluorescence microscopy, spinal

cord was washed three times with phosphate-buffered saline (PBS)

post-fixation, dehydrated through ascending ethanol, cleared in

xylene, and infiltrated with Paraplast wax (Surgipath, Richmond,

IL, USA). Six mm transverse sections were collected on Superfrost/

Plus slides (Fisher Scientific Morris Plains, NJ, USA) and melted at

56�C for 20 min, then deparaffinized in xylene and rehydrated

through descending alcohols.

For cryostat sections, lumbar spinal cord was cryoprotected by

overnight immersion in 30% sucrose at 4�C prior to embedding in

OCT cryostat mounting medium (Miles Inc, Kankakee, IL, USA).

After freezing in 2-methylbutane cooled with liquid nitrogen, trans-

verse 10 mm cryostat sections were mounted on poly-L-lysine coated

slides.

Spinal cord immunohistologyThe primary antibodies used in this study are listed in Table 1.

Paraffin sections were deparaffinized and treated with 10% (v/v)

Triton X-100 in PBS for 30 min, then immunostained with

mouse monoclonal antibodies (mAbs) using a mouse-on-mouse

kit (Vector Labs Burlingane, CA, USA) as per the manufacturer’s

instructions. Binding of the biotinylated secondary layer to the prim-

ary antibody was detected with rhodamine-conjugated streptavidin

(diluted 1 : 100, Jackson Immunoresearch Labs, West Grove, PA,

USA). For combined detection of CD11b+ microglia/macrophages

or CD40+ lymphocytes and TUNEL (terminal deoxynucleotidyl

transferase-mediated dUTP nick end labelling) histochemistry for

intranuclear DNA nicking (Gavrieli et al., 1992; Ray et al., 2000),

frozen sections were incubated overnight with rat anti-CD11b or

goat anti-CD40 in block A [minimum essential medium containing

15 mM HEPES buffer, 10% foetal bovine serum (FBS) and 0.05%

sodium azide].

Detection of primary antibody binding was performed using

rhodamine-conjugated second antibodies. Then we applied a

minor modification of the TUNEL procedure (Gavrieli et al., 1992),

substituting Pacific blue-conjugated streptavidin for streptavidin

peroxidase (Yasuda et al., 1995). Following immunolabelling with

primary and secondary antibodies, all sections, both paraffin and

cryostat, were post-fixed with �20�C methanol, counterstained

(with the exception of the slides used for TUNEL) with the nuclear

1878 Brain (2005), 128, 1877–1886 P. G. Bannerman et al.

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dye Hoechst 33258 (2 mg/ml in PBS, Sigma Chemical Co., St Louis,

MO, USA) for 3 min, and mounted in Vectashield.

Skeletal muscle immunohistologyCryostat sections (10 mm) of tibialis anterior obtained from anaes-

thetized mice prior to perfusion were fixed in 100% methanol for

20 s, pre-incubated in vehicle (10% FBS, 1· PBS, 0.1% Triton X-100)

for 1 h, and then incubated with primary antibodies diluted in vehicle

for 1 h, all at room temperature. Antibodies used were rabbit poly-

clonal AP5B3 (1 : 750) raised against alpha-actinin 3, which specif-

ically stains type 2B skeletal muscle fibres, and rabbit polyclonal

81864 raised against sacrospan to outline the sarcolemma of indi-

vidual muscle fibres (1 : 2500). The slides were then washed 3· for

20 min each with 1· PBS, and incubated with an Alexa fluor-labelled

secondary antibody at room temperature for 1 h in the dark. After

three additional washes, the slides were mounted in Vectashield.

Imaging and image analysisFluorescent images were captured with a Hamamatsu Orca-ER

digital camera mounted on a Leica DMR upright microscope or

by confocal microscopy with a Leica DM IRE2 HC fluo TCS 1-B-

UVmicroscope coupled to a Leica TCS SP2 spectral confocal system.

To evaluate numbers and sizes of motor neurons in EAE versus

control animals, laser scanning confocal microscopic scans were

performed on paraffin sections corresponding to spinal cord levels

L5 through L6. The areas of ventral horn YFP positive cell bodies

were measured using a Leica software program developed specifically

for the confocal system (Leica Microsystems, Heidelberg, Germany).

The area of ventral horn investigated mapped to motor neurons

innervating the following muscle groups: iliopsoas/sartorius,

quadriceps, adductors, hamstrings, gluteal and other proximal

muscles of the thigh, posterior compartment of the distal hind

limb (Vanderhorst and Holstege, 1997), i.e. the major muscle groups

controlling hind limb mobility. To standardize the measurements,

only perikarya in which the nucleus and nucleolus were in the plane

of the section were counted and measured (McPhail et al., 2004).

We devised a semi-quantitative scale to score the extent of motor

neuron dendritic pathology, employing microtubule-associated

protein 2a (MAP2a) immunostaining with the AP14 antibody.

The scorer was not provided with information as to whether a spe-

cimen was from an EAE or a control mouse; nor, in the EAE mice, as

to the duration and severity of EAE. A score of 0 (normal) was

assigned when most or all motor neuron dendrites were of normal

thickness and length. A score of + was assigned when the majority

of motor neuron dendrites were thinner than normal, a score

of ++ when the majority of motor neuron dendrites were shortened

or fragmented, and a score of +++ when the majority of motor

neuron dendrites were lost.

ResultsClinical features of MOG peptide-EAEControl thy1-YFP transgenic mice (n = 20) did not demon-

strate weight loss or neurological deficits at any point follow-

ing CFA injection. Thirty-four mice were immunized with

MOG peptide in CFA. Of these, four did not show weight loss

or neurological deficits at any point during the next 98 days;

these four were excluded from further analysis. In the remain-

ing 30 MOG peptide-injected mice, neurological deficits first

appeared 12–14 days post-immunization, preceded by 1 or

2 days of weight loss which averaged 10% of pre-symptomatic

body weight.

Five EAE mice and four CFA controls were sacrificed on

day 14 post-immunization. Clinical severity in the remaining

EAE mice peaked between days 19 and 21 post-immunization

(Fig. 1). Fifteen EAE mice and eight CFA controls were

sacrificed on day 21. In the remaining 10 EAE mice, clinical

deficits then subsided to a plateau level. However, in three of

these 10, a relapse occurred between day 21 and day 98 post-

immunization, defined as an increase in severity of neurolo-

gical deficits of one grade or more that persisted for �3 days.

Two of the 10 EAE mice had become entirely asymptomatic

by day 98 post-MOG peptide. These remaining 10 EAE mice

and eight CFA controls were sacrificed on day 98.

Lumbosacral spinal cord inflammation,demyelination and axonal fragmentation inMOG peptide-EAEAccumulations of inflammatory cells in spinal cord were

prominent on day 14 after administration of MOG peptide

in CFA, but were less apparent by days 21 and 98, and were

not present in CFA control mice. Combined immunofluor-

escence/TUNEL histochemistry demonstrated frequent

CD11b+ or CD40+ cells undergoing apoptosis on day 14

post-MOG peptide, as has previously been reported by

Ray et al. (2000). Demyelination, as visualized by MBP

immunoreactivity, was most evident in mice sacrificed

Table 1 Antibodies used in this study

Antibody specificity Name Titre used Source

Hypophosphorylated NF-H SMI-32 ascites 1 : 800 Sternberger Monoclonals Inc., Lutherville, MD, USAHypophosphorylated NF-H RMDO9.5 ascites 1 : 100 V.M-Y. Lee PhD, Pathology, University of Pennsylvania, PA, USAMyelin basic protein SMI-99 ascites 1 : 1000 Sternberger Monoclonals Inc.CD11b antigen M1/70 10 mg/ml Serotec, Raleigh, NC, USACD40 antigen sc-975 10 mg/ml Santa Cruz Biotechnology, Inc., Santa Cruz, California, USAMicrotubule-associated AP14 ascites 1 : 200 V.M-Y. Lee PhD, Pathology, University of PennsylvaniaProtein 2a (MAP2a) alpha-actinin 3 AP5B3 1 : 750 Dr A. BeggsSarcospan 81864 1 : 2500 Dr L. Kunkel

Motor neurons in EAE Brain (2005), 128, 1877–1886 1879

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21 days post-MOG peptide. Substantial remyelination had

occurred by day 98 post-MOG peptide. Disrupted and bal-

looned axons, visualized by hypophosphorylated neurofila-

ment heavy (hypoP-NF-H) immunohistology (Sternberger

and Sternberger, 1983; Lee et al., 1987), were present in spinal

cord white matter on day 14 post-MOG peptide, and had

become more prominent by day 21 (data not shown).

Spinal cord grey and white matteratrophy occur in MOG peptide-EAESpinal cord atrophy has been well documented in multiple

sclerosis (Edwards et al., 1999; Lin et al., 2003, 2004; Ukkonen

et al., 2003). To determine whether spinal cord atrophy also

occurs in MOG peptide-EAE, we compared the areas of grey

and white matter in paraffin-embedded cross sections of L5,6

spinal cord from 98-day post-MOG peptide and 98-day post-

CFA control mice. There were �20% smaller cross-sectional

areas of both greymatter (EAE= 0.9486 0.137mm2; control=

1.230 6 0.204 mm2, mean 6 SD, P < 0.01) and white matter

(EAE = 0.891 6 0.178 mm2; control = 1.106 6 0.206 mm2,

mean6SD; P < 0.05) in the MOG peptide-EAE mice.

Motor neurons survive in MOGpeptide-EAELoss of spinal cord ventral horn neurons has previously been

noted in rats with MBP-induced EAE (Smith et al., 2000).

Motor neurons of the thy1-YFP transgenic mice expressed

both cytoplasmic and nuclear YFP, with most intense fluor-

escence in the nucleus (Fig. 2). This intrinsic YFP fluorescence

facilitated visualization of motor neurons in these mice.

Three lines of evidence indicated that substantial numbers

of motor neurons were not lost as a consequence of MOG

peptide-EAE in these mice. First, we saw no TUNEL+ YFP+

neurons in any of the mice we examined on day 14, 21,

or 98 post-MOG peptide (data not shown). Secondly, there

were no significant differences between densities of YFP+

motor neurons in cross sections of L5,6 spinal cords of

MOG peptide-EAE and CFA control mice at day 98 post-

immunization (MOG peptide-EAE mice = 114 6 24; YFP

control mice = 132620; mean 6 SD, n = 8; P > 0.1). Thirdly,

examination of L5,6-innervated skeletal muscles using poly-

clonal anti-sarcospan antibodies, which outline skeletal mus-

cle sarcolemma, showed neither atrophic nor angulated

skeletal muscle fibres in the day 98 post-MOG peptide EAE

animals; and, using polyclonal anti-alpha-actinin 3 antibodies

which specifically label type 2b skeletal muscle fibres, there

was no fibre type-specific grouping (data not shown). These

immunohistological results indicated that skeletal muscle

fibres were neither denervated nor reinnervated by axonal

collateral sprouting at this late time-point in these mice.

Motor neuron phenotype is altered inMOG peptide-EAEWe documented alterations in motor neuron perikaryal size,

perikaryal cytoskeletal phosphorylation and dendrites in the

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1.50

1.00

0.50

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Clin

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8 11 14 17 20 23 26 29 32 35 38 41 44 47

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50 53 56 59 62 65 68 71 74 77 80 83 86 89 92 95 98

Days post-MOG peptide

Fig. 1 Mice immunized with MOG peptide in CFA show prolonged neurological disability. Days post-MOG peptide administrationare plotted on the x-axis and neurological disability scores on the y-axis. Peak disability was on day 21 post-MOG peptide.Data shown are means 6 SD.


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MOG peptide-EAE mice. First, while the mean L5,6 spinal

cord motor neuron perikaryal cross-sectional area in these

mice was not significantly different from that in the CFA

control mice, the average transverse area of L5,6 motor neu-

ron perikarya in the 98-day post-MOG peptide mice was 15%

smaller than that in simultaneous CFA controls (427 6 75

versus 502 6 48 mm2, respectively, mean 6 SD, P < 0.02).

Secondly, we noted a widespread, but reversible, alteration

in motor neuron perikaryal neurofilament phosphorylation

in the EAE mice at day 21 post-MOG peptide immunization.

As previously reported (Carriedo et al., 1996; Tsang et al.,

2000), motor neurons of CFA control mice expressed abun-

dant perikaryal hypoP-NF-H immunoreactivity. Whereas

>80% of lumbar spinal cord motor neuron perikarya

Fig. 2 Motor neuron perikaryal hypoP-NF-H immunoreactivity is depleted in MOG peptide-EAE. In these transversely orientedsections through L5,6 spinal cord anterior horns, Hoechst nuclear staining is shown in blue, hypoP-NF-H (SMI-32 mAb)immunostaining in red, and YFP fluorescence in green. Panels A, C and E are identical fields from a 21-day post-CFA control mouse. PanelsB, D and F are identical fields from a 21-day post-MOG peptide EAE mouse with a clinical disability score of 3.5. Note that YFPfluorescence was most intense in motor neuron nuclei in both control and EAE mice (A, B). Also note the co-localization of YFP andhypoP-NF-H in motor neuron perikarya of the CFA control (E), resulting in the yellow colour and the paucity of hypoP-NF-H in the YFP+motor neuron perikarya in the MOG peptide-EAE mouse (F). Magnifications are the same in the six panels; the size bar denotes 40 mm.


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expressed abundant hypoP-NF-H immunoreactivity in the

21-day post-CFA control mice (n = 8), this proportion had

fallen to <70% in 21-day post-MOG peptide EAE mice with

mild to moderate clinical deficits (deficit score 2.5 or lower, n

= 6), and to <60% in 21-day post-MOG peptide EAE mice

with severe clinical deficits (deficit score 3 or higher, n = 9) (P

< 0.01, x2 test).

An example of the depletion of hypoP-NF-H immuno-

staining in a mouse with grade 4 clinical deficits is shown

in Fig. 2. In the fields illustrated in this figure, average motor

neuron perikaryal size was larger in the CFA control mouse

than the MOG peptide-EAE mouse, but this was not a con-

sistent finding at this time-point. Even though clinical deficits

persisted beyond 21 days in most mice, motor neuron

perikaryal hypoP-NF-H immunoreactivity had returned to

normal by 98-day post-MOG peptide immunization (data

not shown).

The third abnormality we observed in motor neurons

of the MOG peptide-EAE mice was dendritic thinning, short-

ening and fragmentation. This was evident from both YFP

fluorescence and MAP2a immunofluorescence microscopy

(Papandrikopoulou et al., 1989; Riederer et al., 1995)

(Fig. 3). Note the paucity of motor neuron dendrites in the

14-day post-MOG peptide EAE mouse (clinical deficit score

2.5) in Fig. 3C and D compared with those of the 14-day

CFA control mouse shown in Fig. 3A and B. MAP2a

immunostaining was particularly useful in highlighting this

dendritic abnormality.

To permit semi-quantitation of dendritic alterations in the

30 MOG peptide-EAE mice and 20 CFA control mice that we

autopsied, we devised a semi-quantitative score that reflected

the aggregate severities of motor neuron dendritic shortening,

fragmentation and thinning, assigning grades varying between

normal (0) and very marked dendritic pathology (+++) (see

Methods). An example of each of these scores is shown in

Fig. 4; each panel is a Z-stack of 12 sequential 500-nm con-

focal optical slices through an L5,6 lumbar anterior horn. An

observer without knowledge of the history of individual mice

then scored the motor neuron dendritic pathology in L5,6

ventral horns in each of the 50mice, again using Z-stacks of 12

sequential 500-nm confocal optical sections. Only the red

(MAP2a) channel was viewed in order to avoid potential

bias introduced by the presence or absence of inflammatory

cells in the field. Results are summarized in Fig. 5.

Nineteen of the 20 CFA control mice, but only four of the

30MOG peptide-EAEmice, received a score of 0. Moderate to

severe dendritic pathology (++ or +++) was scored for 17 of

the 30 MOG peptide-EAE mice, but was not seen in any of the

CFA control mice. Dendritic abnormalities were observed in

the majority of theMOG peptide-EAEmice both early (day 14

post-immunization) and late (day 98 post-immunization)

in their illness. Interestingly, the two EAE mice with

Fig. 3 Motor neuron dendrites are disrupted in MOG peptide-EAE. In these transverse paraffin sections through L5,6 spinal cord,nuclear Hoechst staining is shown in blue, MAP2a (AP14) immunostaining in red, and YFP fluorescence in green. Co-localization of YFP(green) and MAP2a (red) in the cytoplasm of motor neurons in (A) and (C) results in the yellow coloration of portions of themotor neuron perikarya and dendrites. (A) and (B) are identical fields from a 14-day post-CFA control mouse, while (C) and (D) areidentical fields from a 14-day post-MOG peptide-EAE mouse with a clinical score of 2.5. Note the depletion of AP14+ dendrites in(C) and (D). Magnifications are the same in the four panels; the size bar denotes 50 mm.


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grade 0 (normal) L5,6 motor neuron dendrites on day 98

post-MOG peptide (see Fig. 5) were the only two in the

group of 10 EAE mice examined at that time-point which

had shown full clinical recovery from their clinical neurolo-

gical deficits.

DiscussionUntil 1998, most clinicians attributed all neurological deficits

in multiple sclerosis to demyelination, with permanent

deficits presumed to result from incomplete remyelination

and nerve action potential conduction block. But the redis-

covery of multiple sclerosis-associated axonal disruption by

confocal microscopy (Trapp et al., 1998), followed by longi-

tudinal in vivo imaging and magnetic resonance spectroscopic

evidences of axonal loss in patients with multiple sclerosis

(De Stefano et al., 1998, 2001; Edwards et al., 1999; Filippi

et al., 2003; Lin et al., 2003, 2004) have led to recognition that

axonopathy is a major cause of progressive and permanent

neurological disability in patients with multiple sclerosis

Fig. 4 Motor neuron dendrite disruption scoring system. In these transverse sections through L5,6 spinal cord, MAP2a (AP14)immunostaining is shown in red. Normal dendrites (0) are shown in a section prepared from a CFA control mouse. Progressively moresevere dendritic disruptions (+, ++ and +++) were prepared from MOG peptide-EAE mice. Each panel is a Z-stack of 12 successive500-nm confocal optical sections. Magnifications are the same in the four panels; the size bar denotes 50 mm.

Fig. 5 Scoring of motor neuron dendritic disruption as a functionof time post-immunization with MOG peptide in CFA or withCFA alone. X denotes results in MOG peptide-EAE mice, andsolid circles denote results in CFA control mice. Days post-immunization (D14, D21 and D98) are shown on the x-axis anddendritic disruption scoring (0, +, ++ or +++) on the y-axis.


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(Bjartmar et al., 2000, 2003). The causes of axonopathy in

multiple sclerosis and EAE have still not been fully established

(Kornek et al., 2001; Craner et al., 2004; Stys, 2004). However,

axonal disruption is most prevalent within plaques of demy-

elination (Trapp et al., 1998; Bjartmar et al., 2003), suggesting

that inflammatory processes centred in or bordering plaques

target axons as well as myelin or, alternatively, that a primary

loss of myelin within plaques causes secondary loss of axonal

integrity.

Most of the surviving mice with EAE in the present study

still demonstrated clinical neurological deficits 98 days after

receiving MOG peptide. At this late time-point, spinal

cord white matter atrophy had developed, presumably as

a consequence of depletion of myelinated axons. While we

are not aware of prior reports of spinal cord white matter

atrophy in EAE, autopsy and in vivoMRI studies have demon-

strated white matter atrophy in patients with progressive

multiple sclerosis (Charcot, 1877; Edwards et al., 1999; Lin

et al., 2003, 2004; Ukkonen et al., 2003).

Neuronal apoptosis occurs in the spinal cord and retina in

EAE (Ahmed et al., 2002; Diem et al., 2003; Hobom et al.,

2004) and in multiple sclerosis plaques that involve cerebral

cortical grey matter (Peterson et al., 2001). The observation

that a substantial number of ventral horn neurons are lost

within 2 weeks after immunization of Lewis rats with myelin

basic protein (Smith et al., 2000), together with reports of

spinal cord motor neuron dysfunction, focal amyotrophy and

generalized weakness and fatigue in some patients with

multiple sclerosis (Petajan, 1982; Fisher et al., 1983; Shefner

et al., 1992; de Haan et al., 2000; Chaudhuri and Behan, 2004),

motivated us to evaluate whether neuronal perikarya are

targeted in MOG peptide-EAE. Based on the report by

Smith et al. (2000), we anticipated that YFP fluorescence

imaging might reveal substantial depletion of motor neurons

in the MOG peptide-EAE mice. In fact, however, these

mice did not show a statistically significant loss of motor

neurons.

In accord with this result, TUNEL histochemistry did not

demonstrate motor neuron apoptosis in the MOG peptide-

EAE mice and, 98 days after MOG peptide administration,

these mice did not show immunohistological evidences of

skeletal muscle fibre denervation or re-innervation. The dis-

crepancy between the observations of Smith et al. (2000) and

our own may reflect the divergent species and immunogens

used in the two studies, but differences in the neuron labelling

protocols may also have contributed. Smith et al. (2000)

counted all ventral horn neurons, including interneurons,

labelled by cresyl violet, a Nissl stain, whereas we determined

the density in L5,6 spinal cord of YFP+ motor neurons.

Thus, it is possible that either ventral horn interneurons

are selectively susceptible to death in EAE or the use by

Smith et al. (2000) of cresyl violet resulted in substantial

motor neuron undercounts in their EAE rats, as has been

reported when this stain has been used in other circumstances

where there is motor neuron perikaryal atrophy (McPhail

et al., 2004).

Late in the course of EAE in the thy1-YFP transgenic mice,

the mean cross-sectional area of lumbar spinal cord motor

neuron perikarya was 15% below that in CFA control mice

of the same strain and age. Motor neuron atrophy has not

previously been documented in EAE or multiple sclerosis, nor

has atrophy of this magnitude been reported in other neuronal

subsets in these disorders (Miller et al., 2002), though a

slight diminution in the size of parvocellular neurons in

the lateral geniculate body was noted in patients with multiple

sclerosis (Evangelou et al., 2001). Motor neuron perikaryal

atrophy in theMOGpeptide-EAEmice is unlikely to have been

a consequence of motor neuron axotomy (McPhail et al.,

2004), since there were no evidences in these mice of skeletal

muscle denervation or re-innervation. Other possible causes

for motor neuron shrinkage in these mice include a loss of

necessary trophic input owing to EAE-induced motor neuron

dendritic pathology (Zhu et al., 2003, and the present study)

or a deleterious effect of chronic exposure to pro-

inflammatory cytokines (Villarroya et al., 1997).

Much of the NF-H in normal motor neuron perikarya is

hypophosphorylated, and becomes hyperphosphorylated

only after transport into the axon (Carriedo et al., 1996;

Sun et al., 1996; Veeranna et al., 1998; Brownlees et al.,

2000; Tsang et al., 2000; Ackerley et al., 2004). In the

MOG peptide-EAE mice, the proportion of motor neurons

that displayed perikaryal hypoP-NF-P immunoreactivity had

fallen substantially by day 21 post-MOG peptide. This peri-

karyal cytoskeletal phosphorylation alteration was most

widespread in mice with the most severe clinical deficits.

Our studies do not shed light on the cause of this temporary

alteration in perikaryal neurofilament phosphorylation.

However, prior studies have shown that increased phos-

phorylation of neuronal perikaryal NF-H can be elicited by

raising the extracellular concentration of glutamate (Ackerley

et al., 2000) and perturbed CNS extracellular glutamate

homeostasis has been documented in EAE and multiple

sclerosis (Hardin-Pouzet et al., 1997; Matute et al., 2001;

Werner et al., 2001).

Using both YFP fluorescence and MAP2a immunofluores-

cence, we observed persistent alterations in the architecture

of motor neuron dendrites in the MOG peptide-EAE mice,

including proximal dendritic thinning, shortening and

fragmentation. Even by stacking multiple confocal optical

slices, we could not visualize all of the proximal dendrites

of individual motor neurons accurately; this would best be

achieved using in vivo cholera toxin or post-mortem DiI

retrograde tracing techniques (Snider and Palavali, 1990;

Ritz et al., 1992; Zhu et al., 2003). However, using a semi-

quantitative scoring system to grade abnormalities visualized

byMAP2a immunostaining, we were able to document motor

neuron dendritic abnormalities throughout the course of

MOG peptide-EAE. In addition, while the number of mice

with EAE we examined at day 98 was small (n = 10), our

observations suggested a correlation between full remission

of clinical illness and restoration of normal dendritic

architecture.


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We are not aware of prior reports of persistent dendritic

abnormalities in motor neurons in multiple sclerosis or EAE.

However, acute reversible beading of distal motor neuron

dendrites in spinal cord white matter has been documented

by both MAP2 immunostaining and in vivo cholera toxin

retrograde labelling techniques in Lewis rats with MBP-

induced EAE (Zhu et al., 2003).

In summary, we have documented perikaryal atrophy and

disruption of dendritic architecture in lumbosacral motor

neurons of mice with MOG peptide-induced EAE. Morpho-

metric studies will be required to determine the relative

extents to which motor neuron perikaryal atrophy and dend-

ritic disruption contribute to late spinal cord grey matter

atrophy in these mice. Further investigation is also needed

to evaluate whether similar motor neuron abnormalities

occur in multiple sclerosis.

AcknowledgementsWe wish to thank J. Golden, R. Kalb and J. Grinspan for their

critical reading of this manuscript. This work was supported

by the National Multiple Sclerosis Society, Muscular

Dystrophy Association, and NIH grant NS25044.

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