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Brain Research Bulletin 74 (2007) 307–316

Research report

FTY720 sustains and restores neuronal function in the DA rat modelof MOG-induced experimental autoimmune encephalomyelitis

Balazs Balatoni a,1, Maria K. Storch b,2, Eva-M. Swoboda a,3, Vinzenz Schonborn a,3,Agnieszka Koziel a,3, George N. Lambrou c,4, Peter C. Hiestand c,5,

Robert Weissert d,6, Carolyn A. Foster a,∗a Novartis Institutes for BioMedical Research, Brunner Strasse 59, A-1235 Vienna, Austria

b Department of Neurology, University of Graz, Auenbruggerplatz 22, A-8036 Graz, Austriac Novartis Institutes for BioMedical Research, CH-4002 Basel, Switzerland

d Hertie Institute for Clinical Brain Research, Otfried-Muller-Strasse 27, D-72076 Tubingen, Germany

Received 19 May 2007; accepted 28 June 2007Available online 30 July 2007

bstract

FTY720 (fingolimod) is an oral sphingosine 1-phosphate (S1P) receptor modulator under development for the treatment of multiple sclerosisMS). To elucidate its effects in the central nervous system (CNS), we compared functional parameters of nerve conductance in the DA rat modelf myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) after preventive and therapeuticreatment. We demonstrate that prophylactic therapy protected against the emergence of EAE symptoms, neuropathology, and disturbances toisual and somatosensory evoked potentials (VEP, SEP). Moreover, therapeutic treatment from day 25 to 45 markedly reversed paralysis instablished EAE and normalized the electrophysiological responses, correlating with decreased demyelination in the brain and spinal cord. The

ffectiveness of FTY720 in this model is likely due to several contributing factors. Evidence thus far supports its role in the reduction of inflammationnd preservation of blood-brain-barrier integrity. FTY720 may also act via S1P receptors in glial cells to promote endogenous repair mechanismshat complement its immunomodulatory action.

2007 Elsevier Inc. All rights reserved.

eywords: EAE; Evoked potential; Fingolimod; FTY720; Multiple sclerosis; Sphing

1

∗ Corresponding author. Tel.: +43 1 86634 685; fax: +43 1 86634 582.E-mail addresses: balazs.balatoni@novartis.com (B. Balatoni),

aria.storch@meduni-graz.at (M.K. Storch), eva.swoboda@novartis.comE.-M. Swoboda), vinzenz.schoenborn@novartis.com (V. Schonborn),gnieszka.koziel@novartis.com (A. Koziel), gnlambrou@hotmail.comG.N. Lambrou), peter.hiestand@novartis.com (P.C. Hiestand),obert.weissert@merckserono.net (R. Weissert), carolyn.foster@novartis.comC.A. Foster).1 Present address: Novartis Hungary Healthcare, P.O. Box 453/80, H-1537udapest, Hungary. Tel.: +36 209 323433.2 Tel.: +43 316 385 81785.3 Tel.: +43 1 86634 261.4 Present address: Athens Glaucoma Center, Patriarchou Ioakeim 54, GR-0676 Athens, Greece.5 Tel.: +41 61 329339.6 Tel.: +49 171 531 4748.

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361-9230/$ – see front matter © 2007 Elsevier Inc. All rights reserved.oi:10.1016/j.brainresbull.2007.06.023

osine-1 phosphate

. Introduction

Classical concepts of multiple sclerosis (MS) view relapsess the clinical expression of acute inflammatory demyelina-ion, whereas progression reflects neurodegenerative aspectsnvolving chronic demyelination, gliosis, and axonal loss [16].merging evidence suggest that these pathogenic processes mustot occur sequentially but rather can proceed simultaneously,ased on the detection of cortical atrophy early in disease andxon transection in inflammatory lesions [9,30]. Furthermore,europathology does not support the concept that neurodegen-rative components develop independently from inflammation,et the nature of the inflammatory response does differ between

he acute and progressive stages of MS [32]. Brain, spinal cordnd optic tract nerves in the central nervous system (CNS)re all vulnerable to demyelination, resulting in conductioneficits that reflect overt functional loss or underlying disease in

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linically silent lesions. Impaired nerve impulses can be quan-ified by the amplitude and latency of evoked potential (EP)esponses to sensory stimuli [3]. Visual evoked potential (VEP)nalyses, for example, suggest that delayed latency is morendicative of focal demyelination in the optic nerve, whereas

decrease in amplitude indicates the extent of axonal dam-ge [12,54]. Changes in electrophysiological parameters andisability measures in MS patients underscore the potentialf multimodal EP in monitoring disease evolution, as well asurrogate end points in clinical trials [33]. Investigations ofxperimental autoimmune encephalomyelitis (EAE), the pri-ary model for MS, have likewise helped to elucidate the

athogenic events involving demyelination and axonal loss,specially applying electrophysiology techniques coupled withlinical evaluation and histopathology.

FTY720 (fingolimod) is an oral sphingosine 1-phosphateS1P) receptor modulator that is highly effective in EAE7,17,27,44,57] and is currently in Phase 3 development for MS26]. The in vivo efficacy of FTY720 is mainly reliant on itshosphate ester metabolite (FTY720-P), which acts as a high-ffinity ligand for the G-protein-coupled receptors S1P1 and1P3–5 [7,35]. The signature feature of FTY720 is a reduction

n blood lymphocyte counts as a consequence of S1P1-mediatedetention in the peripheral lymph nodes [8,37], thereby prevent-ng the migration of autoreactive T cells into sites such as theNS [21]. On the other hand, its mode of action in MS and EAEay be related to additional, direct effects in the CNS sinceTY720 and FTY720-P are both present in the brain (C.A.F.nd Andreas Billich, submitted for publication), which containslial cells that express S1P receptors and respond to FTY720-P23,25].

Starting FTY720 treatment at the time of EAE immunizationr at the peak of established disease prevents further develop-ent of neurological deficits [7,17,27,44,57]. However, less is

nown about the disease-modifying potential of FTY720 dur-ng later phases of ongoing neurodegeneration. Because visualnd sensory abnormalities are common manifestations of MS,ur aim was to evaluate EP responses of the somatosensorynd optic pathways in the Dark Agouti (DA) rat model of EAEollowing immunization with myelin oligodendrocyte glycopro-ein (MOG). We demonstrate for the first time that FTY720 canrevent electrophysiological disturbances and restore nerve con-uctance patterns in an established demyelinating disease whichhows striking similarities to MS.

. Materials and methods

.1. Animals and antigens

Female DA/OlaHsd rats from Harlan Winkelmann (Borchen, Germany) weresed at 8–10 weeks of age and kept under standardized light- and climate-ontrolled conditions with free access to food and water. All procedures werepproved by the Austrian health authorities in compliance with internationalnimal welfare standards according to the European Communities Council

irective and the guidelines set forth in the NIH Guide for the Care and Use ofaboratory Animals.

Recombinant rat MOG, corresponding to the N-terminal Ig-like extracellularomain (amino acids 1–125), was expressed in Escherichia coli and purifiedo homogeneity by chelate chromatography as described [2]. The protein was

dL−(±

Bulletin 74 (2007) 307–316

issolved in 6 M urea, dialyzed against phosphate buffered saline (PBS), andtored at −20 ◦C [58].

.2. EAE induction and clinical scoring

Animals were lightly anesthetized by isoflurane inhalation (0.5% Forane®;bbott, Vienna, Austria) and given a single intradermal 200 �l inoculation in

he dorsal base of the tail root. The inoculum consisted of 50–75 �g MOG inBS emulsified 1:1 in complete Freund’s adjuvant (CFA) containing 200 �geat-inactivated Mycobacterium tuberculosis (strain H37 RA; DIFCOTM, BDiagnostics, Oxford, UK) as described [58]. Negative controls were injectedith CFA alone. The rats were weighed every other day and scored daily foreurological signs as follows: 0, no symptoms; 1, complete loss of tail tonus; 2,imb weakness or ataxia; 3, full paralysis of hind or forelimbs; 4, tetraparalysisr moribund; 5, death. Animals with a score of 4 were sacrificed if weight lossndicated little chance of recovery, in accordance with animal welfare standards.

ortality due to sacrifice or spontaneous EAE-related death was recorded as aon the given day; this death score continued to be included in the clinical

ssessment, but body weight measurements were not carried forward.

.3. Comparison of EP recordings in anesthetized versus freelyoving rats

EP responses are generally performed in transversal studies whereby thenimal must be kept under deep anesthesia to ensure that electrophysiologicalignals remain constant. Alternatively, longitudinal analysis in freely movingnimals permits serial VEP recordings without the use of analgesics, coupledith the opportunity for real-time evaluation of electrophysiological parameters.ince the anesthetic choice and dose can alter EP patterns, especially in theisual cortex [3,46], it was necessary to first identify the optimal recordingethod for EAE. We compared flash-induced VEP in conscious rats which

arried an implanted red light emitting diode (LED) to visual stimulation innimals that were anesthetized with ketamine and the �2-adrenergic agonistylazine. Results obtained under anesthesia did not significantly differ fromhose in the longitudinal study. Therefore, EP recordings were generated inwake and sedated animals following prophylactic and therapeutic treatmentith FTY720, respectively.

.4. Transversal and longitudinal study design

FTY720 (2-amino-2-[2-(4-octylphenyl)ethyl]propane-1,3-diol hydrochlo-ide) was provided as a powder (Novartis Pharma AG, Basel, Switzerland) andissolved in water, hereafter designated as vehicle. The drug was freshly pre-ared and given per os once daily by gavage at a dosing volume of 5 ml/kg bodyeight. Age-matched DA rats were acclimatized for a minimum of 1 week beforeistribution into four experimental groups: naıve, CFA/vehicle, MOG/vehicle,nd MOG/FTY720. For preventive therapy, oral dosing with 0.4 mg/kg FTY720r vehicle started on day 0 at immunization and continued for 2–3 weeks. Forherapeutic treatment in fully established EAE, vehicle or FTY720 dosing begann day 25 and continued until day 45. EP recordings were performed prior toacrifice in the transversal studies, or at weekly intervals in freely moving ratsor the longitudinal study.

.5. Surgical procedure for evoked potential recordings

Animals were anesthetized with isoflurane (Abbott), and the head wasorizontally positioned in a stereotaxic instrument (Precision Stereotaxic Sys-em 430005-S-1, Technical Scientific Equipment, Bad Homburg, Germany).n incision in the median sagital plane was made to remove the overlying

ascia and muscles from the skull. Four burr holes were drilled for bilaterallacement of cortical and cerebellar electrodes (0.8 mm) using stereotaxic coor-

inates [42]. Screw electrodes were implanted over the primary visual cortex (A:amda −1.0 mm; L: ±1.0 mm) and the somatosensory cortical areas (A: Bregma1.0 mm; L: −2.0 mm) for registration of VEP and SEP, respectively. Frontal

A: Bregma +5.0 mm; L: ±1.5 mm) and cerebellar (A: Lamda −2.5 mm; L:1.5 mm) reference electrodes were placed over the appropriate cortical areas.

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lectrodes were soldered to connecting sockets and covered by dentacrylate.or flash-induced retrobulbar stimulation, a LED (B1-B6334 SQD, Bright LEDlectronics Corp., Taipei Hsien, Taiwan) was implanted subcutaneously above

he left eye as described [51].

.6. Recording of electrophysiological signals

After a 1 week recovery from surgery, longitudinal EP recordings were takent baseline and at weekly intervals; VEP tracings were performed in freelyoving rats whereas SEP recordings were taken from animals anaesthetized

ntraperitoneally (i.p.) with a mixture of ketamine hydrochloride (25 mg/kg) andylazine (12.5 mg/kg). For transversal EP analysis, epidural electrodes weremplanted on the day of recording in anaesthetized animals. VEP latency in

illiseconds (ms) at the P1 peak was similar between the longitudinal andransversal recording modes at baseline (65.04 ± 1.68 and 66.80 ± 2.12 ms,espectively).

For data acquisition, each animal was placed in a Plexiglas box inside aaraday cage, isolated from noise and light, and covered to maintain body

emperature within normal limits (38 ◦C). Rats were connected via flexibleables to a CED 1902 amplifier (Cambridge Electronic Design, Cambridge,K); the LED was remotely controlled by another cable. Somatosensory stim-lation was elicited using electrodes (multistrand copper wire, 3 mm diameter)ooped around the left foot. P1 peaks were selected to characterize peak latenciesince they were the most stable; amplitudes were measured between the P1 and2 peaks. The EP response was induced by square wave pulses, differentially

mplified and filtered (0.03–100 Hz bandpass, sampling at 300 Hz with 50 msre-stimulus). The signals were passed into a Micro 1401 Acquisition InterfaceCED) and captured with Signal 2.10 software (CED).

.7. Histopathology

For perfusion fixation, animals were euthanized with a 0.5 mL overdose ofodium pentobarbital i.p. (60 mg/mL Nembutal®; Serva Feinbiochemica, Hei-elberg, Germany) and allowed to enter respiratory arrest before transcardialerfusion. Using a peristaltic pump (MityFlex® Series 913; Anko Products Inc.,radenton, FL, USA), rats were infused via the left ventricle and bled via the righttrium. CNS tissue was rapidly removed, postfixed and embedded in paraffin asescribed [50,55].

Serial 4 �m-thick cross-sections prepared from the spinal cord and brainere stained with hematoxylin and eosin (HE), luxol fast blue (LFB), andielchowsky’s silver impregnation (BIEL) as reported [50] to assess inflam-ation, demyelination, and axonal loss, respectively. Immunocytochemical

taining was performed on adjacent sections using a peroxidase-conjugatedtreptavidin–biotin method as described [55] against the following targets:D1 (macrophages/activated microglia; Serotec, Oxford, UK), W3/13 (T cells;erotec), 2′-3′-cyclic nucleotide phosphodiesterase (CNPase; SMI-91; Stern-erger Monoclonals, Lutherville, MD, USA), myelin basic protein (anti-MBP;igma–Aldrich, Saint Louis, MO, USA), proteolipid protein (anti-PLP; Serotec)nd �-amyloid precursor protein (anti-APP; Chemicon, Temecula, CA, USA).egative control sections were incubated with irrelevant mAb of the appropri-

te Ig isotype or with non-immune serum. Sections were lightly counterstainedn hematoxylin. Photographs were taken with an Axioplan 2 microscope (Carleiss GmbH, Jena, Germany).

To evaluate the inflammatory index, we calculated the mean number oferivascular inflammatory infiltrates from an average of 15 complete spinalord cross-sections per rat, as described [50]. The degree of demyelination wascored semi-quantitatively: 0.5, traces of perivascular or subpial demyelination;, marked perivascular or subpial demyelination; 2, confluent perivascular orubpial demyelination; 3, massive confluent demyelination (half of spinal cordr one complete optic nerve); 4, extensive demyelination (at least half of cere-ellar white matter or both optic nerves). The investigator (MKS) who read thelides was blinded to the clinical and electrophysiological results.

.8. Statistical analysis

A one-way analysis of variance (ANOVA) was used to compare area underhe curve (AUC) values of body weight and clinical grade EAE scores during the

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Bulletin 74 (2007) 307–316 309

ntire prophylactic treatment period or from values measured after the initiationf therapeutic dosing; ANOVA also was used to compare the electrophysiologi-al data. Differences between groups were analyzed using the post hoc Tukey testor pairwise multiple comparison. Histopathological results were evaluated withhe non-parametric Mann–Whitney U test. Calculation of correlation coefficientR value) was used to analyze the temporal relationship between EP parametersnd clinical scores.

. Results

DA rats given a single injection of MOG develop anscending and chronic paralytic disease beginning 8–12 daysost-immunization, accompanied by weight loss. The kineticsnd severity of MOG-induced EAE are known to be dependentn the rat MHC haplotype and immunization protocol [50,58]. Ingreement with such reports, our studies showed that higher con-entrations of MOG invoked a sustained and often fatal diseaseourse while lower protein levels induced a more protracted orelapsing EAE with less mortality. We utilized these experimen-al settings to assess the efficacy of FTY720 oral monotherapyased on clinical disease progression compared to electrophys-ological parameters and histopathological analysis of the CNSissue.

.1. MOG-induced electrophysiological dysfunction in DAat model

EAE induction resulted in a prolongation of somatosensoryvoked potential (SEP) and VEP latencies, as well as a reductionn SEP amplitude (Fig. 1). VEP amplitudes were more vari-ble compared to the other cortical EP responses, in agreementith observations in the guinea pig [4], Lewis rat [15] and rhe-

us monkey [19]; significant alterations in the mean P1 to N2eaks were rarely seen (data not shown). However, SEP andEP latency delays based on the P1 peak positively correlatedith increasing disease severity (Fig. 1A and C) and temporalevelopment of EAE (Fig. 1B and D). A peak-to-peak decreasen SEP amplitude from P1 to N2 was inversely related to theppearance and severity of clinical symptoms (Fig. 1E). MOGoncentrations ≥ 60 �g elicited the most consistent and robustP responses in the DA rat, regardless of whether a transversalr longitudinal analysis was performed.

.2. FTY720 prophylaxis prevents evoked potentialisturbances in MOG-EAE

Oral prophylactic dosing with FTY720 (0.4 mg/kg), start-ng at immunization and continuing for 2 weeks, significantlyrotected against disease-related mortalities and weight lossven in a very severe form of EAE where rats were injectedith 75 �g MOG (Fig. 2A). FTY720 completely prevented the

mergence of neurological deficits during treatment, quantifiedy the cumulative disease score ± S.E.M. for FTY720 (0 ± 0)ompared to vehicle (14.3 ± 1.2) from day 0 to 13.

To serially monitor clinical and electrophysiological param-ters in the same animal, recordings of SEP and VEP wereaken at weekly intervals (Fig. 2A, arrowheads along x-axis).he cortical response to peripheral or visual stimulation was

310 B. Balatoni et al. / Brain Research Bulletin 74 (2007) 307–316

Fig. 1. Electrophysiological alterations correlate with EAE onset and disease severity. (A–E) Nerve conductance pattern in DA rats injected on day 0 with adjuvantalone (�; n = 8) or MOG at about 60 �g (�; n = 6). Mean EP values ± standard error mean (S.E.M.) are plotted as log 10 on the y-axis against a linear scale for clinicalscores and logit for days after immunization on the x-axis; latencies are shown in millisecond (ms) and amplitudes in microvolt (�V). Significant prolongation of EPlatencies, based on the P1 peak, occurred soon after EAE onset as depicted on day 15 for SEP (B) and VEP (D). Latency delays for SEP (A) and VEP (C) positivelycorrelated with increasing paralysis in MOG-injected animals, indicated by R values (p = 0.004 and 0.025, respectively). Reduction of the SEP amplitude (E) fromP 3 fora not sie and D

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uantified by a delay in the P1 peak compared to longitudi-al measurements in adjuvant-injected animals (Fig. 2B) ando baseline recordings on day 0. Significant prolongation ofEP and VEP latencies in the MOG/vehicle controls positivelyorrelated with EAE onset and high disease severity arounday 10 (Fig. 2C and D, respectively). Reduction of the SEPmplitude from P1 to N2 (Fig. 2B) also showed a tight tem-oral relationship with the emergence of clinical symptomsn the vehicle group (R = −0.97, data not shown); the VEPmplitude did not significantly differ between the experimentalroups.

FTY720 significantly prevented electrophysiological distur-ances in both the SEP and VEP responses, demonstrated byatency measurements taken during and after the 2-week treat-

ent (Fig. 2C and D). As illustrated by tracings on day 24Fig. 2B), the EP latency and amplitude under FTY720 werelmost identical to those of the negative (adjuvant) controls,hich were injected with CFA and vehicle-treated.

.3. Maintenance of nerve conduction correlates withreservation of CNS structural integrity

Preventive therapy with FTY720 0.4 mg/kg, starting at 50 �gOG immunization and continuing for 3 weeks, fully blocked

he development of EAE symptoms (Fig. 3A). The animals weretill protected against wasting and the emergence of paralysisfter FTY720 discontinuation, in contrast to sustained diseasen the vehicle group. To evaluate the nerve conductance velocityn a transversal study utilizing 50 �g MOG, EP recordings wereaken at weekly intervals throughout the disease course. Vehicle-reated animals exhibited a significant delay in SEP and VEPatencies by day 33 (Fig. 3B and C, respectively), in addition to

reduction in the SEP amplitude (Fig. 3D). Consistent with its

ffectiveness on clinical symptoms, FTY720 significantly pre-ented EP disturbances even up to 2 weeks after drug cessationFig. 3B–D). To compare the clinical and electrophysiological

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R value), with a maximum decrease already occurring by about day 15 whengnificantly altered in animals injected with adjuvant alone (A–E). p-Values are) using one-way ANOVA, followed by a post hoc Tukey test.

rofile with CNS histology, the animals were sacrificed on day3 following the EP analysis (Fig. 4).

MOG-immunized animals that received vehicle from day 0 to1 developed chronic-protracted EAE (Fig. 3A), accompaniedy extensive perivascular infiltrates throughout the CNS andonfluent demyelination in the optic chiasms, optic nerves andpinal cords (Fig. 4A–C and I and J). All vehicle-treated animalshowed marked axonal loss, as indicated by a clear reduction ofielschowsky silver impregnation in the optic nerves and spinalord (Fig. 4D and K); some animals exhibited additional lesionst other predilection sites of MS. In sharp contrast, we found noetectable pathological changes in the CNS under FTY720 pro-hylactic treatment, based on the light microscopic evaluationf inflammation, demyelination and axonal integrity (Fig. 4E–Hnd L–N).

.4. FTY720 therapeutic treatment restores neuronalunction in fully established EAE

To explore the therapeutic potential of FTY720 under morelinically relevant and stringent conditions, we administered therug as rescue therapy in fully established EAE. Three-weekreatment with FTY720 at 0.4 mg/kg, starting 25 days after0 �g MOG-immunization and after the second bout of com-lete paralysis, rapidly reversed symptoms to a residual deficitn tail tonus (Fig. 5A). This was accompanied by a significantncrease in body weight under FTY720 therapy (Fig. 5B). Inontrast, the positive controls continued to exhibit sustainedaraplegia with severe wasting.

EAE induction with MOG at 60 �g provides an optimalxperimental condition for evaluating therapeutic treatmentodalities since disease symptoms are sustained at near max-

mum levels by day 25 without extensive mortality (Fig. 5).oreover, alterations in nerve conductance have reached peak

evels by day 25, as demonstrated in Fig. 1 using a comparablemmunization protocol. For these reasons we sought to deter-

B. Balatoni et al. / Brain Research Bulletin 74 (2007) 307–316 311

Fig. 2. FTY720 prophylaxis prevents impairment of EP responses in severe MOG-EAE. (A) Mean change in body weight ± S.E.M., along with EAE-related deaths(†), during disease course in DA rats immunized with 75 �g MOG. Animals received daily treatment per os with vehicle (�; n = 7) or FTY720 0.4 mg/kg (�; n = 7) for2 weeks, starting from immunization on day 0. Negative controls were injected with CFA alone and vehicle-treated (*; n = 6). FTY720 significantly prevented clinicalsymptoms, disease-related mortalities and weight loss (A) during treatment and up to almost 2 weeks after drug cessation. Level of significance was determined byone-way ANOVA of area under the curve (AUC) values from day 0 to 24, followed by a post hoc Tukey test for multiple comparison of the three experimental groups.(B–D) Serial recordings of cortical EP taken at weekly intervals (arrowheads in A) in freely moving rats, starting on day 0. (B) Representative SEP (left) and VEP(right) tracings from individual animals on day 24. FTY720 protected against peak latency (P1) delays and amplitude (P1 to N2) reductions compared to the vehiclecontrol, resulting in a similar response to that recorded in adjuvant-injected animals. (C and D) Longitudinal analysis of mean EP responses ± S.E.M. Prolongation ofSEP (C) and VEP (D) latencies coincided with EAE onset and high disease severity around day 10 in the vehicle group (�; R = 0.99 and 0.97, respectively). FTY720(�) significantly prevented MOG-induced latency delays for both SEP (C) and VEP (D), as shown on days 12–24. EP responses did not differ between FTY720 andthe adjuvant ( ). p-Values are expressed as the multiple comparison of latency values in the negative (adjuvant) and positive (vehicle) controls vs. FTY720-treated(C and D) using one-way ANOVA, followed by a post hoc Tukey test.

Fig. 3. Transversal electrophysiological analysis of MOG-EAE using FTY720 prophylactic treatment. (A) Mean disease score ± S.E.M. during a 5 week studywhere DA rats were immunized with 50 �g MOG on day 0 and given vehicle (�) or FTY720 (�) at 0.4 mg/kg for 3 weeks. Negative controls were immunizedwith CFA alone and given vehicle; n = 12 for each group. Vehicle-treated animals exhibited sustained neurological deficits throughout the study (A). In contrast,FTY720 totally prevented clinical symptoms, even after drug cessation. Level of significance was determined by one-way ANOVA of AUC values from day 0 to 33,followed by a post hoc Tukey test for pairwise comparison of the two groups. There were no deaths with either treatment. (B–D) Transversal EP recordings weretaken at weekly intervals during the disease course. From day 18 onwards, mean SEP (B) and VEP (C) latencies ± S.E.M. tended to be progressively delayed in thevehicle group (�), with significant prolongation by day 33 (data not shown for days 12, 18 and 24). A significant reduction in mean SEP amplitude ± S.E.M. alsooccurred by day 33 in vehicle-treated animals (D). FTY720 (�), on the other hand, effectively protected against alterations in the EP responses (B–D) for at least2 weeks after treatment ended, showing a similar pattern to naıve (×) and adjuvant-injected animals ( ). VEP amplitudes did not differ between the three groups(data not shown). p-Values are expressed as the comparison of EP values in naıve rats, negative (adjuvant) and positive (MOG) controls vs. FTY720-treated (B–D)using one-way ANOVA, followed by a post hoc Tukey test.

312 B. Balatoni et al. / Brain Research Bulletin 74 (2007) 307–316

Fig. 4. Representative optic nerve and spinal cord histology from above study (Fig. 3A) where MOG-immunized rats received vehicle or FTY720 from day 0 to 21.Following EP recordings and fixation on day 33, serial cross-sections of the brain and spinal cord were stained with hematoxylin and eosin (HE), anti-ED1, luxolfast blue (LFB), and Bielschowsky (BIEL) silver impregnation. (A–D, I–K) EAE controls exhibited widespread inflammation (A) of the optic nerves, both of whichwere mainly infiltrated by activated ED1+ macrophages (B) and were completely demyelinated (C), coupled with a reduction of axonal density (D). In the spinalc uction of axonal density (K) in the dorsal, lateral and ventral column; arrows denotel -treated animals, in contrast, showed no pathological alterations in the brain, optict , depicted in I).

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Table 1Summary of CNS demyelination and inflammation from the FTY720 rescuetherapy study (Figs. 5 and 6) where DA rats were immunized with 60 �g MOGand treated with 0.4 mg/kg FTY720 from day 25 to 45

Animal Demyelination index Inflammatory indexa

Brain Spinal cord Spinal cord

Control-1 3.0 3.5 0Control-2 4.0 3.5 0.53Control-3 0 3.0 0Control-4 4.0 2.5 0

2.8 ± 1.9* 3.1 ± 0.5* 0.13 ± 0.3

FTY720-1 0 2.0 0.17FTY720-2 0 2.0 0FTY720-3 0 0.5 0FTY720-4 0 2.0 0FTY720-5 0 0.0 0

0 ± 0* 1.3 ± 1.0* 0.03 ± 0.1

Brain and spinal cord samples were taken on day 53 post-immunization afterthe final EP recordings. Aggregate results for both groups are expressed as

ord, inflammation (I) was associated with confluent demyelination (J) and redesions in serial spinal cord-sections from EAE controls. (E–H, L–N) FTY720ract, or spinal cord. Scale bar = 400 �m (A–H, depicted in A) and 800 �m (I–N

ine if FTY720 could also rescue the animals from ongoingeurophysiological disturbances, based on EP recordings takenn day 53 post-immunization.

Therapeutic intervention with FTY720, starting on day 25,ot only reversed established clinical symptoms within the-week treatment period (Fig. 5) but also restored the SEPerve conductance rate to that measured in age-matched naıveats (Fig. 6). Even 1 week after drug cessation, the FTY720-reated animals exhibited a significant normalization of SEPatencies (Fig. 6A), as well as a marked increase in the SEPmplitude (Fig. 6B). The positive controls continued to exhibitlear latency prolongation and amplitude reduction, comparedo naıve (Fig. 6) and adjuvant-injected animals (Fig. 1). Sub-equent to the EP recordings, both experimental groups wereacrificed for histopathological analysis (Table 1).

.5. FTY720 rescue therapy reduces neuropathologicalamage in the CNS

In agreement with our previous observations of MOG-nduced EAE in the DA rat [49,50,58], clinical disability isssociated with extensive CNS lesions and large plaques ofemyelination by day 25 when FTY720 rescue therapy was ini-

iated, as shown in the cerebellum (Fig. 7); neuropathologicalamage is also depicted on day 33 in the optic nerves and spinalord (Fig. 4A–D and I–K). Histological evaluation of brain andpinal cord samples taken on day 53, following transversal EP

mean ± S.D.a Score 0 signifies the presence of macrophages but no perivascular inflam-

matory infiltrate.* p < 0.05 by Mann–Whitney U test between demyelination index for controls

and FTY720-treated in both brain and spinal cord samples.

B. Balatoni et al. / Brain Research Bulletin 74 (2007) 307–316 313

Fig. 5. FTY720 rescue therapy reverses established EAE symptoms. (A and B)Mean clinical scores and body weights ± S.E.M. during a 7.5 week EAE studywhere DA rats were immunized with 60 �g MOG on day 0. Daily oral treatmentwith vehicle (�; n = 4) or 0.4 mg/kg FTY720 (©; n = 5) started on day 25 after thesecond disease bout and continued for 3 weeks. Negative controls (*; n = 7) wereimmunized with CFA alone and given vehicle. Clinical symptoms under FTY720therapy were significantly reversed to background levels (A), concomitant withawp

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Fig. 6. (A and B) Transversal electrophysiological recordings taken on day 53in the above study (Fig. 5) where rats were immunized with 60 �g MOG andtreated with FTY720 from day 25 to 45. SEP values are depicted as mean latencyand amplitude ± S.E.M. for age-matched naıve rats ( ; n = 5), positive controls(�; n = 4) and FTY720-treated animals (�; n = 5). The control group exhibitedpronounced latency prolongation (A) and amplitude reduction (B). Therapeutictreatment with FTY720 significantly reversed the latency response to levelsapproximating those seen in naıve animals (A) and likewise normalized the SEPaif

wads

iluttrAalesions of positive control animals and was primarily localized

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progressive gain in body weight (B) compared to the positive controls. One-ay ANOVA was performed on AUC values from day 25 to 53, followed by aost hoc Tukey test. No deaths occurred in any experimental group.

ecordings and 1 week after drug cessation (Fig. 5), clearlyemonstrated that therapeutic treatment with FTY720 signif-cantly reduced the extent of demyelination. As summarizedn Table 1, 75% of the positive control animals continued toxhibit severe demyelination which was especially evident inhe medulla oblongata and cerebellar white matter; the patho-ogical damage was indistinquishable from that seen on day 25

n the cerebellum (Fig. 7A). Demyelinated areas were accom-anied by inflammation throughout the entire lesion and mainlyonsisted of activated ED1+ macrophages, the extent of which

toa

ig. 7. (A–C) Representative brain histology on day 25 post-immunization in DA ratshich is exemplified by the absence of MBP-positive axons (A). Widespread inflaf demyelination (arrows), shown in a serial cross-section stained by HE (B). Periacrophages (C). Scale bar = 120 �m for A and B and 50 �m for C.

mplitude (B). p-Values are expressed as the multiple comparison of EP valuesn naıve rats, positive (MOG) controls and FTY720 using one-way ANOVA,ollowed by a post hoc Tukey test.

as similar to that observed on day 25 (Fig. 7B and C). Inctively demyelinating lesions, macrophages contained degra-ation products positive for CNPase and PLP (M.K.S., data nothown).

All FTY720-treated animals were strikingly devoid of lesionsn the brain and showed significantly less demyelination in allevels of the spinal cord (Table 1). As expected, almost no resid-al T cells were found within the spinal cord of either group,aking into account that perivascular inflammation is knowno largely resolve by this late stage of MOG-EAE in the DAat and predominately comprises ED1+ macrophages [50,58].cute axonal damage, as assessed by immunocytochemistry for

myloid precursor protein (APP), was pronounced in the brain

o demyelinated areas in the cerebellar white matter and medullablongata. In contrast, there was no evidence for APP positivexons in the brain under FTY720 treatment. Animals in both

diseased with EAE. The cerebellar white matter shows extensive demyelination,mmation occurred throughout the entire lesion and this coincided with areasvascular infiltrates and demyelinated areas mainly contained activated ED1+

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roups showed equivalent signs of early remyelination, as evi-ent by fibers positive for myelin proteins within the lesion andlongside areas of demyelination.

. Discussion

To more efficiently manage progressive disability in MSatients, it will be necessary to identify therapeutic strategies thatrevent and curtail ongoing pathogenesis as well as repair theesidual damage that has already occurred. While considerablerogress has been made with the former through the develop-ent of anti-inflammatory and immunomodulatory agents, there

re currently no effective repair therapies that are routinely usedn MS patients [13,34]. Various MOG-EAE studies have high-ighted the striking similarities of this experimental disease inats to the clinical course and pathology in MS [29,39,40,50,58],einforcing the usefulness of preclinical models in drug discov-ry and in the ongoing search for novel neuroprotective agents.

By applying electrophysiology techniques to the DA ratodel, we have now provided further insight into the pathogenic

rocesses underlying EAE development. Our findings demon-trate that MOG immunization elicits marked prolongation ofEP and VEP latencies, assessed by the P1 peak, and reduc-

ion of SEP amplitude from P1 to N2. These changes inerve conductance velocity were significantly associated withhe development and severity of clinical symptoms. Concor-ance with previous studies was most consistently seen in therolongation of SEP latency, as observed in Lewis rats afterdoptive-transfer of EAE [20] or upon active immunization14]. A delay in VEP latency also was seen in the guineaig [4], monkey [19] and Lewis rat [15]. Some EAE studies,owever, reported no strict relationship between electrophysio-ogical parameters and the clinical state [18] while others onlyoted a change in SEP latency but not in VEP [11]. MOG-AE in the DA rat is regarded as one of the most robust MSodels. Therefore, it is likely that some disparity in the EP

esponse using different EAE-induction protocols is related totrain susceptibility and disease severity.

Our results, along with previous studies in other MS models7,17,27,44,57], clearly demonstrate that prophylactic treat-ent with FTY720 at clinically relevant concentrations prevents

he onset and development of neurological disability. We havextended these findings and show for the first time that functionallectrophysiological parameters, namely SEP and VEP, are pre-ictive for the efficacy of FTY720 in DA rats immunized withOG and that this correlates with the CNS histopathological

rofile. Notwithstanding, the more critical mechanistic stud-es that will impact future management of MS revolve aroundherapeutic treatment modalities. To identify and develop moreffective drugs for this indication, it will be necessary to bet-er understand the pathogenesis of acute versus progressive MSelative to the respective roles of inflammation, demyelination,xonal injury and restoration of neuronal function [1,28,48].

Here we show that delayed initiation of FTY720 therapyuring established MOG-EAE markedly attenuated clinicalymptoms, which was confirmed by a concomitant gain in bodyeight. Reversal of severe paralysis coincided with almost nor-

tlot

Bulletin 74 (2007) 307–316

al somatosensory responses by day 53, corresponding to aboutweek after drug cessation. Histopathological analysis of brain

nd spinal cord revealed several key features that distinguishhese FTY720-treated animals from the positive controls andhat would account, at least in part, for improvements seen inoth the SEP recordings and clinical scores. The most obvi-us amelioration under FTY720 treatment included significantlyess demyelination in the spinal cord and no detectable lesions inhe brain parenchyma. In contrast, 75% of the positive controlshowed a high level of demyelination in association with acutempairment of axonal transport in the cerebellum and medullablongata, the latter assessed by the extent of APP+ axons.

As suggested 100 years ago [36], remyelination of primaryemyelinated lesions is now considered a normal response dur-ng the early stages of MS [13,16], even in some patients withrogressive disease [41], and a consistent feature in experimentalodels [29,39,40]. In the present study, we detected a similar

xtent of early remyelination in both the positive control andTY720 groups by day 53. The absence of residual demyeli-ation in brain samples from all animals therapeutically treatedith FTY720, coupled with about 50% reduction of demyeli-ating lesions in the spinal cord, may provide a mechanisticxplanation for the observed reversal of clinical deficits and nor-alization of somatosensory responses. Since both the FTY720

nd positive control animals displayed a comparable level ofemyelination, we cannot exclude the possibility that FTY720-ediated efficacy is simply due to a profound anti-inflammatory

ffect which results in a restoration of nerve conductance andreversal of clinical symptoms. Nonetheless, it is tempting to

peculate that FTY720-P additionally interacts with its cognate1P receptors on glial cells to exert a centralized effect in theNS. The molecular events leading to functional restoration oferve conduction are complex and not fully understood, yett is becoming increasingly clear that cross talk between neu-ons and glia plays an essential role. For example, signals fromligodendrocytes and astrocytes are required for axonal survivalnd profoundly influence neuronal excitability, axonal transport,lustering of ion channels and synaptic transmission [43,47].eciprocal signals provided by the axon in turn help to regulate

he proliferation, survival and differentiation of glial cells.We favor the hypothesis that FTY720 is able to sustain and

estore nerve function in MOG-induced EAE by complementaryctions involving S1P receptor modulation in the periphery andithin the CNS, based on the following paradigms: (a) inter-alization of S1P1 on lymphocytes [10] prevents the entry ofncephalogenic T cells into the brain and spinal cord, (b) stabi-ization of vascular endothelium by translocation of cadherinsnd catenins [6,31,45] preserves integrity of the blood-brain-arrier, and (c) signaling of glial and/or neuronal cells promotesndogenous repair mechanisms. Regarding the latter, it is note-orthy that the nervous system is a major locus for constitutive1P receptor expression in glial cells and neurons [22,23,53].our of the five known S1P receptor subtypes display a dis-

inct distribution pattern within specific brain regions and cellineages. CNS expression of S1P5, for example, is restricted toligodendrocytes [52] and expressed throughout development tohe mature myelin-forming cell [24]. Subsequent to the discov-

earch

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B. Balatoni et al. / Brain Res

ry that S1P acts as an important regulator of cell growth [59],t has become increasingly clear that this sphingolipid mediator

ay induce the survival of such cells in the CNS upon receptorigation [22,23,53]. Indeed, recent studies have demonstratedhat FTY720-P promotes the survival of oligodendroglial lin-age cells [25]. A key consideration behind these in vitro resultss the finding that FTY720 can distribute to the CNS [38], whichontains sphingosine kinase-2 [5] for efficient phosphorylationf FTY720 in the DA rat brain (C.A.F. and Andreas Billich,ubmitted for publication).

Additional studies are necessary to further elucidate theolecular events that culminate in the remarkable efficacy

bserved with FTY720 therapy, not only in experimental modelsf demyelination but more importantly in MS patients [26], whoontinue to benefit from this novel oral agent. Key questions thatemain to be addressed would include the effect of S1P receptorodulation on neuronal plasticity and regeneration, particularly

nvolving remodeling of axonal connections, neurotrophin pro-les, ion channel rearrangement, mitochondrial dysfunction and

mpaired electron transport [28,56].

cknowledgements

We thank Hans Lassmann for reviewing this manuscript,hris Linington for kindly providing the MOG plasmid, Marijkeefzger for statistical consultation, Sam C. Wilkerson III for IT

ssistance, and Franz Hammerschmid for protein analysis. Allxperiments were supported by Novartis Pharma AG.

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