Garth Bigelow - CDS - Combined therapy with methylprednisolone anderythropoietin in a model of multiple sclerosis

Hope for Future C.D.S. Sufferers

Page 1

doi:10.1093/brain/awh365

Brain Page 1 of 11

Combined therapy with methylprednisolone and

erythropoietin in a model of multiple sclerosis

Ricarda Diem,

Muriel B. Sättler,

Doron Merkler,

Iris Demmer,

Katharina Maier,

Christine Stadelmann,

Hannelore Ehrenreich

and Mathias Bähr

Correspondence to: Ricarda Diem, Neurologische

Universitätsklinik, Robert-Koch-Strasse 40,

D-37075 Göttingen, Germany

E-mail: rdiem@gwdg.de

Neurologische Universitätsklinik,

Institut für

Neuropathologie and

Max-Planck-Institut für

Experimentelle Medizin, Göttingen, Germany

Summary

Neurodegenerative processes determine the clinical dis-

ease course of multiple sclerosis, an inflammatory autoim-

mune CNS disease that frequently manifests with acute

optic neuritis. None of the established multiple sclerosis

therapies has been shown to clearly reduce neurodegenera-

tion. In a rat model of experimental autoimmune encepha-

lomyelitis, we recently demonstrated increased neuronal

apoptosis under methylprednisolone therapy, although

CNS inflammation was effectively controlled. In the pre-

sent study, we combined steroid treatment with applica-

tion of erythropoietin to target inflammatory as well as

neurodegenerativeaspects.Afterimmunizationwithmyelin

oligodendrocyte glycoprotein (MOG), animals were ran-

domly assigned to six treatment groups receiving different

combinations of erythropoietin and methylprednisolone,

or respective monotherapies. After MOG-induced experi-

mental autoimmune encephalomyelitis became clinically

manifest, optic neuritis was monitored by recording

visual evoked potentials. The function of retinal ganglion

cells, the neurons that form the axons of the optic nerve,

was measured by electroretinograms. Functional and

histopathological data of retinal ganglion cells and optic

nerves revealed that neuron and axon protection was most

effective when erythropoietin treatment that was started at

immunization was combined with high-dose methyl-

prednisolone therapy given from days 1 to 3 of MOG-

induced experimental autoimmune encephalomyelitis. In

contrast, isolated neuronal or axonal protection without

clinical benefit was achieved under monotherapy with

erythropoietin or methylprednisolone, respectively.

Keywords: methylprednisolone; erythropoietin; experimental autoimmune encephalomyelitis; neuroprotection;

visual evoked potentials

Abbreviations: AMPA = a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; BDNF = brain-derived neurotrophic

factor; CFA = complete Freund’s adjuvant; EAE = experimental autoimmune encephalomyelitis; Epo = erythropoietin;

ERG = electroretinogram; FG = fluorogold; IGF = insulin-like growth factor; MAPK = mitogen-activated protein

kinase; MBP = myelin basic protein; MOG = myelin oligodendrocyte glycoprotein; MPred = methylprednisolone;

RGC = retinal ganglion cell; VEP = visual evoked potential

Received July 21, 2004. Revised October 26, 2004. Accepted October 28, 2004

Introduction

Multiple sclerosis is an autoimmune CNS disease that has

long been thought to be primarily characterized by inflam-

mation and demyelination. In the last few years, histopatho-

logical and MRI studies (Losseff et al., 1996; Trapp et al.,

1998; Peterson et al., 2001) as well as data from animal

models (Smith et al., 2000; Meyer et al., 2001) have

reintroduced the presence of axonal and neuronal degenera-

tion in multiple sclerosis. This neurodegenerative aspect

of multiple sclerosis has the strongest impact on the

development of permanent neurological deficits in patients

(Trapp et al., 1999).

Experimental autoimmune encephalomyelitis (EAE) is the

principal model of multiple sclerosis (Wekerle et al., 1994).

By the use of different agents and modes of immunization and

of different animal strains, EAE models mimicking the whole

histopathological spectrum of the human disease could be

established (Storch et al., 1998; Kornek et al., 2000). Pre-

viously, we demonstrated that EAE induced by immunization

Brain

Brain Advance Access published December 15, 2004

Page 2

of female Brown Norway rats with recombinant myelin oli-

godendrocyte glycoprotein (MOG) strongly reflects the neuro-

degenerativeaspectsofmultiplesclerosis.Inthismodel,severe

optic neuritis that occurs in 80–90% of the animals leads to

acute axonal degeneration of the optic nerve and consecu-

tive apoptosis of retinal ganglion cells (RGCs), the neurons

that form its axons (Meyer et al., 2001; Hobom et al., 2004).

Clinically established strategies for the treatment of multi-

ple sclerosis mainly target the autoimmune response by

using anti-inflammatory, immunomodulatory and immuno-

suppressive agents. Although these substances have been

proved to be beneficial in terms of modifying the clinical

disease course (Noseworthy et al., 1999), for none of them

have clear neuroprotective properties been demonstrated.

In many studies, the concept of achieving neuroprotection

as a secondary phenomenon resulting from the treatment

of inflammation and autoimmunity was not confirmed

(Hickman et al., 2003; Parry et al., 2003). Whereas common

pharmacological multiple sclerosis therapies are well charac-

terized with respect to theautoimmune reaction,little isknown

about direct effects on neuronal survival. For methyl-

prednisolone (MPred), the standard therapy of acute multiple

sclerosisrelapses(BrusaferriandCandelise,2000),weshowed

negative effects on RGC survival during MOG-induced optic

neuritis, although inflammatory infiltration of the optic nerve

was reduced (Diem et al., 2003). This pro-apoptotic action of

the steroid was caused by inhibition of an endogenous

neuroprotective pathway involving the phosphorylation of

mitogen-activated protein kinases (MAPKs).

Erythropoietin (Epo), the main regulator of erythropoiesis

in mammals (Jelkmann, 1992), has shown neuroprotective

properties in models of brain injury, such as experimental

ischaemia, trauma and epilepsy (Bernaudin et al., 1999;

Brines et al., 2000). Beneficial effects on the clinical disease

course were demonstrated in EAE induced by myelin basic

protein (MBP) in rats (Brines et al., 2000). Compared with the

effects of MPred during autoimmune optic neuritis (Diem

et al., 2003), we recently observed that Epo acts in an ant-

agonistic way by up-regulating active MAPKs in RGCs

without influencing inflammatory infiltrates, demyelination

or axonal damage of the optic nerve (Sättler et al., 2004).

In the present study we tested the hypothesis raised from

our previous data that a combination of MPred and Epo might

act synergistically during MOG-induced optic neuritis and

thereby protect RGC bodies as well as their axons. By com-

paring different treatment protocols, we show that early appli-

cation of a neuroprotective agent such as Epo together with

steroid therapy during the acute stage of the disease is an

effective strategy to prevent axonal and neuronal degenera-

tion in inflammatory autoimmune CNS diseases.

Material and methods

Rats

Female Brown Norway rats 8–10 weeks of age were used in all

experiments. They were obtained from Charles River (Sulzfeld,

Germany) and kept under environmentally controlled conditions

without the presence of pathogens.

All experiments that involved animal use were performed in

compliance with the relevant laws and institutional guidelines.

These experiments have been approved by the local authorities of

Braunschweig, Germany.

Immunogen

Recombinant rat MOG, corresponding to the N-terminal sequence

of rat MOG (amino acids 1–125), was expressed in Escherichia coli

and purified to homogeneity by chelate chromatography (Weissert

et al., 1998). The purified protein in 6 M urea was then dialysed

against 0.01 M sodium acetate, pH 3, to obtain a preparation that was

stored at À20

Induction and evaluation of EAE

The rats were anaesthetized by inhalation of diethylether and inject-

ed intradermally at the base of the tail with a total volume of 100 ml

inoculum, containing 50 mg MOG in saline emulsified (1 : 1) with

complete Freund’s adjuvant (CFA) (Sigma, St Louis, MO, USA)

containing 200 mg heat-inactivated Mycobacterium tuberculosis

(strain H 37 RA; Difco Laboratories, Detroit, MI, USA). Rats

were scored for clinical signs of EAE and weighed daily. The

signs were scored as follows: grade 0.5, distal paresis of the tail;

grade 1, complete tail paralysis; grade 1.5, paresis of the tail and

mild hind leg paresis; grade 2.0, unilateral severe hind leg paresis;

grade 2.5, bilateral severe hind limb paresis; grade 3.0, complete

bilateral hind limb paralysis; grade 3.5, complete bilateral hind limb

paralysis and paresis of one front limb; grade 4, complete paralysis

(tetraplegia), moribund state, or death. Rats were followed until day 8

of the disease (end of study). Statistical significance was assessed

using Bonferroni corrected one-way analysis of variance (ANOVA)

followed by Duncan’s test.

Retrograde labelling of RGCs

Two weeks before immunization, adult Brown Norway rats were

anaesthetized with intraperitoneal chloral hydrate (0.42 mg/kg body

weight), the skin was incised mediosagittally, and holes were drilled

into the skull above each superior colliculus (6.8 mm dorsal and 2 mm

lateral from bregma). We injected stereotactically 2 ml of the fluor-

escent dye fluorogold (FG; 5% in normal saline) (Fluorochrome,

Englewood, CO, USA) into both superior colliculi.

Electrophysiological recordings

The rats were anaesthetized by intraperitoneal injection of 10%

ketamine (0.65 ml/kg; Atarost, Twistringen, Germany) together with

2% xylazine (0.35 ml/kg; Albrecht, Aulendorf, Germany) and

mounted on a stereotaxic device. During the experiment, body

temperature was maintained between 35

and 37

C with a heating

pad, and the electrocardiogram was monitored continuously on an

oscilloscope. For recording of visual evoked potentials (VEPs) from

the primary visual cortex, two gold screw electrodes with a tip dia-

meter of 1 mm were placed 3–4 mm lateral to the interhemispheric

fissure and 1 mm frontal to the lambda fissure. Reference electrodes

were placed 1 mm lateral to the midline and 1 mm before bregma.

The electroretinogram (ERG) was recorded with chlorinated silver

ball electrodes as described (Meyer et al., 2001). Visual stimuli were

presented on a 17-inch monitor (Acer View 76i) positioned 20 cm in

Page 2 of 11

R. Diem et al.

Page 3

front of the eye. The display was centred on a position $40

medially

from the pupil axis. Light flashes of 20 ms duration were used at a

rate of 1 Hz, and bar stimulation consisted of vertical gratings of

variable spatial frequency, alternating in phase with a temporal

frequency of 1.8 Hz at 66% Michelson contrast (constant mean

luminance, 15 cd/m

). Signals were amplified 10 000-fold and

bandpass-filtered between 0.1 and 100 Hz, and 128 events were

averaged to improve the signal-to-noise ratio. Amplitudes of pattern

ERG and pattern VEP were determined as described earlier (Meyer

et al., 2001). Assessment of visual acuity was also described earlier

(Meyer et al., 2001). VEP and ERG measurements were performed

at clinical onset of the disease. To monitor the disease course

and to investigate therapeutic effects of the different treatment

protocols, a second measurement of VEPs and ERGs was done on

day 8 of the disease.

Treatment of animals

Animals were randomly assigned to the different treatment groups

(n = 6 animals or n = 12 eyes for each group). Four eyes in each

treatment group were used for western blot analysis and did not

undergo electrophysiological assessment. Groups one (‘early Epo’)

and two (‘late Epo’) received daily intraperitoneal injections of

5000 units recombinant human Epo (Janssen Cilag, Neuss,

Germany)/kg bodyweight in 1 ml of 0.9% NaCl from the day of

immunization onwards or started at disease onset, respectively. In

addition to systemic application of Epo (given from immunization or

disease manifestation onwards), group three (‘early Epo+ MPred’)

and four (‘late Epo + MPred’) were treated with intraperitoneal injec-

tions of MPred (20 mg/kg; Urbason

; Hoechst Marion Roussel,

Frankfurt/Main, Germany) on days 1–3 of the disease. Group five

(‘MPred’) received MPred alone from day 1 to day 3 of MOG-EAE.

Group six (‘vehicle’) was treated with daily intraperitoneal injections

of 1 ml 0.9% NaCl from the day of immunization onwards. Pre-

experiments with application of vehicle over different treatment

periods (from immunization or disease manifestation on) showed

no differences concerning clinical outcome, electrophysiological

or histopathological data.

Quantification of RGC density

At the end of the second recording session, the rats received an

overdose of chloral hydrate and were perfused via the aorta with

4% paraformaldehyde in phosphate-buffered saline. The brain, the

optic nerves and both eyes were removed, and the retinas were

dissected and flat-mounted on glass slides. They were examined by

fluorescence microscopy (Axiophot 2; Zeiss, Göttingen, Germany)

using an UV filter (365/397 nm), and RGC densities were determined

by counting labelled cells in three areas (62 500 mm

) per retinal

quadrant at eccentricities of 1/6, 3/6 and 5/6 of the retinal radius. Cell

counts were performed by two independent investigators follow-

ing a blind protocol. Statistical significance was assessed using

Bonferroni-corrected one-way ANOVA followed by Duncan’s test.

Histopathology

Animals were perfused 8 days after disease onset and postfixed

overnight in 4% paraformaldehyde. Optic nerves were removed and

embedded in paraffin. Histological evaluation was performed on

4 mm thick slices stained with haematoxylin and eosin, Luxol-fast

blue, and Bielschowky’s silver impregnation to assess inflammation,

demyelination and axonal pathology. Axonal densities were

determined in vertical sections of the optic nerves stained by

Bielschowsky’s silver impregnation. Overview photographs (2003

magnification) and high-magnification photographs (10003

magnification) were made with a CCD camera (Color View II; Soft

imaging System

). The number of axons in each optic nerve was

counted in at least 14 standardized microscopic fields of 2500 mm

(Brück et al., 1997). Mean axon density was calculated for each optic

nerve. The surface area of the optic nerve was measured using the

analySIS

Docusoftware(SoftimagingSystem).Demyelinatedareas

were determined as a percentage of the whole optic nerve cross-

section. The investigators who performed neuropathological exam-

inations were blinded to the electrophysiological results of the study.

Statistical significance was assessed using Bonferroni-corrected one-

way ANOVA followed by Duncan’s test.

Western blotting

In each treatment group, retinal protein lysates were prepared at day 3

of manifest EAE, 6 h after the last dose of vehicle, MPred and/or

Epo was given. The western blot analysis was performed as described

elsewhere (Diem et al., 2001). After incubation with the primary

antibody against phospho-p44/42 MAPK [Thr180/Tyr182; New

England Biolabs, Schwalbach, Germany; 1:200 in 1% skim milk in

0.1% Tween 20 in phosphate-buffered saline (PBS-T)], membranes

were washed in PBS-T and incubated with horseradish peroxidase

(HRP)-conjugated secondary antibodies against rabbit IgG (Santa

Cruz Biotechnology, Santa Cruz, CA, USA; 1:3000 in PBS-T).

Labelled proteins were detected using the ECL-plus reagent

(Amersham, Arlington Heights, IL, USA).

p44/42 MAPK protein levels were detected using a primary

antibody (sc-93-G; Santa Cruz Biotechnology) diluted 1 : 500 in 1%

skim milk in PBS-T, and an HRP-conjugated secondary antibody

against goat IgG (Santa Cruz Biotechnology; 1 : 3000 in PBS-T).

Results

Clinical disease course

Rats were randomly assigned into six treatment groups con-

taining six animals each. The day of disease manifestation did

not significantly differ between the groups. Vehicle-treated

animals developed symptoms at day 15.9 6 2.0 after

immunization. In the early Epo treatment group (daily

application of Epo from the day of immunization until day 8

of the disease; 5000 units/kg), the first neurological symptoms

occurredatday15.660.8.Diseaseonsetwasatday17.062.6

in the late Epo group (treatment with Epo given from disease

manifestation on until day 8 of EAE; 5000 U/kg). The early

Epo + MPredgroup(20mg/kgMPredgivenfromday1today3

of EAE in addition to the early Epo treatment protocol)

developed first signs of EAE at day 17.1 6 1.9. In the late

Epo + MPred group (20 mg/kg MPred given from day 1 to

day 3 of EAE in addition to the late Epo treatment protocol),

disease onset occurred 17.3 6 0.8 days after immunization.

Animals treated with MPred as a monotherapy from day 1 to

day 3 of MOG-EAE (20 mg/kg) showed the first neuro-

logical symptoms at day 15.6 6 1.5 after immunization.

In addition, the severity of symptoms at disease mani-

festation was similar in the different groups (vehicle group,

mean clinical score 1.1 6 0.4; early Epo, 1.0 6 0.2; late Epo,

Methylprednisolone and erythropoietin in EAE

Page 3 of 11

Page 4

0.8 6 0.1; early Epo + MPred, 0.8 6 0.3; late Epo + MPred,

1.3 6 0.3; MPred, 0.8 6 0.3). Whereas the clinical score

remained stable until day 8 of MOG-EAE in vehicle-treated

animals (1.0 6 0.2), in the group treated with MPred as a

monotherapy (0.9 6 0.3), as well as in late Epo-treated

animals (0.6 6 0.2), significant improvement was observed

in both groups that received combination therapies. Mean

clinical score in the early Epo + MPred group dropped to

0.1 6 0.1 (P < 0.008). Rats treated according to the late

Epo + MPred protocol performed normally at day 8 of

MOG-EAE (mean clinical score, 0). Animals that received

Epo from the day of immunization onwards showed a

tendency to a better clinical outcome, which was, however,

not statistically significant (mean clinical score of 0.3 6 0.3

at day 8 of MOG-EAE).

Functional assessment of optic nerves and

retinal ganglion cells

To diagnose optic neuritis in vivo and to monitor the function

of RGCs, the neurons that form the axons of the optic nerve,

we performed VEP and ERG measurements in response to

flash and pattern stimulation. Flash VEP experiments were

performed to test the axonal signalling of the optic nerve

whereas pattern VEP recordings were used to estimate the

animal’s visual acuity. ERG measurements in response to

flash stimulation indicate the intact function of the whole

retina. In contrast, pattern ERG is a specific electrophysiolo-

gical marker for RGCs. Recently, we have shown that healthy

sham-immunized rats have visual acuity values of 1.31 6 0.16

cycles/

determined by pattern VEP recordings and 1.10 6

0.13 cycles/

in the pattern ERG measurements (Meyer et al.,

2001). The electrophysiological results of the different

treatment groups in our present study are summarized in

Table 1. VEP and ERG recordings in each animal were

performed at day 1 and day 8 of clinically manifest EAE.

Compared with all other treatment protocols, only application

of Epo from the day of immunization onwards combined

with high-dose MPred therapy (early Epo + MPred) led to

partial recovery of VEP responses to pattern stimulation and

thereby to regaining of visual acuity on day 8 of MOG-EAE

(Table 1; Fig. 1A–F). Also the electrophysiological responses

to flash stimulation were improved in this group: at day 8 of the

disease, eight out of eight tested eyes responded to flash light.

Compared with pattern VEP responses, electrophysio-

logical responses to flash stimulation showed a certain

variability during clinical disease course that seemed to

be partly independent of therapeutic intervention. Under

vehicle treatment, three out of eight tested eyes recovered

their response to flash light, whereas spontaneous recovery

in response to pattern stimulation could not be observed

(Table 1).

In the flash ERG measurements, most of the animals (42 out

of 48 tested eyes at day 1 or 45 out of 48 tested eyes at day 8 of

EAE) showed intact function of the whole retina (Table 1).

Recordings of ERGs in response to pattern stimulation

revealed significantly better RGC function at days 1 and 8

of MOG-EAE in both groups that received Epo (alone or in

combination with MPred) from the day of immunization on

(early Epo, early Epo + MPred) (Table 1; Fig. 2E, F) when

compared with all other treatment groups. Both of these early

Epo-treated groups showed a decrease in visual acuity

determined by pattern ERG from day 1 to day 8 of the

disease, independent of cotreatment with MPred (Table 1).

In none of the other treatment groups were ERG potentials in

response to pattern stimulation recorded at disease onset

(Table 1; Fig. 2A–D). Spontaneous recovery of pattern ERG

responses under vehicle treatment was also not observed

(Table 1; Fig. 2A, B).

Histopathological evaluation of optic nerves

To correlate the results from VEP measurements with histo-

pathological data of the optic nerves, we analysed axonal

density, demyelination and inflammation. Representative

optic nerve sections of the different treatment groups stained

Table 1 Results of VEP and ERG recordings (obtained on days 1 and 8 of MOG-EAE) of the different treatment groups

VEP day 1

(flash)

VEP day 8

(flash)

VEP day 1

(pattern)

VEP day 8

(pattern)

ERG day 1

(flash)

ERG day 8

(flash)

ERG day 1

(pattern)

ERG day 8

(pattern)

Vehicle

0/8

3/8

—

8/8

—

Early Epo

3/8

1/8

—

8/8

0.35 6

0.06 cyc/

0.30 6

0.05 cyc/

Late Epo

4/8

—

7/8

8/8

—

Early Epo +

MPred

4/8

8/8

—

0.16 6

0.03 cyc/

8/8

0.37 6

0.04 cyc/

0.29 6

0.06 cyc/

Late Epo +

MPred

0/8

4/8

—

8/8

7/8

—

MPred

0/8

2/8

—

3/8

6/8

—

Control

(CFA-immunized)

8/8

(day 18 p.i.)

1.31 6

0.16 cyc/

(day 18 p.i.)

8/8

(day 18 p.i.)

1.10 6

0.13 cyc/

(day 18 p.i.)

Each group contained eight tested eyes. The number of eyes with detectable potentials to flash stimulation is given as the number out of eight

tested eyes. The responses to pattern VEP and ERG are given as visual acuity values calculated from VEP or ERG amplitudes and the spatial

frequency of the pattern stimulation. Control rats immunized with CFA were measured on day 18 after immunization (p.i.).

Page 4 of 11

R. Diem et al.

Page 5

with Bielschowsky’s silver impregnation, Luxol–fast blue and

haematoxylin–eosin are given in Fig. 3. Mean optic nerve

surface areas of the six treatment groups did not differ

significantly (vehicle, 0.27 6 0.07 mm

; early Epo, 0.25 6

0.02 mm

; late Epo, 0.28 6 0.01 mm

; early Epo + MPred,

0.28 6 0.07 mm

; late Epo + MPred, 0.27 6 0.04 mm

;

MPred, 0.26 6 0.06 mm

). In accordance with the functional

results, the animal group which was treated using the early

Epo + MPred protocol had significantly higher axon counts

(32.73267585axons/mm

;mean6SEM;P<0.008)(Fig.4A)

when compared with vehicle-treated animals (10.539 6

3113 axons/mm

). Also, the extent of myelin damage deter-

mined as the percentage of the demyelinated area with respect

to the whole optic nerve cross-section differed significantly

when compared with vehicle treatment (early Epo + MPred,

6.1 6 2.92% versus vehicle 66.1 6 15.28%; mean 6 SEM;

P < 0.001) (Fig. 4B). Inflammatory infiltrates were markedly

reduced in all three animal groups which received MPred

(as monotherapy or cotreatment), whereas Epo alone had no

influence on the extent of inflammatory infiltration.

An increased number of remaining axons was also

detected in the group which received MPred as a monotherapy

(25.856 6 6516 axons/mm

; P < 0.008 when compared with

vehicle) (Fig. 4A). In this group, a trend towards an increased

amount of remaining myelin was observed (41.4 6 13.02

versus 66.1 6 15.2% in vehicle-treated animals) (Fig. 4B),

which was not statistically significant. In the group that was

treated according to the late Epo + MPred protocol, both

axonal density (13.426 6 5302 versus 10.539 6 3113 axons/

in vehicle-treated animals) (Fig. 4A) and demyelination

(33.9 6 14.33 versus 66.1 6 15.2% for vehicle) (Fig. 4B)

showed a trend towards a better outcome, which was not

statistically significant.

Histopathological results in both treatment groups that

received Epo as monotherapy (early Epo, late Epo) did not

differ from those of the vehicle-treated animals (Fig. 4A, B).

The numbers of remaining axons/mm

in early Epo- and late

Epo-treated animals were in the range of 6348 6 1033 and

7600 6 909, respectively. The extent of demyelination was

also similar to that of the vehicle-treated group. The mean

percentage of the demyelinated area was 67.28 6 8.47% in

rats treated with Epo from the day of immunization onwards,

and 48.5 6 14.6% when Epo therapy was started at disease

onset (vehicle, 66.1 6 15.2%).

Fig. 1 Combined therapy of Epo started on the day of immunization and MPred given from day 1 to day 3 of MOG-EAE leads to

recovery of VEP responses in rats with severe optic neuritis. Representative examples of VEP recordings from different treatment

groups at day 1 (A, C, E) and day 8 (B, D, F) of MOG-EAE are given. In each group, recordings from days 1 and 8 were taken from the

same individual animal and the same eye, respectively. Flash light stimuli are indicated by arrows. (A, B) No VEP responses to

flash light stimulation were obtained from an animal of the vehicle-treated group on days 1 (d1 EAE veh) and 8 (d8 EAE veh)

of MOG-EAE. (C, D) After treatment with MPred, no recovery of VEPs was seen at day 8 of MOG-EAE (d8 EAE after mpred) when

compared with recordings from the same animal on day 1 of the disease (d1 EAE before mpred). (E, F) Under treatment with Epo

from the day of immunization onwards, no potentials in response to flash light stimulation were produced on the day of clinically manifest

EAE (d1 EAE early epo before mpred). After combination with MPred, clear potentials following flash light stimulation and in

response to large-pattern bar stimulation (three alternating bars) were recorded (d8 EAE early epo + mpred). The stimulating pattern is

indicated on top of the respective VEP recording sequence.

Methylprednisolone and erythropoietin in EAE

Page 5 of 11

Page 6

Retinal ganglion cell counts

To compare the functional data from RGCs obtained by

pattern ERG recordings with their survival rates, we counted

FG-positive RGCs at day 8 of MOG-EAE in the different

treatment groups. In control retinas of healthy CFA-

immunized rats, mean RGC density was 2730 6 145 cells/

(mean 6 SEM; n = 9) (Diem et al., 2003). Previously, we

demonstrated that in our MOG-EAE model RGCs degenerate

during severe optic neuritis (Meyer et al., 2001) and that this

apoptotic RGC death was augmented under high-dose MPred

therapy (Diem et al., 2003). Furthermore, we observed that

Epo as a monotherapy started on the day of immunization

protects RGCs from secondary cell death following

autoimmune inflammation of the optic nerve (Sättler et al.,

2004). In the present study comparing different treatment

periods and combination protocols, Epo therapy started on

the day of immunization was superior to all other therapeutic

regimens. At day 8 of MOG-EAE, RGC counts in the early

Epo-treated animal group were in the range of 1499 6

41 cells/mm

(mean 6 SEM; n = 8; P < 0.008 when com-

pared with vehicle treatment) (Fig. 5C, E). RGC counts in

vehicle-treated animals dropped to 775 6 112 until day 8 of

MOG-EAE (n = 8) (Fig. 5A, E). In contrast to the early Epo

treatment protocol, Epo given as a monotherapy started at

disease onset (late Epo) had no significant effect on RGC

survival when compared with vehicle treatment (960 6 60

versus 775 6 112 RGCs/mm

; n = 8 each) (Fig. 5E). In

agreement with our previous results, MPred given from day

1 to day 3 of MOG-EAE decreased the number of surviving

RGCs to 441 6 61/mm

(n =8; P < 0.008 when compared with

vehicle treatment) (Fig. 5B, E). This effect was completely

abolished under combined treatment with Epo if Epo therapy

was started at disease onset (late Epo + MPred, 760 6

85 RGCs/mm

; n = 8; P < 0.008 when compared with

MPred alone) (Fig. 5E). If MPred therapy was combined

with application of Epo following the early Epo treatment

protocol, the survival-promoting effect of the cytokine

predominated. Under this combination therapy, RGC

survival at day 8 of MOG-EAE was still promoted to 1211

6 43 cells/mm

(n = 8; P < 0.008 when compared with

MPred alone or when compared with vehicle-treated

animals) (Fig. 5D, E).

Signal transduction in retinal ganglion cells

Recently, we observed that MPred or Epo can regulate intra-

cellular concentrations of phosphorylated MAPKs in RGCs in

Fig. 2 Improved RGC function under treatment with Epo started on the day of immunization remains stable following MPred pulse

therapy. Representative examples of pattern ERG recordings from different treatment groups on day 1 (A, C, E) and day 8 (B, D, F) of

MOG-EAE are given. In each group, recordings from days 1 and 8 were taken from the same individual animal and the same eye,

respectively. The stimulating pattern is indicated on bottom of the left panel. (A, B) No ERG responses to pattern bar stimulation

(three alternating bars) were recorded in an animal from the vehicle-treated group on days 1 (d1 EAE veh) and 8 (d8 EAE veh) of

MOG-EAE. (C, D) Treatment with MPred did not lead to recovery of ERG potentials in response to pattern bar stimulation (3 alternating

bars) at day 8 of MOG-EAE (d8 EAE after mpred) when compared with recordings from day 1 of the disease (d1 EAE before mpred).

(E, F) Under daily treatment with Epo given from the day of immunization onwards, clear ERG potentials in response to three alternating

bars were obtained on day 1 of MOG-EAE (d1 EAE early epo before mpred). After additional application of MPred from day 1 to day 3 of

the disease, stable ERG responses to pattern bar stimulation were recorded on day 8 of the disease in the same animal (d8 EAE early

epo + mpred).

Page 6 of 11

R. Diem et al.

Page 7

an antagonistic way (Diem et al., 2003; Sättler et al., 2004).

The functional relevance of this pathway for RGC survival in

our MOG-EAE model has been shown by experiments using

a pharmacological inhibitor of MAPK kinase, the upstream

kinase, which in turn phosphorylates and thereby activates

MAPKs (Diem et al., 2003). To investigate whether the extent

of MAPK phosphorylation under the different treatment

regimens in the present study correlates with RGC survival,

we performed western blot analysis on phosphorylated and

unphosphorylated forms of p44/42-MAPKs (data not shown).

These experiments revealed that Epo-induced phosphory-

lation of MAPKs does not depend on the different treatment

intervals used in this study. In both animal groups which

received Epo as a monotherapy (early Epo, late Epo), protein

concentrations of phospho-p44/42-MAPKs were increased

when compared with vehicle treatment, whereas levels of the

unphosphorylated forms of both proteins were unchanged. In

accordance with our previous results, high-dosage MPred

therapy led to downregulation of phospho-p44/42-MAPKs

in RGCs. This effect was abolished under combined

application of MPred and Epo if Epo therapy was

administered according to the late Epo protocol. If Epo as a

combination therapy was started on the day of immunization,

protein levels of phosphorylated MAPKs were still higher

than those under vehicle treatment, indicating that Epo

given early has a stronger influence than MPred on the

regulation of this neuroprotective pathway.

Discussion

Recently, we demonstrated that MPred has direct pro-

apoptotic effects on RGCs during MOG-induced optic

Fig. 3 Histopathological analysis of optic nerves obtained on day 8 of MOG-EAE after different treatment regimes. Differences in surface

areas of the optic nerves presented result from individual selection but do not reflect general differences between the treatment groups.

(A–C) Vehicle treatment. (D–F) Early Epo (daily application of Epo started on the day of immunization). (G–I) Early Epo + MPred

(combined application of Epo started on the day of immunization and MPred given from day 1 to day 3 of MOG-EAE). (J–L) Treatment

with MPred from day 1 to day 3 of MOG-EAE. (A, D, G, J) Haematoxylin and eosin staining shows extensive cellular infiltration (purple

spots) in the vehicle- (A) and in the early Epo (D)-treated optic nerve, whereas cellular infiltrates were reduced after treatment with MPred

alone (J) or in combination with Epo (G). (B, E, H, K) Representative optic nerves stained with Luxol–fast blue reveal pronounced myelin

preservation (blue) in the early Epo + MPred-treated optic nerve (H). After vehicle treatment (B), the whole optic nerve cross-section

appears demyelinated (purple). The optic nerve treated with Epo as monotherapy from the day of immunization on (E) looks almost

completely demyelinated. The blue area at the left side (low-magnification image) indicates a small part containing preserved myelin.

Arrowheads indicate macrophages filled with myelin degradation products showing ongoing demyelination (high-magnification image).

After treatment with MPred (K), large parts of the optic nerve show intact myelin (blue). (C, F, I, L) Bielschowsky silver impregnation

shows marked axonal preservation in animals treated with MPred alone (L) or in combination with Epo (I). Remaining axons are indicated

by green arrows; blue arrowheads indicate infiltrating macrophages. Scale bars: 100 or 20 mm for haematoxylin and eosin or Luxol–fast

blue staining; 20 mm for Bielschowsky silver impregnation.

Methylprednisolone and erythropoietin in EAE

Page 7 of 11

Page 8

neuritis by inhibiting MAPK phosphorylation in these

neurons, although inflammatory infiltrates of the optic

nerve were reduced (Diem et al., 2003). In contrast, Epo

was observed to promote phosphorylation of MAPKs in

RGCs

without

exerting

any

positive

effects

histopathological changes in the optic nerve (Sättler et al.,

2004). Under the hypothesis that protective influences might

be complementary to each other if both substances are

combined, we tested different treatment protocols and

combination therapies of Epo and MPred in our present study.

Application of Epo was performed systemically due to

its ability to cross the blood–brain barrier under physiological

and pathophysiological conditions (Brines et al., 2000). As

revealed by functional, electrophysiological data as well as

histopathological results from retinas and optic nerves, we

show that combination of Epo started on the day of

immunization and MPred given from day 1 to day 3 of

MOG-EAE was most effective in protecting RGCs as well

as their axons. Compared with this protocol, Epo given as

monotherapy from the day of immunization onwards was

superior with respect to pure neuronal cell body protection

but failed to achieve any axonal rescue. Functionally, this

was reflected by improved results from pattern ERG

measurements without any improvement in VEP data. From

theratmodelofsurgicalaxotomyoftheopticnerve,itisknown

that apoptotic cell death of 80–90% of RGCs occurs following

optic nerve transection (Isenmann et al., 1997; Diem et al.,

2001). In this model, it has also been demonstrated that

neuroprotective therapies delay apoptotic cell death of RGCs

rather than resulting in permanent rescue or even promoting

neuronal regeneration (Kermer et al., 1999). From these

observations, it can be concluded that, also during auto-

immune optic neuritis, classical neuroprotective therapies

alone exert ‘cosmetic’ effects on RGC survival that must

remain transient as long as axon degeneration continues.

Accordingly, from the very few successfully tested neuro-

protective substances in EAE models, stable effects on

neuronal cell body survival were produced only by those that

influenced axonal pathology as well. It has been shown that

antagonists of the a-amino-3-hydroxy-5-methyl-4-isoxazole-

propionic acid (AMPA)/kainate type of glutamate receptor

rescued motoneurons in the lumbar spine during MBP-

induced EAE (Smith et al., 2000) and protected spinal cord

axons from EAE-associated degeneration in an adoptive

transfer model in rodents (Pitt et al., 2000). Neuron- as well

as axon-protective effects in EAE might also be expected from

substances with combine anti-inflammatory and neuro-

protective properties. Minocycline, for instance, has been

shown to reduce inflammation in EAE (Brundula et al.,

2002) and to slow down disease progression in a model of

amyotrophic lateral sclerosis (Kriz et al., 2002). At least

parts of the neuroprotective effects of this tetracycline

derivate seem to be mediated through inhibition of glutamate

excitotoxicity (Darman et al., 2004).

We demonstrate in our present study that Epo does not have

any significant effect on neuronal survival when monotherapy

is started at disease onset of EAE. This is explained by obser-

vations from our previous work showing that apoptotic cell

death of RGCs starts around 1 week before clinical manifesta-

tion of EAE. At disease onset, rats have lost 54% of their

RGCs (Hobom et al., 2004), indicating that effective neuro-

protective therapies should cover subclinical periods of

Fig. 4 Combined treatment with Epo started at immunization and

MPred given from day 1 to day 3 of MOG-EAE reduces axonal

damage and demyelination of the optic nerve. (A) Data are

mean 6 SEM of Bielschowsky-stained optic nerve axons/mm

day 8 of MOG-EAE. veh, vehicle treatment; mpred, treatment

with MPred from day 1 to day 3 of MOG-EAE; epo early + mpred,

combined application of Epo starting on the day of immunization

and MPred given from day 1 to day 3 of MOG-EAE; epo

late + mpred, combined treatment with Epo starting at disease

onset and MPred given from day 1 to day 3 of MOG-EAE; epo

early, daily application of Epo starting on the day of

immunization; epo late, daily application of Epo starting at disease

onset. *Statistically significant compared with vehicle treatment

(P < 0.008; Bonferroni-corrected one-way ANOVA followed by

Duncan’s test). (B) The extent of demyelination is given as the

percentage of the whole optic nerve cross-section stained with

Luxol–fast blue. Data are mean 6 SEM obtained at day 8 of

MOG-EAE. **Statistically significant compared with vehicle

treatment (P < 0.001; Bonferroni-corrected one-way ANOVA

followed by Duncan’s test).

Page 8 of 11

R. Diem et al.

Page 9

autoimmune CNS inflammation. Transferring this to the

human disease where the starting point of autoimmunity can-

not be determined, it might be useful to extend neuroprotec-

tive treatment for a longer period than that of acute

neurological deterioration. In support of this notion, recent

MRI studies in patients with relapsing–remitting or primary

progressive multiple sclerosis revealed the existence of

neurodegenerative processes independent of inflammation

and clinically manifest disease activity (De Stefano et al.,

2003a, b). However, late, short-duration application of Epo

in our present study was beneficial when treatment was

combined with high-dose MPred therapy. Under these

RGCs/mm

Fig. 5 Daily application of Epo alone starting at immunization or in combination with MPred given from day 1 to day 3 of MOG-EAE

promotes RGC survival during optic neuritis. (A) Representative whole-mount area at three-sixths of the retinal radius from a

vehicle-treated animal at day 8 of MOG-EAE. RGCs are labelled with FG. (B) When compared with vehicle treatment, the number of

FG-positive RGCs is reduced after MPred monotherapy. (C) Epo treatment started at immunization increases the number of surviving

RGCs counted at day 8 of MOG-EAE. (D) Under combined treatment with Epo starting on the day of immunization and MPred given from

day 1 to day 3 of MOG-EAE, RGC counts are still increased when compared with vehicle treatment. Scale bar, 100 mm. (E) Data are

mean 6 SEM of retrogradely labelled RGCs/mm

. veh, vehicle treatment; mpred, treatment with MPred from day 1 to day 3 of MOG-EAE;

epo early + mpred, combined application of Epo started at the day of immunization and MPred given from day 1 to day 3 of MOG-EAE;

epo late + mpred, combined treatment with Epo started at disease onset and MPred given from day 1 to day 3 of MOG-EAE; epo early, daily

application of Epo started at the day of immunization; epo late, daily application of Epo started at disease onset. *Statistically significant

when compared with vehicle treatment (P < 0.008); **statistically significant when compared with MPred monotherapy (P < 0.008;

Bonferroni-corrected one-way ANOVA followed by Duncan’s test).

Methylprednisolone and erythropoietin in EAE

Page 9 of 11

Page 10

conditions, Epo completely abolished negative steroid effects

on neuronal survival by antagonistic regulation of phospho-

MAPKs, as shown by western blot analysis. These data reveal

that Epo treatment started simultaneously with steroid pulse

therapy is effective in protecting neurons from acute MPred-

induced augmentation of cell death but fails to significantly

influence the underlying, advanced EAE-associated processes

of neurodegeneration.

Comparing our results from VEP measurements with his-

topathological data on the optic nerves, good functional out-

come was obtained only in the animal group that was treated

according to the early Epo + MPred protocol, although bene-

ficial effects of MPred on axon counts were also seen if MPred

was given as monotherapy. This can be explained by the

severe reduction of RGCs after application of MPred alone.

It is known that RGC loss can occur before alterations of VEP

potentials are detectable. But after RGC death has reached a

certain threshold, it is not only ERG potentials that are affec-

ted (Hobom et al., 2004), severe functional deficits of VEP

responses can also be the consequence (Hood and Greenstein,

2003). From a functional point of view, these data again argue

for the necessity of therapeutically targeting both neuronal

cell bodies and axons to avoid effects that do not result in any

clinically relevant benefit. Comparing histopathological

changes in the optic nerves between our different steroid-

treated rat groups, protection from axonal damage and demyel-

ination in animals treated according to the early Epo+ MPred

protocol was better than under MPred therapy alone. In

contrast, no additional effects on histopathological changes

of the optic nerves were seen when Epo treatment was started

simultaneously with MPred. In a study on MBP-EAE in

Lewis rats, it was shown that Epo treatment started at day 3

after immunization decreased CNS tissue levels of tumour

necrosis factor-a and interleukin-6 (Agnello et al., 2002).

For both of these proinflammatory cytokines, relevant

influences on the development of demyelination and axonal

damage have been described in different EAE models

(Okuda et al., 1999; Probert et al., 2000). Although Epo

alone did not improve histopathological changes of the

optic nerves in our study, it is conceivable that its

suppressing effect on the production of proinflammatory

cytokines early during development of EAE synergistically

enhances MPred-induced tissue protection.

In a recent study, it has been demonstrated that simultan-

eoustreatmentwithananti-inflammatoryantibody,anAMPA/

kainate receptor antagonist and the N-terminal tripeptide

of insulin-like growth factor (IGF) reverses EAE in mice

(Kanwar et al., 2004). The concept of ‘benign auto-

immunity’ might help to explain the effectiveness of

combining anti-inflammatory treatment with application of

neurotrophin-like substances, such as IGF and Epo, as

shown in our EAE model. According to this hypothesis,

autoimmune inflammatory conditions can promote neuronal

survival via increased secretion of neurotrophic factors by

cells of the immune system. High levels of brain-derived

neurotrophic factor (BDNF) and other glial cell line-derived

neurotrophins were detected in T cells in the spinal cord from

animalswithMBP-EAE,whichinturnledtoincreasedneuronal

survival of spinal motoneurons after additional axotomy in this

model (Hammarberg et al., 2000). Production of BDNF upon

antigenstimulationwasshowninastudyonT-celllinesspecific

for myelin autoantigens, such as MBP and MOG

(Kerschensteiner et al., 1999). In this study, immune-cell-

derived BDNF was demonstrated to support the survival of

sensory neurons in vitro. BDNF immunoreactivity was also

detected in inflammatory cells in lesional areas within the

brain of multiple sclerosis patients (Kerschensteiner et al.,

1999). Neurotrophic factors in turn activate a variety of pro-

tective intracellular neuronal pathways, such as MAPK phos-

phorylation, which has been described in many studies

(Yamada et al., 2001; Barnabe-Heider et al., 2003). With

this background, delivery of Epo as an exogenous

neurotrophin-like substance might compensate for the lack

of endogenous neurotrophic factor support resulting from

anti-inflammatory treatment of EAE or multiple sclerosis.

In summary, we present evidence for beneficial effects of

combined anti-inflammatory and neuroprotective treatment

in a rat model of multiple sclerosis that especially reflects

neurodegenerative aspects of the disease. Significant differ-

ences in functional outcome resulting from alternative pro-

tocols point up the importance of exactly selecting treatment

intervals for the therapeutic agents and thoroughly consider-

ing their interactions concerning intracellular signal transduc-

tion. Compared with the Epo-induced effects in this study,

a similar activation of intracellular signalling steps or

neuronal cell body protection might also be achieved by

classical neurotrophins. However, limitations concerning

their application mode make Epo and its recently developed

carbamylated derivate (Leist et al., 2004) more promising

candidates for testing in the human disease as well.

Acknowledgements

This work was supported by the Medical Faculty of the

University of Göttingen, Germany ( junior research group;

R. D., D. M., C. S.), Biogen Idec and the Gemeinnützige

Hertie Stiftung. Erythropoietin was kindly provided by

Janssen Cilag, Neuss, Germany. We thank Inna Boger for

expert technical assistance. We also thank F. Staub for crit-

ically reading this manuscript.

References

Agnello D, Bigini P, Villa P, Mennini T, Cerami A, Brines ML, et al.

Erythropoietin exerts an anti-inflammatory effect on the CNS in a

model of experimental autoimmune encephalomyelitis. Brain Res 2002;

952: 128–34.

Barnabe-Heider F, Miller FD. Endogenously produced neurotrophins regulate

survival and differentiation of cortical progenitors via distinct signaling

pathways. J Neurosci 2003; 23: 5149–60.

Bernaudin M, Marti HH, Roussel S, Divoux D, Nouvelot A, MacKenzie ET,

et al. A potential role for erythropoietin in focal permanent cerebral ische-

mia in mice. J Cereb Blood Flow Metab 1999; 19: 643–51.

Page 10 of 11

R. Diem et al.

Page 11

Brines ML, Ghezzi P, Keenan S, Agnello D, de Lanerolle NC, Cerami C,

et al. Erythropoietin crosses the blood-brain barrier to protect against

experimental brain injury. Proc Natl Acad Sci USA 2000; 97: 10526–31.

Brück W, Bitsch A, Kolenda H, Brück Y, Stiefel M, Lassmann, H. Inflam-

matory central nervous system demyelination: correlation of magnetic

resonance imaging findings with lesion pathology. Ann Neurol 1997;

42: 783–93.

Brundula V, Rewcastle NB, Metz LM, Bernard CC, Young VW. Targeting

leukocyte MMPs and transmigration: minocycline as a potential therapy

for multiple sclerosis. Brain 2002; 125: 1297–308.

Brusaferri F, Candelise L. Steroids for multiple sclerosis and optic neuritis: a

meta-analysis of randomized controlled clinical trials. J Neurol 2000; 247:

435–42.

Darman J, Backovic S, Dike S, Maragakis NJ, Krishnan C, Rothstein JD

et al. Viral-induced spinal motor neuron death is non-cell-autonomous

and involves glutamate excitotoxicity. J Neurosci 2004; 24: 7566–75.

De Stefano N, Guidi L, Stromillo ML, Bartolozzi ML, Federico A. Imaging

neuronal and axonal degeneration in multiple sclerosis. Neurol Sci Suppl

2003a; 5: 283–6.

De Stefano N, Matthews PM, Filippi M, Agosta F, De Luca M, Bartolozzi

ML, et al. Evidence of early cortical atrophy in MS: relevance to white

matter changes and disability. Neurology 2003b; 60: 1157–62.

Diem R, Meyer R, Weishaupt JH, Bähr M. Reduction of potassium currents

and phosphatidylinositol 3-kinase-dependent Akt phosphorylation by

tumor necrosis factor-a rescues axotomized retinal ganglion cells from

retrograde cell death in vivo. J Neurosci 2001; 21: 2058–66.

Diem R, Hobom M, Maier K, Weissert R, Storch MK, Meyer R, et al.

Methylprednisolone increases neuronal apoptosis during autoimmune

CNS inflammation by inhibition of an endogenous neuroprotective

pathway. J Neurosci 2003; 23: 6993–7000.

Hammarberg H, Lidman O, Lundberg C, Eltayeb SY, Gielen AW, Muhallab S,

et al. Neuroprotection by encephalomyelitis: rescue of mechanically

injured neurons and neurotrophin production by CNS-infiltrating T and

natural killer cells. J Neurosci 2000; 20: 5283–91.

Hickman SJ, Kapoor R, Jones SJ, Altmann DR, Plant GT, Miller DH.

Corticosteroids do not prevent optic nerve atrophy following optic neuritis.

J Neurol Neurosurg Psychiatry 2003; 74: 1139–41.

Hobom M, Storch MK, Weissert R, Maier K, Radhakrishnan A, Kramer B,

et al. Mechanisms and time course of neuronal degeneration in experi-

mental autoimmune encephalomyelitis. Brain Pathol 2004; 14: 148–57.

Hood DC, Greenstein VC. Multifocal VEP and ganglion cell damage: appli-

cations and limitations for the study of glaucoma. Prog Retin Eye Res

2003; 22: 201–51.

Isenmann S, Wahl C, Krajewski S, Reed JC, Bähr M. Up-regulation of Bax

protein in degenerating retinal ganglion cells precedes apoptotic cell death

after optic nerve lesion in the rat. Eur J Neurosci 1997; 9: 1763–72.

Jelkmann W. Erythropoietin: structure, control of production, and function.

Physiol Rev 1992; 72: 449–89.

Kanwar JR, Kanwar RK, Krissansen GW. Simultaneous neuroprotection

and blockade of inflammation reverses autoimmune encephalomyelitis.

Brain 2004; 127: 1313–31.

Kermer P, Klöcker N, Bähr M. Long-term effect of inhibition of ced 3-like

caspases on the survival of axotomized retinal ganglion cells in vivo. Exp

Neurol 1999; 158: 202–5.

Kerschensteiner M, Gallmeier E, Behrens L, Leal VV, Misgeld T,

Klinkert WE, et al. Activated human T cells, B cells, and monocytes

produce brain-derived neurotrophic factor in vitro and in inflammatory

brain lesions: a neuroprotective role of inflammation? J Exp Med 1999;

189: 865–70.

Kornek B, Storch MK, Weissert R, Wallstroem E, Stefferl A, Olsson T, et al.

Multiple sclerosis and chronic autoimmune encephalomyelitis: a compar-

ative quantitative study of axonal injury in active, inactive, and remyeli-

nated lesions. Am J Pathol 2000; 157: 267–76.

Kriz J, Nguyen MD, Julien JP. Minocyline slows disease progression in a

mouse model of amyotrophic lateral sclerosis. Neurobiol Dis 2002; 10:

268–78.

Leist M, Ghezzi P, Grasso G, Bianchi R, Villa P, Fratelli M, et al. Derivates of

erythropoietin that are tissue protective but not erythropoietic. Science

2004; 239–42.

Losseff NA, Wang L, Lai HM, Yoo DS, Gawne-Cain ML, McDonald WI,

et al. Progressive cerebral atrophy in multiple sclerosis. A serial MRI

study. Brain 1996; 119: 2009–19.

Meyer R, Weissert R, Diem R, Storch MK, de Graaf KL, Kramer B, et al.

Acute neuronal apoptosis in a rat model of multiple sclerosis. J Neurosci

2001; 21: 6214–20.

Noseworthy JH, Gold R, Hartung HP. Treatment of multiple sclerosis:

recent trials and future perspectives. Curr Opin Neurol 1999; 12:

279–93.

Okuda Y, Sakoda S, Fujimura H, Saeki Y, Kishimoto T, Yanagihara T. IL-6

plays a crucial role in the induction phase of myelin oligodendrocyte

glucoprotein 35–55 induced experimental autoimmune encepha-

lomyelitis. J Neuroimmunol 1999; 101: 188–96.

Parry A, Corkill R, Blamire AM, Palace J, Narayanan S, Arnold D, et al. Beta-

interferon treatment does not always slow the progression of axonal injury

in multiple sclerosis. J Neurol 2003; 250: 171–8.

Peterson JW, Bö L, Mörk S, Chang A, Trapp BD. Transected neurites, apop-

totic neurons, and reduced inflammation in cortical multiple sclerosis

lesions. Ann Neurol 2001; 50: 389–400.

Pitt D, Werner P, Raine CS. Glutamate excitotoxicity in a model of multiple

sclerosis. Nat Med 2000; 6: 67–70.

Probert L, Eugster HP, Akassoglou K, Bauer J, Frei K, Lassmann H, et al.

TNFR1 signalling is critical for the development of demyelination and

the limitation of T-cell responses during immune-mediated CNS disease.

Brain 2000; 123: 2005–19.

Sättler MB, Merkler D, Maier K, Stadelmann C, Ehrenreich H, Bähr M, et al.

Neuroprotective effects and intracellular signaling pathways of erythro-

poietin in a rat model of multiple sclerosis. Cell Death Differ 2004;

11 (Suppl 2); S181–92.

Smith T, Groom A, Zhu B, Turski L. Autoimmune encephalomyelitis ame-

liorated by AMPA antagonists. Nat Med 2000; 6: 62–6.

Storch MK, Stefferl A, Brehm U, Weissert R, Wallstrom E, Kerschensteiner

M, et al. Autoimmunity to myelin oligodendrocyte glycoprotein in rats

mimics the spectrum of multiple sclerosis pathology. Brain Pathol 1998; 8:

681–94.

Trapp BD, Peterson J, Ransohoff RM, Rudick R, Mörk S, Bö L. Axonal

transection in the lesions of multiple sclerosis. N Engl J Med 1998;

338: 278–86.

Trapp BD, Ransohoff R, Rudick R. Axonal pathology in multiple sclerosis:

relationship to neurologic disability. Curr Opin Neurol 1999; 12: 295–302.

Weissert R, Wallstrom E, Storch MK, Stefferl A, Lorentzen J, Lassmann H,

et al. MHC haplotype-dependent regulation of MOG-induced EAE in

rats. J Clin Invest 1998; 102: 1265–73.

Wekerle H, Kojima K, Lannes-Vieira J, Lassmann H, Linington, C. Animal

models. Ann Neurol Suppl 1994; 36: S47–53.

Yamada M, Tanabe K, Wada K, Shimoke K, Ishikawa Y, Ikeuchi T, et al.

Differences in survival-promoting effects and intracellular signaling

properties of BDNF and IGF-1 in cultured cerebral cortical neurons.

J Neurochem 2001; 78: 940–51.

Methylprednisolone and erythropoietin in EAE

Page 11 of 11