behavioral testing strategies in a localized animal model of multiple sclerosis

www.elsevier.com/locate/jneuroim

Journal of Neuroimmunology 153 (2004) 158–170

Behavioral testing strategies in a localized

animal model of multiple sclerosis

Bigna S. Buddeberg, Martin Kerschensteiner 1, Doron Merkler2,Christine Stadelmann2, Martin E. Schwab*

Brain Research Institute, University of Zurich and Department Biology Swiss Federal Institute of Technology,

Winterthurerstrasse 190, CH-8057, Zurich, Switzerland

Received 16 January 2004; received in revised form 26 April 2004; accepted 21 May 2004

Abstract

To assess neurological impairments quantitatively in an animal model of multiple sclerosis (MS), we have used a targeted model of

experimental autoimmune encephalomyelitis (EAE), which leads to the formation of anatomically defined lesions in the spinal cord. Deficits

in the hindlimb locomotion are therefore well defined and highly reproducible, in contrast to the situation in generalized EAE with

disseminated lesions. Behavioral tests for hindlimb sensorimotor functions, originally established for traumatic spinal cord injury, revealed

temporary or persistent deficits in open field locomotion, the grid walk, the narrow beam and the measurement of the foot exorotation angle.

Such refined behavioral testing in EAE will be crucial for the analysis of new therapeutic approaches for MS that seek to improve or prevent

neurological impairment.

D 2004 Elsevier B.V. All rights reserved.

Keywords: EAE; Multiple sclerosis; Behavior; Locomotion

1. Introduction

Multiple sclerosis (MS) is a major inflammatory, demy-

elinating disease of the central nervous system (CNS) that

leads to severe neurological disabilities (Noseworthy et al.,

2000). Similar disabilities can be mimicked in experimental

autoimmune encephalomyelitis (EAE), the most commonly

used animal model to study MS in rats and mice. The

classical EAE model is characterized by disseminated

lesions which give rise to a high variability in disease

course and behavioral outcome. Behavioral symptoms in

EAE animals may be related to a brainstem lesion or to a

lesion located in the spinal cord or the cerebellum. In

addition, EAE animals often have multiple lesions adding

even more to the complexity of the observed disease course.

Therefore, sensitive behavioral testing is hard to carry out in

a classical EAE model and the disease course of animals

0165-5728/$ - see front matter D 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.jneuroim.2004.05.006

* Corresponding author. Tel.: +41-1-635-3330; fax: +41-1-635-3303.

E-mail address: [email protected] (M.E. Schwab).1 Department of Anatomy and Neurobiology, Washington University

School of Medicine, St. Louis, USA.2 Institute of Neuropathology, University of Gottingen, Germany.

with such diverse symptoms might be difficult to compare.

This is the reason why the only behavioral analysis carried

out in classical EAE so far is an assessment of general over-

ground locomotion with a rough five-step scoring system

(Meyer et al., 2001).

In contrast, the field of traumatic spinal cord injury has

put much effort on the elaboration of behavioral tests

allowing sensitive and specific assessment of locomotor

function (Kunkel-Bagden et al., 1993; Metz et al., 2000;

Muir and Webb, 2000). Hindlimb movements are dependent

on the integrity of central pattern-generating networks

situated in the lumbar spinal cord and reflex pathways under

the control of descending motor tracts from the brainstem

and cortex (Pearson, 1976; Raineteau and Schwab, 2001). A

well-chosen combination of tests can therefore reveal the

extent of damage to these different tract systems after spinal

cord injury.

For example, the BBB open field locomotion score

(Basso et al., 1995) measures general over-ground locomo-

tion, which is influenced by various descending tract sys-

tems as well as intraspinal networks. The footprint analysis

visualizes the pattern of hindlimb locomotion with special

regard to the foot rotation angle, toe spreading, stride length,

B.S. Buddeberg et al. / Journal of Neuroimmunology 153 (2004) 158–170 159

and base of support (De Medinaceli et al., 1982; Kunkel-

Bagden et al., 1993; Metz et al., 2000). Integrity of supra-

spinal input to spinal cord circuits can be measured by the

grid walk test (Kunkel-Bagden et al., 1993; Merkler et al.,

2001; Metz et al., 2000). This paradigm requires fine

sensorimotor control and precise foot placing, and therefore

relies partly on an intact corticospinal tract (CST). The same

has been described for the narrow beam test in which

animals are required to cross elevated beams of varying

width and shape (Hicks and D’Amato, 1975; Metz et al.,

2000). However, these tests are not well applicable in

conventional EAE models due to the disseminated nature

of the inflammatory lesions and consequently the high

variability of the functional impairment.

Recently, we succeeded in establishing a new, targeted

EAE model (Kerschensteiner et al., 2004). Rather than

mimicking the disseminated disease course of MS, this

model localizes the disease to one single inflammatory

lesion at a defined location of the spinal cord. In the present

study, we targeted the inflammatory lesion to the dorsal

thoracic spinal cord at a level of T8 resulting in an affection

of the CST and other dorsal and lateral tract systems.

Therefore, the animals exhibit predictable and comparable

deficits.

The aim of the present study is to expand the initial

observations, to evaluate systematically, which of the estab-

lished tests used in the traumatic spinal cord injury field

would be transferable to the targeted EAE model, and how

these tests correlate with the standard EAE clinical score. A

quantitative and precise assessment of these functional

deficits is of crucial importance for the evaluation of

therapeutic strategies.

2. Materials and methods

2.1. Animals

A total of 51 adult female Lewis rats (180–220 g)

obtained from Harlan (Horst, The Netherlands) were used.

45 animals underwent the sensitization procedure and spinal

cord injection and 6 animals were used as controls and did

not receive any sensitization or injection. The rats were

housed in groups of five or six under a 12:12-h light/dark

cycle with food and water ad libitum. All experiments were

approved by the Veterinary Department of the Canton of

Zurich.

2.2. Induction of the localized EAE lesion

Rats were anaesthetized by inhalation anaesthesia. In-

duction of localized EAE was performed as previously

described (Kerschensteiner et al., 2004). Briefly, recombi-

nant myelin oligodendrocyte glycoprotein (MOG; 10–25

Ag diluted in saline) was emulsified in incomplete Freund’s

adjuvant (IFA; Sigma) and a total volume of 100 Al was

injected subcutaneously into the base of the tail. This

sensitization protocol leads to the induction of an anti-

MOG immune response without the development of clinical

disease (Kerschensteiner et al., 2004). About 18–22 days

after sensitization, the development of a localized EAE

lesion at a predetermined location of the spinal cord was

induced by the injection of cytokines. Sensitized rats were

anaesthetized with a combination of Dormicum [midazo-

lam, 0.6 mg/100-g body weight intraperitoneally (i.p.);

Roche; Basel, Switzerland] and Hypnorm (fentanyl, 0.02

mg/100-g body weight; Janssen-Cilag; Beerse, Belgium).

Two microliters of a cytokine solution containing 250 ng of

recombinant rat TNF-a (R&D Systems; Abingdon, UK) and

150 U of recombinant rat IFN-g (Pepro Tech; London, UK)

dissolved in PBS were injected into the spinal cord at the

thoracic level 8, with a minimal invasive stereotactic tech-

nique as previously described (Kerschensteiner et al., 2004).

2.3. Histology

Histological analysis of the EAE lesions were performed

in some rats after the behavioral analysis was concluded 28

days postinjection. For this purpose, rats were killed by a

pentobarbital overdose (50 mg/100 g) and perfused trans-

cardially with Ringer solution containing 100000 IU/l hep-

arin and 0.25% NaNO2, followed by 4% paraformaldehyd

in 0.1 M phosphate buffer with 5% sucrose. The spinal

cords were dissected and postfixed over night. After im-

mersion for 4–6 days in a 30% sucrose solution, the

segments of the spinal cord containing the EAE lesion were

cut on a vibratom in serial 50-Am cross-sections. The

sections were mounted, air dried, stained with cresyl violet

and were cover-slipped with Eukitt (Kindler; Freiburg,

Germany). Lesions were analysed with a light microscope

(Zeiss, Germany).

In addition, histological evaluation was performed on 1-

Am sections of representative spinal cords as previously

described (Kerschensteiner et al., 2004). Sections were

stained with Luxol fast blue and Bielschowsky silver

impregnation to assess demyelination and axonal pathology,

respectively. The extent of macrophage influx was visual-

ized by immunohistochemistry with antibodies against

macrophages/activated microglia (clone ED1, Serotec; Ox-

ford, UK). Detection of amyloid precursor protein (APP,

clone 22C11; Chemicon, USA) as a marker for acute axonal

damage was performed on adjacent sections.

2.4. Behavioral analysis

The disease course of all animals was evaluated with the

classical EAE-scoring scale (Kerschensteiner et al., 2004;

Meyer et al., 2001). With subgroups of the animals, we

performed a number of tests specifically designed to assess

hindlimb motor function. Performed tests can be divided

into two major groups: basic locomotor tests and tests to

assess specific aspects of locomotion. In addition, we

B.S. Buddeberg et al. / Journal of Neuroimmunology 153 (2004) 158–170160

addressed the open field exploration of the animals with an

activity measuring system in order to determine the impact

of the disease course on the spontaneous locomotor activity.

If not mentioned differently, tests were performed before

induction of the localized lesion (baseline evaluation) and

then at 3, 7, 14, 21 and 28 days postlesion induction (D3,

D7, D14, D21 and D28).

2.4.1. Spontaneous locomotor activity

To measure the spontaneous open field activity and

inquisitiveness, the activity measuring system TSE Acti-

Mot/MoTil was used. The rat is placed in a 48� 48-cm box

with four pairs of light–barrier strips. The light–barrier

strips are arranged at right angles to each other. Two pairs

are installed at the bottom of the box to determine the X and

Y coordinates of the animal during normal locomotion. The

other two pairs of light–barrier strips are arranged at a

higher lever so that the rat only interferes with the light

beams when rearing. A few days before the first testing

session, the animals were placed into the activity box for a

period of 10 min to make them familiar with the testing

situation. At the beginning of every testing session, we gave

the animals 1 min for habituation before the pattern of

movement was recorded during 29 min. We evaluated the

following parameters: total distance which the animal cov-

ered in the time given, number of rearings and number of

rotations.

2.4.2. Basic locomotor tests

Basic locomotor tests were carried out to measure the

recovery of the general hindlimb locomotor performance.

2.4.2.1. EAE clinical score. For the evaluation of the

disease course, all rats were examined daily from the

sensitization on and up to 28 days after the cytokine

injection. An adapted EAE-scoring scale was used (Ker-

schensteiner et al., 2004; Meyer et al., 2001):

1. Score = 0, no clinical disease;

2. Score = 0.5, partial tail weakness or slight loss of

muscle tone;

3. Score = 1.0, tail weakness;

4. Score = 1.5, slightly clumsy gait;

5. Score = 2.0, hindlimb paresis;

6. Score = 2.5, marked hindlimb paresis and partial

dragging of the hindlimbs;

7. Score = 3, hindlimb paralysis;

8. Score = 3.5, hindlimb paralysis and forelimb paresis;

9. Score = 4.0, complete paralysis (tetraplegy);

10. Score = 5.0: moribund or dead.

Due to the localized nature of the lesion, the animals did not

score any higher than 3.

2.4.2.2. BBB open field locomotion score. The BBB open

field locomotion score was described by Basso et al. (1995)

to measure recovery of hindlimb movements after spinal

cord contusion injury in rats during free open field locomo-

tion. For examination, rats are placed one by one in a

70� 70-cm transparent Plexiglas box with walls of 30-cm

and a pasteboard-covered nonslippery floor. Two examiners

observe each rat during a period of 3–5 min. The nonlinear

scoring scale (score 0 to 21) covers a broad range of

parameters assessing a sequence of locomotor recovery,

starting with no spontaneous movement of the hindlimbs

up to plantar stepping, weight support, forelimb–hindlimb

coordination and trunk stability. A score of 21 indicates

unimpaired locomotion as observed in unlesioned rats. We

used a slightly modified version of the BBB score, as

previously described (Metz et al., 2000). When the sequence

of recovering motor features was not the same as described

in the original score, points for the single features were

added independently.

2.4.3. Tests for specific aspects of locomotion

2.4.3.1. Grid walk. Deficits in descending motor control

were examined by assessing the ability to cross a 1-m-long

runway with irregularly spaced bars (1-4 cm) elevated 1 m

above the ground as previously described (Merkler et al.,

2001; Metz et al., 2000; Metz and Whishaw, 2002). A

defined sector of 13–20 bars was chosen for analysis.

In order to cross the runway, accurate placement of the

limbs on the bars is required. Animals were trained for 5

days before baseline evaluation. For baseline and postoper-

ative testing, each animal had to cross the runway at least

twice. Evaluation was done on video recordings of the trials.

A drop of the foot below the plane of the grid was

considered as a footfall. The total number of footfalls as

well as the total number of steps was counted and then the

percentage of footfalls per steps was calculated. Animals

which were not able to support their bodyweight with the

hindlimbs would make errors with every step and were thus

assigned 100% of footfalls.

2.4.3.2. Narrow beam. This paradigm was performed to

assess the ability of the animals to balance across a 1-m-

long wooden beam which was elevated 30 cm above the

ground. Animals were tested on three different beams: a

rectangular 2.3-cm-wide beam, a rectangular 1.2-cm-wide

beam and a round beam with a diameter of 1.5 cm. Thus, the

difficulty to balance across increased from beam to beam.

Animals were trained for 5 days before baseline evaluation.

For baseline and postoperative testing, animals had to cross

each beam three times and were then evaluated by a scoring

system previously described (Metz et al., 2000). For tra-

versing each beam with full weight support and normal

plantar paw placing, a score of 2 was applied. If plantar

placing of the paw was only partly possible, the animal

scored 1.5. A score of 1 was given if the animal could

traverse the entire beam but without plantar placing of the

hind paws. When only half of the beam could be crossed, a


score of 0.5 was assigned, and for complete inability to

cross the beam, a score of 0 was applied. For each beam, the

average score was calculated. Average scores of all three

beams were then added up so that a maximum of 6 points

could be reached.

2.4.3.3. Footprint analysis. Footprint analysis was modi-

fied from Metz et al. (2000) and De Medinaceli et al.

(1982). The animal’s hind paws were inked and footprints

were made while the animal walked along a 1-m-long and 7-

cm-wide runway with a paper-covered floor. The runway

ensured that the animal was walking along a straight line. A

sequence of at least six consecutive steps was chosen for

evaluation. For each time point, three measurements of the

angle of foot rotation, the base of support and the stride

length on the left and the right sides were taken. Then, the

average value for each parameter was calculated.

The base of support was determined as the distance

between the central pads of the hind paws. Stride length

was determined as the distance between the central pads of

two consecutive footprints on the same side. As a measure-

ment of the limb rotation, the angle between the lines

through the third digit and the central pad of each paw

was determined. In baseline testing, footprint analysis was

performed with all animals. After spinal cord injection,

footprint recording could be carried out only once the

animals were able to support the weight of their hind body

properly. Animals with incomplete weight support would

smear the fresh ink prints with their dragging body.

2.5. Statistical analysis

For ordinal-scaled tests (EAE and BBB scores and

narrow beam), data are presented as the median; first and

third quartiles are shown as well. The results of the interval-

scaled tests (activity parameters, grid walk and footprint

parameters) are presented as the meanF standard error

(S.E.M.). The correlations between the EAE score and other

behavioral tests were calculated with the Spearman’s rank

test (Spearman’s, r). Analyses of variances (ANOVAs) with

repeated measurements and following contrast (repeated)

were carried out using SPSS for Windows to calculate the

significance between measurements of individual time

points. P values of V 0.05 were considered significant;

values of V 0.01 were considered highly significant.

3. Results

3.1. Histology

In all the animals examined, a single, well-circumscribed

lesion site was observed in the dorsal half of the spinal cord.

The lesions varied in size. As shown earlier, the lesion

volume correlates significantly with the maximal behavioral

deficits measured with several locomotor tasks (Kerschen-

steiner et al., 2004). In this study, we show representative

lesions of one animal with a severe disease course (maximal

EAE score: 2.5) and one animal with a milder disease course

(maximal EAE score: 1.5) at D28. Animals with a higher

maximal deficit have a larger extent of myelin destruction

(Fig. 1a,b; LFB/PAS staining) and macrophage infiltration

(Fig. 1c,d; ED1 staining). In addition, Bielschowsky staining

(Fig. 1e–h) and APP staining (Fig. 1i,j) demonstrate a greater

loss of axons including CSTaxons in the animal with a severe

clinical deficit. These results are in full agreement with the

more extensive histological description in this lesion model

given in our previous study (Kerschensteiner et al., 2004).

3.2. Behavioral analysis

3.2.1. Spontaneous locomotor activity

It has been shown that classical EAE leads to a decrease

in many aspects of general activity such as food intake and

exploration, a phenomenon commonly referred to as the

EAE behavioral syndrome (Pollak et al., 2000, 2003a,b). To

rule out the possibility that the general locomotor activity of

the rats with targeted EAE decreases at early stages of

disease, and therefore would interfere with the locomotor

performance in the individual tests, we studied the sponta-

neous activity performance of nine animals with localized

EAE, in comparison to the performance of six control

animals which had received no sensitization or surgical

treatment. We recorded three different parameters: the total

distance which the animals covered in the given time, the

number of rearings and the number of rotations as shown in

Fig. 2. The variability in between individual testing sessions

was substantial in both the control and the EAE group. The

performance in the parameters total distance and number of

rearings did not show a significant difference between the

EAE and the control animals after the onset of the disease.

Only the number of rearings was initially reduced but

recovered after D14 in EAE.

3.2.2. Basic locomotor tests

We have previously shown that substantial behavioral

impairments occur in rats which have undergone the sensi-

tization procedure as well as the cytokine injection into the

spinal cord. Sensitization alone followed by an injection of

PBS without cytokines does not lead to the formation of

large inflammatory lesions and is thus not expected to cause

a substantial change in the locomotor performance (Ker-

schensteiner et al., 2004). We therefore focused the current

study only on the behavioral assessment of animals, which

underwent both sensitization and cytokine injection.

By correlating the results from the different tests with the

classic EAE score, we addressed the question whether they

allow the quantification of additional locomotor properties.

3.2.2.1. EAE clinical score. All animals (n = 45) were

scored daily according to an adapted version of the EAE

clinical score as described under Materials and Methods.

Fig. 1. Representative thoracic spinal cord cross sections of an animal with a mild (maximal EAE score 1.5; a, c, e, g and i) and an animal with a more severe

disease course (maximal EAE score 2.5; b, d, f, h and j) at D28. The animal with a higher maximal deficit displays a more extended area of demyelination (b;

LFB/PAS staining) including the region of the dorsal CST and infiltration of ED1 positive macrophages is more widespread (d). Bielschowsky staining (e– f)

shows the marked reduction of axons in the dorsal funiculus of both animals, including the dorsal region of the CST (g–h). In contrast to the animal with a

milder disease course (i), acute axonal damage reflected by APP-positive axonal spheroids is found in the region of CST (j) in the animal with more severe

EAE. Arrowheads in panel j indicate APP-positive axons.


Fig. 2. Time course of three different parameters of locomotor activity for nine EAE (n) and six control (w ) animals. (a) Overall distance covered in the given

time (29 minutes), (b) number of rearings and the (c) number of rotations. Data are shown as the meanF S.E.M. Although the EAE rats score slightly lower,

variability in both groups is high and no significant change in activity, which could be attributed to the disease, as was observed except at D3 for rearings. (11)

indicates that the difference was highly significant ( p< 0.01).


Scoring started at the time point of sensitization and was

performed up to 28 days after cytokine injection. Results are

shown in Fig. 3a. In the period after sensitization but before

cytokine injection, the animals showed no signs of clinical

disease. The first signs of clinical disease usually became

Fig. 3. Tests, which assess general aspects of locomotion: EAE score (a–b) and

animals) starting at the time of sensitization up to 28 days postcytokine injection. T

animal with a high (w ) and an animal with a low (.) EAE score at D3. (c) Time co

third quartile. Animals were tested once before cytokine injection (BL), once at the

injection (D28). (d) Correlation between the EAE and the BBB scores at D3 (r =

significant ( p< 0.05), it is indicated with 1; when it was highly significant ( p<

apparent on the first day after the cytokine injection. Three

days postinjection (D3), the animals normally reached their

maximal clinical score (median value, 2; first quartile, 2;

third quartile, 2.5) which they kept for about 1 week.

Afterwards, a slow recovery resulting in a consistent decline

BBB score (c–d). (a) Time course of the median of the EAE score (n= 45

hin lines indicate the first and the third median. (b) Typical time course of an

urse of the median BBB score of 18 animals. Thin lines indicate the first and

peak of disease (D3) and then on a weekly basis until 28 days after cytokine

� 0.79; p< 0.01). If the difference between two following time points was

0.01), it is indicated with 11.


of the clinical score was observed. At the end of the testing

period (D28), the majority of the animals had recovered

completely to a score of 0. From the 18 animals with an

initial EAE score of 2.5 or 3, 10 showed a remaining deficit

after 28 days, 8 animals had fully recovered. From the 27

animals with an initial score of 2 or less, 24 animals had

fully recovered after 28 days, whereas 3 animals showed a

remaining deficit. This indicates that the severity of the

initial disease course influences the long-term deficits.

Fig. 3b shows the typical disease courses of an animal

with a high and an animal with a low initial score.

3.2.2.2. BBB open field locomotion score. A more detailed

analysis of open field locomotion using the BBB score was

performed with 18 animals (Fig. 3c). At the baseline

evaluation, all animals obtained the maximum score of 21

points. At D3, the animals showed their maximal locomotor

deficit reflected by a median score of 12 (first quartile, 9.25;

third quartile, 13). A score of 12 corresponds to consistent

weight-supporting plantar steps and occasional forelimb–

hindlimb coordination. In the following days, the locomotor

performance substantially improved and reached a plateau at

D21, with a median of 19 (first quartile, 18; third quartile,

20). At this time point, animals showed consistent plantar

stepping and consistent forelimb–hindlimb coordination.

Some animals still revealed a slightly rotated foot position

at liftoff, whereas others had a parallel paw position all of

the time. Animals with a score below 8 (no weight support)

at D3 (n = 2) recovered only incompletely up to a BBB score

of 15 points, whereas in animals with an initial score of 8 or

more (n = 16), an almost full recovery (up to 19–21 points)

could be observed.

Seven animals did not follow the sequence of recovery

described in the BBB score and showed a ‘tail-up’ position

before they reached a score of 19. Therefore, an additional

point was given as described under Materials and Methods.

Recovery of both hindlimbs usually occurred in parallel.

Only in one animal, we were able to observe a difference in

the locomotor performance of the two hindlimbs at early

timepoints (D3 and D7). In this case, we scored the two

hindlimbs separately, and then assigned the animal the mean

of the two scores. Along with the locomotor recovery, the

difference between the limbs vanished, and at later time-

points, both limbs scored equally.

Table 1

Correlations between EAE score and different behavioral tests

D3 D7

BBB � 0.73265** � 0.67167**

Grid walk 0.54613** 0.61859**

Narrow beam � 0.81944** � 0.45384

Footprints: rotation � 0.07297

Footprints: base of support 0.46027

Footprints: stride length 0.13392

Correlations between the EAE score and the different behavioral tasks over the e

*p< 0.05.

**p< 0.01.

The BBB score revealed a significant correlation with the

EAE score over the entire time course (Table 1), indicating

that the BBB score measures similar aspects as the EAE

score. The correlation between the BBB and the EAE scores

at D3 [shown in Fig. 3d (r=� 0.733; p < 0.01)] illustrates that

every score point on the EAE scale covers a broad range of

score points on the BBB scale. The BBB score therefore

allows a more sensitive discrimination than the EAE score.

3.2.3. Tests for specific aspects of locomotion

3.2.3.1. Grid walk. This test requires that the animals are

able to control and place their hindlimbs precisely and

depends on supraspinal input partly mediated by the CST.

We tested a total number of 25 EAE animals. The results

are shown in Fig. 4a. During baseline measurements, the

animals crossed the grid with a mean error percentage of

5F 1%. At D3, the error percentage mounted to the max-

imum observed during the testing period (67F 7%) and

then declined rapidly. The most pronounced improvement

took place between D7 and D14. At D7, the animals made a

mean of 54F 8% mistakes and recovered to a mean of

19F 4% at D14. Further recovery after D14 was consider-

ably slower and less pronounced. At D21, the mean error

percentage reached 16F 5%, and at D28, the mean error

percentage was 16F 4%.

At D3, 10 animals and at D7, 6 animals were unable to

support their body weight with the hindlimbs. Therefore,

these animals were dragging themselves with the use of the

forelimbs only over the grid and fell with the hindlimbs into

every gap. They were therefore assigned an error percentage

of 100%. These animals tended to show a remaining deficit

in their grid walk performance at D28 compared to those 15

animals which showed weight support at early stages of the

disease. At later time points (D14, D21 and D28), all

animals were able to walk over the grid.

The correlation between the grid walk and the EAE score

at D3 is shown in Fig. 4b (r = 0.546; p < 0.01). The corre-

lation is highly significant over the entire time course (Table

1), indicating that the EAE score and the grid walk assess

similar locomotor mechanisms. As for the BBB score, each

point on the EAE scale covers a broad range of grid error

percentage, showing that the grid walk allows for a refine-

ment in behavioral assessment compared to the EAE score.

D14 D21 D28

� 0.34936 � 0.56684** � 0.49544*

0.52025** 0.61931** 0.80815**

� 0.44420 � 0.82297** � 0.76671**

0.39632 0.46841 � 0.39313

0.37963 0.30739 � 0.10808

� 0.2904 � 0.17650 � 0.63560

ntire time course shown as r-values.

Fig. 4. Behavioral paradigms which address specific aspects of locomotion: grid walk and narrow beam. All tests were performed once before the cytokine

injection (BL), once at the peak of the disease (D3) and then on a weekly basis until 28 days after cytokine injection (D28). (a) Time course of the grid walk

paradigm. The mean percentage of footfalls of 25 animals is shown with the S.E.M. Animals revealed maximum deficit at D3. The most pronounced recovery

could be observed within the first 2 weeks. (b) Correlation between the EAE score and the grid error percentage at D3 (r = 0.55; p< 0.01). (c) Time course of

the narrow beam performance of 18 animals presented as the median (thick line). Thin lines indicate the first and the third quartile. As seen for the grid walk,

the peak deficits were observed at D3, and the major recovery took place within the first 2 weeks. (d) Correlation between the EAE score and the narrow beam

score at D3 (r =� 0.82; p< 0.01). If the difference between two following time points was significant ( p< 0.05), it is indicated with 1; when it was highly

significant ( p< 0.01), it is indicated with 11.


3.2.3.2. Narrow beam. With this test, we investigated the

ability of 18 animals to navigate across a 1-m-long beam of

varying width and shape. Like the grid walk, this test relies

on supraspinal input partly mediated by the CST.

The results are shown in Fig. 4c. At baseline testing, all

animals were able to cross all three beams with proper

plantar steps and less than one or two footfalls; thus, all of

them reached the maximum score of 6. At D3, the median

score was 3.34 (first quartile, 2.21; third quartile, 4.79). As

in the grid walk test, the most pronounced improvement

took place within the first 14 days. By D7, the median score

was 4.83 (first quartile, 2.79; third quartile, 5.42). By D14,

the median was 5.59 (first quartile, 4.63; third quartile,

5.96). In the following 2 weeks, the improvement slowed

down considerably. By D21, a median of 5.67 (first quartile,

5.25; third quartile, 5.83), and by D28, a median of 5.83

(first quartile, 5.71; third quartile, 6) was reached. At D28,

8 animals had fully recovered to a score of 6, whereas 10

animals still showed a remaining deficit.

The correlation between the narrow beam and the EAE

score over the entire time course is shown in Table 1. The

correlation between the narrow beam and the EAE score at

D3 is shown in Fig. 4d (r =� 0.819; p< 0.01). As for the BBB

score and the grid walk, the correlation is significant and each

score point in the EAE score covers a broad range of score

points in the narrow beam score showing the refinement in

behavioral analysis achieved with the narrow beam score.

3.2.3.3. Footprint analysis. Footprint analysis reflects

aspects of hindlimb balance (exorotation of feet, broad base

of support) and kinetics (stride length).

We analysed the footprints of 12 EAE animals. Only

animals with a good locomotor performance (complete

weight support) could be included in the analysis. At D3,

many animals did not show enough weight support to

deliver proper footprints. We therefore started footprint

measurements only at D7 and then carried them out once

a week as the other test paradigms.

Baseline evaluation revealed a mean foot exorotation

angle of 19.1F1.4j. After the lesion induction, the rotation

angle increased to a mean of 37.7F 2.4j at D7 and remained

at this increased level for the rest of the testing period (Fig.

5a). The base of support showed a baseline value of

1.7F 0.1 cm. It increased to a mean value of 2.0F 0.2 cm

at D7 and then dropped down to a subbaseline value of

1.4F 0.1 by D14 (Fig. 5b). The baseline value for the stride

length was 12.7F 0.3 cm. No significant alteration could be

observed after the cytokine injection (Fig. 5c).

As already mentioned before, we took three measure-

ments of every parameter at every time point and then

calculated the mean. Individual measurements of the base of

support showed a high variability, sometimes, exceeding

more than 100%.

None of the footprint parameters investigated correlated

with the EAE score (Table 1) indicating that footprint

Fig. 5. (a–c) Result of three different parameters obtained from the analysis

of footprints. Measurements were taken once before cytokine injection (BL)

and on a weekly basis after the cytokine injection starting at D7. Results are

shown as the meanF S.E.M. (a) Angle of exorotation of the feet increased

after the cytokine injection and did not recover. (b) The base of support

increased only initially. (c) Stride length measurements did not change

significantly.


analysis includes aspects of locomotion which are not

covered by the EAE score.

Comparing the footprints of different time points led

to the following additional observation. At baseline

evaluation, the animals delivered proper prints, putting

down their entire foot. However, after the onset of the

disease, the animals started to tiptoe slightly so that their

heel was not visible any more on the prints. This

tiptoeing behavior did not cease over the time course

and was still observed at D28. In animals, which under-

went sensitization but then received an injection of PBS

only without cytokines, tiptoeing could not be observed.

Fig. 6 shows baseline footprints and footprints from the

time points D7 and D28 from the same animal to

illustrate the tiptoeing. Additionally, the footprints of a

control animal (sensitization followed by PBS injection)

from the time point D7 are shown to demonstrate that

this animal did not tiptoe.

4. Discussion

The recently introduced targeted EAE model leads to

anatomically well localized, defined inflammatory lesions

and to reproducible functional deficits (Kerschensteiner et

al., 2004). In the present study, we applied a range of

established behavioral tests for hindlimb locomotion to the

localized EAE model with a lesion targeted to the dorsal

funiculus at a level of T8. Our data show that the targeted

EAE lesion did not substantially influence the spontaneous

exploratory and locomotor activity. On the other hand, the

basic hindlimb locomotor function, as well as more specific

aspects such as precision movements, balance and foot

exorotation, was affected to various degrees. The use of

specific behavioral tests enables us to quantify the affected

aspects of hindlimb locomotion and follow their recovery

over time in a much more precise way than by the

conventional EAE clinical score.

4.1. Spontaneous locomotor activity

Monitoring spontaneous locomotor activity is a strategy

widely used to quantify changes in overall activity. Activity

can be measured in various ways, e.g., the exploration of the

animals can be observed by eye (Metz et al., 2000), moni-

tored by video recordings (Castanon et al., 2001) or mea-

sured in activity-recording boxes (Ferguson and Cada, 2003;

Mikolajczak et al., 2002). Using the TSE ActiMot/MoTil

system, we found that the performance of the EAE animals

was not significantly different from that of the untreated

control group, with the exception of the number of rearings at

D3. At this time point, a significant decrease of rearings

could be observed, which completely recovered thereafter.

This deficit occurs most likely because many EAE animals

lose weight support at early stages of the disease and

therefore are not able to stand on their hind limbs.

In rodents with classical disseminated EAE, a significant

alteration in spontaneous behavior has been observed (Pol-

lak et al., 2000). This so called EAE-associated behavioral

syndrome results in reduced social exploration and de-

creased food intake. Symptoms appear before the onset of

the disease and vanish before the neurological symptoms

cease. Such sickness behavior has been reported in a wide

range of inflammatory and infectious diseases (Larson,

2002). Activation of the immune system leads to high levels

of proinflammatory cytokines which result in changes of

behavior and motivation. A decrease in the general activity

as seen during the sickness behavior would of course

interfere with the locomotor performance in specific tests

so that the neurological deficits would be overlaid by

systemic symptoms. Therefore, the locomotor abilities

would be underestimated.

For induction of targeted EAE, a subclinical stimulation

of the immune system is required. Our results indicate that

this immune stimulation is insufficient to cause the sickness

behavior typical for systemic EAE. In line with the activity

Fig. 6. Footprints of one EAE animal at three different time points (BL, D7, D28) and of one control animal at time point D7. During baseline measurements,

the EAE animal put down the entire foot. After the cytokine injection, the heel was not visible any more on the prints due to slight tip toeing. In the control

animal, which received an injection of PBS only after the sensitization procedure, such tiptoeing was not observed during the entire time course.


data is the following observations: we controlled the body

weight of the animals every second day and we could

observe only a slight decrease in weight around the onset

of the disease, which was substantially less than the pro-

nounced weight loss seen in classical EAE models (data not

shown). These results indicate that in the targeted EAE

model, alterations of the locomotor activity due to EAE-

associated behavioral alterations are much less likely to

interfere with behavioral testing of motor function than in

conventional EAE models.

4.2. Basic locomotor tests

4.2.1. EAE clinical score

The 5-point EAE clinical score is a qualitative scoring

system widely used in the field of EAE to assess basic

locomotion (Kerschensteiner et al., 2004; Meyer et al.,

2001). Sensitization with MOG alone did not lead to any

clinical signs of disease. Three days after the injection of the

cytokines into the spinal cord, the animals developed

neurological impairments reflected by EAE scores of 1.5–

3. The scores returned to low baseline levels 3–4 weeks

later. A major advantage of the EAE scoring system is that it

can be performed relatively quickly and thus can be carried

out daily in order to provide a good overview over the entire

disease process. In addition, this scoring system is well

suited for the variable clinical symptoms typical for con-

ventional, disseminated EAE models. However, it provides

only a very imprecise read-out for the specific locomotor

abilities of the animals. We therefore consider it crucial to

refine these results by more specific and precise behavioral

tests.

4.2.2. BBB open field locomotion score

The BBB open field locomotion score measures hind-

limb use during over-ground locomotion. The animals

showed a pronounced deficit in the BBB score right after

the onset of the disease, which recovered in parallel with

the EAE score almost completely within the testing

period. Accordingly, EAE and BBB scores showed a

strong correlation over the entire time course of the

disease.


The BBB open field locomotion score has been devel-

oped to assess over-ground locomotion in rats with a

spinal cord contusion lesion (Basso et al., 1995). Over-

ground locomotion is influenced by various tract systems

of the dorsal, lateral and ventral columns of the spinal

cord (Loy et al., 2002; Muir and Webb, 2000; Schucht et

al., 2002). For normal coordinated stepping, it is essential

that local spinal pattern generating networks are activated

properly by descending tracts. The ventrally running

reticulospinal tract probably plays the most important role

in activating these networks (Grillner, 1996; Jordan,

1998). In agreement with our results, animals with a

surgical lesion restricted to the dorsal columns showed

an almost complete recovery of basic locomotor function

(Loy et al., 2002; Metz et al., 2000; Schucht et al., 2002).

In an earlier EAE study, we have shown that only animals

with a lesion extension well into the lateral and the ventral

part of the spinal cord showed a remaining BBB deficit

after 28 days (Kerschensteiner et al., 2004). Animals with

an EAE lesion restricted to the dorsal column recovered

fully.

One disadvantage of the BBB scale is that it is not linear.

In its lower rating range, it measures rather gross aspects of

locomotion, whereas in the upper range of the scale, the

focus is set on discrete aspects of fine movements. We found

that in the lower rating range, the locomotor features of our

EAE animals recovered according to the order on the

scale. In the upper rating range, however, some features

improved at earlier stages than described by the scale. This

observation has already been made previously by studies

applying the BBB score to a hemisection rather than a

contusion model of spinal cord injury in rats (Metz et al.,

2000). As suggested by this group, this problem can be

solved by adding points independently for single features

like tail position or toe clearance when they occur at an

earlier stage.

With this adaptation, we consider the BBB score a very

valuable test for EAE animals. The BBB scoring system

requires more time than the EAE scoring system but

delivers a more exact picture of an animal’s locomotor

status and recovery.

4.3. Tests for specific aspects of locomotion

4.3.1. Grid walk

The animals with a local EAE lesion targeted to the

dorsal column revealed a substantial deficit in the grid walk

performance at early time points (D3 and D7). The grid

walk paradigm assesses deficits in the descending fine

motor control. It requires voluntary fine movement control

which is predominantly mediated by the corticospinal tract,

as well as forelimb–hindlimb coordination mediated by

ventrolateral tracts, and a proper initiation of the stepping

rhythm controlled mainly by the reticulospinal system

(Kunkel-Bagden et al., 1993; Metz et al., 2000). Thus, the

test is more specific for measuring lesions in the dorsal

columns than a general test such as the EAE or BBB score.

In line with these findings, we have previously found a good

correlation between the persistent deficits in the grid walk

task and the damage to the corticospinal tract system in

animals with targeted EAE lesions (Kerschensteiner et al.,

2004). An important advantage of this test system is its

parametric nature and objectiveness which allows interla-

boratory comparisons.

In summary, the grid walk test provides a valuable option

for the evaluation of precise hindlimb placing and thus the

descending motor control in targeted EAE animals.

4.3.2. Narrow beam

The results obtained from the narrow beam test resemble

the grid walk results: animals showed a high deficit at D3,

and the pronounced recovery took place within the sequent

2 weeks. In contrast to the grid walk, also animals with a

high initial deficit can be tested on the beams.

Beam walk abilities rely on descending motor control of

the vestibulospinal tract and of the CST (Hicks and

D’Amato, 1975; Metz et al., 2000). Additionally, it has

been seen in cats that their performance on the narrow

beam was impaired after the transection of the supraspinal

control to the tail only (Walker et al., 1998), indicating that

the integrity of the tail function is crucial to fulfil the task.

At early stages of EAE and in some animals with more

severe disease also at later stages, the tail position and

function is hampered. Thus, we considered this test well

suited.

A special benefit of the test is that the use of beams with

different width and shape can alter the difficulty of the task.

With a wide rectangular beam, gross deficits in locomotor

control can be detected, whereas a round narrow beam

reveals deficits in balance and fine motor control which

would be missed if one looked only at general over-ground

locomotion. Due to the variable levels of difficulty, the time

window in which animals can be tested on the narrow beam

is much wider than in the grid walk paradigm, making it

particularly suitable for testing animals with severe motor

deficits.

Overall the narrow beam provides another valuable

option for the evaluation of balance, tail function and

descending motor control in animals with targeted EAE.

4.3.3. Footprint analysis

By analyzing the footprints of an animal, one can obtain

information about the angle of foot rotation, base of support,

stride length, balance and limb movement kinetics (Kunkel-

Bagden et al., 1993; Metz et al., 2000).

The EAE lesions targeted to the dorsal columns caused

an increased exorotation of the hindfeet, which did not

recover. This is interesting to note as the vast majority of

other behavioral parameters undergo an extensive recovery

process during the study period. Measurements of the

exorotation of the hindfeet are therefore of high value for

the assessment of long-term deficits caused by EAE. Exor-


otation of the hindfeet indicates that the animal tries to

compensate balance problems, which reflect CST or

descending pathway lesions.

Mean values of the base of support also increased initially.

By D14, the mean values of the base of support were,

however, below the baseline, and individual measurements

showed a high variability even in the baseline measurements.

This uneven walking pattern is likely to again reflect balance

problems caused by the EAE lesion.

The tiptoeing, which we were able to observe after the

disease onset, has been reported before in animals with a

demyelinated ventrolateral funiculus leading to a conduc-

tion block in the affected tract system (Loy et al., 2002). At

least in some cases, our lesion did expand along the

meninges into the ventrolateral region of the spinal cord.

It could be that similar processes occurred in our animals as

seen by Loy et al. (2002). Another cause for the tiptoeing

could come directly from the CST or descending pathway

lesions.

In summary, it can be concluded that the footprint

measurements are the only parameters which show no

significant recovery. In addition, footprint analysis is the

only test which does not correlate with the EAE score,

indicating that it addresses different aspects of locomotion

then the other tests.

4.4. Comparison with the traumatic spinal cord injury

model

The major difference between animals with a traumatic

spinal cord lesion and animals with a targeted EAE lesion

is the extent of functional recovery which is substantially

more pronounced in animals with a targeted EAE lesion.

In the traumatic spinal cord injury model, many axons of

the large tracts are transected mechanically, whereas in the

targeted EAE model, many axons lose their function only

temporarily due to the inflammatory milieu; cytokines,

edema and demyelination can cause a temporary conduc-

tion block. Redistribution of sodium channels as well as

remyelination assist these axons in regaining conduction

abilities. Thus, especially when the inflammation is mild,

the initial neurological deficits exceed the persistent def-

icit and the structural damage by far. This is underlined

by the remarkable improvement in the locomotor behavior

which took place within 2–3 weeks after lesion induction

in animals with targeted EAE. This pronounced recovery

indicates that the impairment at early stages can be

mainly attributed to the axonal dysfunction caused by

inflammation. The loss of smaller numbers of axons in a

given tract can also be compensated well by the remain-

ing fibers, at least for relatively global functions. Only

those EAE animals with a substantial amount of structural

damage are bound to develop persistent deficits. In

contrast, animals with a partly transected or contused

spinal cord recover more slowly and to a lesser degree

(Metz et al., 2000).

Regardless of this difference in pathophysiology, our

results show that many of the tests used in the field of spinal

cord injury can be transferred to the targeted EAE model.

4.5. Testing strategy

One of our aims was to establish a testing strategy for the

targeted EAE model which would be refined and sensitive

enough to detect even small improvements occurring spon-

taneously or as a result of therapeutic interventions. We

consider it crucial to combine several tests within one

experiment in order to obtain a broad assessment of the

sensorimotor recovery.

As a crude, fast and basic examination of locomotor

behavior, the broadly used EAE score is a useful tool.

Although imprecise, it is easy and fast to perform. The value

of the EAE scoring system is underlined by our findings that

many of themore specific tests correlated with the EAE score.

The EAE score can therefore be used as a common initial

screen. The results of which should then be refined by the use

of more precise and sensitive behavioral tests.

On a first level of priority, we suggest performing a BBB

score. Although the BBB score measures similar aspects as

the EAE score, it already provides a much more detailed

graduation of the over-ground locomotion deficits. Due to

the broad range of the scale, animals can be judged

adequately from very mild to very severe deficits.

On a second level, one or two further tests addressingmore

specific aspects of locomotor function should be chosen. The

grid walk task is best suited for animals with mild to moderate

deficits in hindlimb motor function (BBB>10), as animals

have to be able to perform weight-supported steps in order to

be analysed on the grid walk. The grid walk provides an

excellent, objective and reproducible way for quantifying

deficits in precise hindlimb placing. The narrow beam task

requires a subjective judgment and provides a less-precise

grading of the outcome parameter. However, it can be applied

in animals of all severity grades and is thus the test of choice

for animals with severe motor deficits. Footprint analysis

provide a parameter, the exorotation of the feet, which does

not recover over time and may thus be an interesting indicator

of permanent deficits.

In conclusion, many of the tests used to assess hindlimb

sensorimotor behavior in traumatic spinal cord injury mod-

els can be transferred reliably to a localized EAE model.

They allow monitoring in detail the functional deficits and

their recovery during the disease course of EAE. They

provide, therefore, a valuable tool for MS related therapeutic

interventions.

Acknowledgements

We wish to thank Eva Hochreutener and Roland Schob

for help with illustrations. We also thank Richard Klaghofer

for help with the statistics.


References

Basso, D.M., Beattie, M.S., Bresnahan, J.C., 1995. A sensitive and reliable

locomotor rating scale for open field testing in rats. J. Neurotrauma 12,

1–21.

Castanon, N., Bluthe, R.M., Dantzer, R., 2001. Chronic treatment with the

atypical antidepressant tianeptin attenuates sickness behavior induced

by peripheral but not central lipopolysaccharide and interleukin-1beta in

the rat. Psychopharmacology 154, 50–60.

de Medinaceli, L., Freed, W.J., Wyatt, R.J., 1982. An index of the func-

tional condition of rat sciatic nerve based on measurements made from

walking tracks. Exp. Neurol. 77, 634–643.

Ferguson, S.A., Cada, A.M., 2003. A longitudinal study of short- and long-

term activity levels in male and female spontaneously hypertensive,

Wistar–Kyoto, Sprague–Dawley rats. Behav. Neurosci. 117, 271–282.

Grillner, S., 1996. Neural networks for vertebrate locomotion. Sci. Am.

274, 64–69.

Hicks, S.P., D’Amato, C.J., 1975. Motor – sensory cortex–corticospinal

system and developing locomotion and placing in rats. Am. J. Anat.

143, 1–42.

Jordan, L.M., 1998. Initiation of locomotion in mammals. Ann. N.Y. Acad.

Sci. 860, 83–93.

Kerschensteiner, M., Stadelmann, C., Buddeberg, B.S., Merkler, D.,

Bareyre, F.M., Anthony, D.C., Linington, C., Bruck, W., Schwab,

M.E., 2004. Targeting EAE lesions to a predetermined axonal tract

system allows for refined behavioral testing in an animal model of

multiple sclerosis. Am. J. Pathol. 164, 1455–1469.

Kunkel-Bagden, E., Hai-Ning, D., Bregman, B.S., 1993. Methods to assess

the development and recovery of locomotor function after spinal cord

injury in rats. Exp. Neurol. 119, 153–164.

Larson, S.J., 2002. Behavioral and motivational effects of immune-system

activation. J. Gen. Psych. 129, 401–414.

Loy, D.N., Talbott, J.F., Onifer, S.M., Mills, M.D., Burke, D.A., Dennison,

J.B., Fajardo, L.C., Magnuson, D.S.K., Whittemore, S.R., 2002. Both

dorsal and ventral spinal cord pathways contribute to overground loco-

motion in the adult rat. Exp. Neurol. 177, 575–580.

Merkler, D., Metz, G.A.S., Raineteau, O., Dietz, V., Schwab, M.E., Fouad,

K., 2001. Locomotor recovery in spinal cord-injured rats treated with an

antibody neutralizing the myelin-associated neurite growth inhibitor

nogo-A. J. Neurosci. 21, 3665–3673.

Metz, G.A.S., Whishaw, I.Q., 2002. Cortical and subcortical lesions impair

skilled walking in the ladder rung walking test: a new task to evaluate

fore- and hindlimb stepping, placing, and co-ordination. J. Neurosci.

Methods 115, 169–179.

Metz, G.A.S., Merkler, D., Dietz, V., Schwab, M.E., Fouad, K., 2000.

Efficient testing of motor function in spinal cord injured rats. Brain

Res. 883, 165–177.

Meyer, R., Weissert, R., Diem, R., Storch, M.K., de Graaf, K.L., Kramer,

B., Bahr, M., 2001. Acute neuronal apoptosis in a rat model of multiple

sclerosis. J. Neurosci. 21, 6214–6220.

Mikolajczak, P., Okulicz-Kozaryn, I., Kaminska, E., Niedopad, L.,

Polanska, A., Gebka, J., 2002. Effects of acamprosate and some

polyamine site ligands of NMDA receptor on short-term memory

in rats. Eur. J. Pharmacol. 444, 83–86.

Muir, G.D., Webb, A.A., 2000. Assessment of behavioral recovery follow-

ing spinal cord injury in rats. Eur. J. Neurosci. 12, 3079–3086.

Noseworthy, J.H., Luchhinetti, C., Rodriguez, M., Weinshenker, B.G.,

2000. Multiple Sclerosis. N. Engl. J. Med. 343, 938–952.

Pearson, K., 1976. The control of walking. Sci Am 235, 72–74, 79–82,

83–86.

Pollak, Y., Ovadia, H., Goshen, I., Gurevich, R., Monsa, K., Avitsur, R.,

Yirmiya, R., 2000. Behavioral aspects of experimental autoimmune

encephalomyelitis. J. Neuroimmunol. 104, 31–36.

Pollak, Y., Ovadia, H., Orion, E., Weidenfeld, J., Yirmiya, R., 2003a. The

EAE-associated behavioral syndrome: I. Temporal correlation with in-

flammatory mediators. J. Neuroimmunol. 137, 94–99.

Pollak, Y., Ovadia, H., Orion, E., Yirmiya, R., 2003b. The EAE-associated

behavioral syndrome: II. Modulation by anti-inflammatory treatments.

J. Neuroimmunol. 137, 100–108.

Raineteau, O., Schwab, M.E., 2001. Plasticity of motor systems after in-

complete spinal cord injury. Nat. Rev. Neurosci. 2, 263–273.

Schucht, P., Raineteau, O., Schwab, M.E., Fouad, K., 2002. Anatomical

correlates of locomotor recovery following dorsal and ventral lesions of

the rat spinal cord. Exp. Neurol. 176, 143–153.

Walker, C., Vierck Jr., C.J., Ritz, L.A., 1998. Balance in the cat: role of

the tail and effects of sacrocaudal transection. Behav. Brain Res. 91,

41–47.

behavioral testing strategies in a localized animal model of multiple sclerosis

Documents