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FTY720 sustains and restores neuronal function in the DA rat model of MOG induced EAE

Brain Research Bulletin74(2007)

307–316

Research report

FTY720sustains and restores neuronal function in the DA rat model of MOG-induced experimental autoimmune encephalomyelitis

Bal′a zs Balatoni a,1,Maria K.Storch b,2,Eva-M.Swoboda a,3,Vinzenz Sch¨o nborn a,3, Agnieszka Koziel a,3,George https://www.doczj.com/doc/fb7654793.html,mbrou c,4,Peter C.Hiestand c,5,

Robert Weissert d,6,Carolyn A.Foster a,?

a Novartis Institutes for BioMedical Research,Brunner Strasse59,A-1235Vienna,Austria

b Department of Neurology,University of Graz,Auenbruggerplatz22,A-8036Graz,Austria

c Novartis Institutes for BioMedical Research,CH-4002Basel,Switzerland

d Herti

e Institute for Clinical Brain Research,Otfried-M¨u ller-Strasse27,D-72076T¨u bingen,Germany

Received19May2007;accepted28June2007

Available online30July2007

Abstract

FTY720(?ngolimod)is an oral sphingosine1-phosphate(S1P)receptor modulator under development for the treatment of multiple sclerosis (MS).To elucidate its effects in the central nervous system(CNS),we compared functional parameters of nerve conductance in the DA rat model of myelin oligodendrocyte glycoprotein(MOG)-induced experimental autoimmune encephalomyelitis(EAE)after preventive and therapeutic treatment.We demonstrate that prophylactic therapy protected against the emergence of EAE symptoms,neuropathology,and disturbances to visual and somatosensory evoked potentials(VEP,SEP).Moreover,therapeutic treatment from day25to45markedly reversed paralysis in established EAE and normalized the electrophysiological responses,correlating with decreased demyelination in the brain and spinal cord.The effectiveness of FTY720in this model is likely due to several contributing factors.Evidence thus far supports its role in the reduction of in?ammation and preservation of blood-brain-barrier integrity.FTY720may also act via S1P receptors in glial cells to promote endogenous repair mechanisms that complement its immunomodulatory action.

?2007Elsevier Inc.All rights reserved.

Keywords:EAE;Evoked potential;Fingolimod;FTY720;Multiple sclerosis;Sphingosine-1phosphate

?Corresponding author.Tel.:+43186634685;fax:+43186634582.

E-mail addresses:balazs.balatoni@https://www.doczj.com/doc/fb7654793.html,(B.Balatoni),

maria.storch@meduni-graz.at(M.K.Storch),eva.swoboda@https://www.doczj.com/doc/fb7654793.html, (E.-M.Swoboda),vinzenz.schoenborn@https://www.doczj.com/doc/fb7654793.html,(V.Sch¨o nborn), agnieszka.koziel@https://www.doczj.com/doc/fb7654793.html,(A.Koziel),gnlambrou@https://www.doczj.com/doc/fb7654793.html,

(https://www.doczj.com/doc/fb7654793.html,mbrou),peter.hiestand@https://www.doczj.com/doc/fb7654793.html,(P.C.Hiestand),

robert.weissert@https://www.doczj.com/doc/fb7654793.html,(R.Weissert),carolyn.foster@https://www.doczj.com/doc/fb7654793.html, (C.A.Foster).

1Present address:Novartis Hungary Healthcare,P.O.Box453/80,H-1537 Budapest,Hungary.Tel.:+36209323433.

2Tel.:+4331638581785.

3Tel.:+43186634261.

4Present address:Athens Glaucoma Center,Patriarchou Ioakeim54,GR-10676Athens,Greece.

5Tel.:+4161329339.

6Tel.:+491715314748.1.Introduction

Classical concepts of multiple sclerosis(MS)view relapses as the clinical expression of acute in?ammatory demyelina-tion,whereas progression re?ects neurodegenerative aspects involving chronic demyelination,gliosis,and axonal loss[16]. Emerging evidence suggest that these pathogenic processes must not occur sequentially but rather can proceed simultaneously, based on the detection of cortical atrophy early in disease and axon transection in in?ammatory lesions[9,30].Furthermore, neuropathology does not support the concept that neurodegen-erative components develop independently from in?ammation, yet the nature of the in?ammatory response does differ between the acute and progressive stages of MS[32].Brain,spinal cord and optic tract nerves in the central nervous system(CNS) are all vulnerable to demyelination,resulting in conduction de?cits that re?ect overt functional loss or underlying disease in

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308 B.Balatoni et al./Brain Research Bulletin74(2007)307–316

clinically silent lesions.Impaired nerve impulses can be quan-ti?ed by the amplitude and latency of evoked potential(EP) responses to sensory stimuli[3].Visual evoked potential(VEP) analyses,for example,suggest that delayed latency is more indicative of focal demyelination in the optic nerve,whereas a decrease in amplitude indicates the extent of axonal dam-age[12,54].Changes in electrophysiological parameters and disability measures in MS patients underscore the potential of multimodal EP in monitoring disease evolution,as well as surrogate end points in clinical trials[33].Investigations of experimental autoimmune encephalomyelitis(EAE),the pri-mary model for MS,have likewise helped to elucidate the pathogenic events involving demyelination and axonal loss, especially applying electrophysiology techniques coupled with clinical evaluation and histopathology.

FTY720(?ngolimod)is an oral sphingosine1-phosphate (S1P)receptor modulator that is highly effective in EAE [7,17,27,44,57]and is currently in Phase3development for MS [26].The in vivo ef?cacy of FTY720is mainly reliant on its phosphate ester metabolite(FTY720-P),which acts as a high-af?nity ligand for the G-protein-coupled receptors S1P1and S1P3–5[7,35].The signature feature of FTY720is a reduction in blood lymphocyte counts as a consequence of S1P1-mediated retention in the peripheral lymph nodes[8,37],thereby prevent-ing the migration of autoreactive T cells into sites such as the CNS[21].On the other hand,its mode of action in MS and EAE may be related to additional,direct effects in the CNS since FTY720and FTY720-P are both present in the brain(C.A.F. and Andreas Billich,submitted for publication),which contains glial cells that express S1P receptors and respond to FTY720-P [23,25].

Starting FTY720treatment at the time of EAE immunization or at the peak of established disease prevents further develop-ment of neurological de?cits[7,17,27,44,57].However,less is known about the disease-modifying potential of FTY720dur-ing later phases of ongoing neurodegeneration.Because visual and sensory abnormalities are common manifestations of MS, our aim was to evaluate EP responses of the somatosensory and optic pathways in the Dark Agouti(DA)rat model of EAE following immunization with myelin oligodendrocyte glycopro-tein(MOG).We demonstrate for the?rst time that FTY720can prevent electrophysiological disturbances and restore nerve con-ductance patterns in an established demyelinating disease which shows striking similarities to MS.

2.Materials and methods

2.1.Animals and antigens

Female DA/OlaHsd rats from Harlan Winkelmann(Borchen,Germany)were used at8–10weeks of age and kept under standardized light-and climate-controlled conditions with free access to food and water.All procedures were approved by the Austrian health authorities in compliance with international animal welfare standards according to the European Communities Council Directive and the guidelines set forth in the NIH Guide for the Care and Use of Laboratory Animals.

Recombinant rat MOG,corresponding to the N-terminal Ig-like extracellular domain(amino acids1–125),was expressed in Escherichia coli and puri?ed to homogeneity by chelate chromatography as described[2].The protein was

dissolved in6M urea,dialyzed against phosphate buffered saline(PBS),and stored at?20?C[58].

2.2.EAE induction and clinical scoring

Animals were lightly anesthetized by iso?urane inhalation(0.5%Forane?;

Abbott,Vienna,Austria)and given a single intradermal200?l inoculation in the dorsal base of the tail root.The inoculum consisted of50–75?g MOG in PBS emulsi?ed1:1in complete Freund’s adjuvant(CFA)containing200?g heat-inactivated Mycobacterium tuberculosis(strain H37RA;DIFCO TM,BD Diagnostics,Oxford,UK)as described[58].Negative controls were injected with CFA alone.The rats were weighed every other day and scored daily for neurological signs as follows:0,no symptoms;1,complete loss of tail tonus;2, limb weakness or ataxia;3,full paralysis of hind or forelimbs;4,tetraparalysis or moribund;5,death.Animals with a score of4were sacri?ced if weight loss indicated little chance of recovery,in accordance with animal welfare standards.

Mortality due to sacri?ce or spontaneous EAE-related death was recorded as a 5on the given day;this death score continued to be included in the clinical assessment,but body weight measurements were not carried forward.

https://www.doczj.com/doc/fb7654793.html,parison of EP recordings in anesthetized versus freely

moving rats

EP responses are generally performed in transversal studies whereby the animal must be kept under deep anesthesia to ensure that electrophysiological signals remain constant.Alternatively,longitudinal analysis in freely moving animals permits serial VEP recordings without the use of analgesics,coupled with the opportunity for real-time evaluation of electrophysiological parameters.

Since the anesthetic choice and dose can alter EP patterns,especially in the visual cortex[3,46],it was necessary to?rst identify the optimal recording method for EAE.We compared?ash-induced VEP in conscious rats which carried an implanted red light emitting diode(LED)to visual stimulation in animals that were anesthetized with ketamine and the?2-adrenergic agonist xylazine.Results obtained under anesthesia did not signi?cantly differ from those in the longitudinal study.Therefore,EP recordings were generated in awake and sedated animals following prophylactic and therapeutic treatment with FTY720,respectively.

2.4.Transversal and longitudinal study design

FTY720(2-amino-2-[2-(4-octylphenyl)ethyl]propane-1,3-diol hydrochlo-ride)was provided as a powder(Novartis Pharma AG,Basel,Switzerland)and dissolved in water,hereafter designated as vehicle.The drug was freshly pre-pared and given per os once daily by gavage at a dosing volume of5ml/kg body weight.Age-matched DA rats were acclimatized for a minimum of1week before distribution into four experimental groups:na¨?ve,CFA/vehicle,MOG/vehicle, and MOG/FTY720.For preventive therapy,oral dosing with0.4mg/kg FTY720 or vehicle started on day0at immunization and continued for2–3weeks.For therapeutic treatment in fully established EAE,vehicle or FTY720dosing began on day25and continued until day45.EP recordings were performed prior to sacri?ce in the transversal studies,or at weekly intervals in freely moving rats for the longitudinal study.

2.5.Surgical procedure for evoked potential recordings

Animals were anesthetized with iso?urane(Abbott),and the head was horizontally positioned in a stereotaxic instrument(Precision Stereotaxic Sys-tem430005-S-1,Technical Scienti?c Equipment,Bad Homburg,Germany).

An incision in the median sagital plane was made to remove the overlying fascia and muscles from the skull.Four burr holes were drilled for bilateral placement of cortical and cerebellar electrodes(0.8mm)using stereotaxic coor-dinates[42].Screw electrodes were implanted over the primary visual cortex(A: Lamda?1.0mm;L:±1.0mm)and the somatosensory cortical areas(A:Bregma ?1.0mm;L:?2.0mm)for registration of VEP and SEP,respectively.Frontal (A:Bregma+5.0mm;L:±1.5mm)and cerebellar(A:Lamda?2.5mm;L:±1.5mm)reference electrodes were placed over the appropriate cortical areas. Edited by Foxit Reader

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B.Balatoni et al./Brain Research Bulletin74(2007)307–316309

Electrodes were soldered to connecting sockets and covered by dentacrylate. For?ash-induced retrobulbar stimulation,a LED(B1-B6334SQD,Bright LED Electronics Corp.,Taipei Hsien,Taiwan)was implanted subcutaneously above the left eye as described[51].

2.6.Recording of electrophysiological signals

After a1week recovery from surgery,longitudinal EP recordings were taken at baseline and at weekly intervals;VEP tracings were performed in freely moving rats whereas SEP recordings were taken from animals anaesthetized intraperitoneally(i.p.)with a mixture of ketamine hydrochloride(25mg/kg)and xylazine(12.5mg/kg).For transversal EP analysis,epidural electrodes were implanted on the day of recording in anaesthetized animals.VEP latency in milliseconds(ms)at the P1peak was similar between the longitudinal and transversal recording modes at baseline(65.04±1.68and66.80±2.12ms, respectively).

For data acquisition,each animal was placed in a Plexiglas box inside a Faraday cage,isolated from noise and light,and covered to maintain body temperature within normal limits(38?C).Rats were connected via?exible cables to a CED1902ampli?er(Cambridge Electronic Design,Cambridge, UK);the LED was remotely controlled by another cable.Somatosensory stim-ulation was elicited using electrodes(multistrand copper wire,3mm diameter) looped around the left foot.P1peaks were selected to characterize peak latencies since they were the most stable;amplitudes were measured between the P1and N2peaks.The EP response was induced by square wave pulses,differentially ampli?ed and?ltered(0.03–100Hz bandpass,sampling at300Hz with50ms pre-stimulus).The signals were passed into a Micro1401Acquisition Interface (CED)and captured with Signal2.10software(CED).

2.7.Histopathology

For perfusion?xation,animals were euthanized with a0.5mL overdose of sodium pentobarbital i.p.(60mg/mL Nembutal?;Serva Feinbiochemica,Hei-delberg,Germany)and allowed to enter respiratory arrest before transcardial https://www.doczj.com/doc/fb7654793.html,ing a peristaltic pump(MityFlex?Series913;Anko Products Inc., Bradenton,FL,USA),rats were infused via the left ventricle and bled via the right https://www.doczj.com/doc/fb7654793.html,S tissue was rapidly removed,post?xed and embedded in paraf?n as described[50,55].

Serial4?m-thick cross-sections prepared from the spinal cord and brain were stained with hematoxylin and eosin(HE),luxol fast blue(LFB),and Bielchowsky’s silver impregnation(BIEL)as reported[50]to assess in?am-mation,demyelination,and axonal loss,respectively.Immunocytochemical staining was performed on adjacent sections using a peroxidase-conjugated streptavidin–biotin method as described[55]against the following targets: ED1(macrophages/activated microglia;Serotec,Oxford,UK),W3/13(T cells; Serotec),2 -3 -cyclic nucleotide phosphodiesterase(CNPase;SMI-91;Stern-berger Monoclonals,Lutherville,MD,USA),myelin basic protein(anti-MBP; Sigma–Aldrich,Saint Louis,MO,USA),proteolipid protein(anti-PLP;Serotec) and?-amyloid precursor protein(anti-APP;Chemicon,Temecula,CA,USA). Negative control sections were incubated with irrelevant mAb of the appropri-ate Ig isotype or with non-immune serum.Sections were lightly counterstained in hematoxylin.Photographs were taken with an Axioplan2microscope(Carl Zeiss GmbH,Jena,Germany).

To evaluate the in?ammatory index,we calculated the mean number of perivascular in?ammatory in?ltrates from an average of15complete spinal cord cross-sections per rat,as described[50].The degree of demyelination was scored semi-quantitatively:0.5,traces of perivascular or subpial demyelination; 1,marked perivascular or subpial demyelination;2,con?uent perivascular or subpial demyelination;3,massive con?uent demyelination(half of spinal cord or one complete optic nerve);4,extensive demyelination(at least half of cere-bellar white matter or both optic nerves).The investigator(MKS)who read the slides was blinded to the clinical and electrophysiological results.

2.8.Statistical analysis

A one-way analysis of variance(ANOV A)was used to compare area under the curve(AUC)values of body weight and clinical grade EAE scores during the entire prophylactic treatment period or from values measured after the initiation of therapeutic dosing;ANOV A also was used to compare the electrophysiologi-cal data.Differences between groups were analyzed using the post hoc Tukey test for pairwise multiple comparison.Histopathological results were evaluated with the non-parametric Mann–Whitney U test.Calculation of correlation coef?cient (R value)was used to analyze the temporal relationship between EP parameters and clinical scores.

3.Results

DA rats given a single injection of MOG develop an ascending and chronic paralytic disease beginning8–12days post-immunization,accompanied by weight loss.The kinetics and severity of MOG-induced EAE are known to be dependent on the rat MHC haplotype and immunization protocol[50,58].In agreement with such reports,our studies showed that higher con-centrations of MOG invoked a sustained and often fatal disease course while lower protein levels induced a more protracted or relapsing EAE with less mortality.We utilized these experimen-tal settings to assess the ef?cacy of FTY720oral monotherapy based on clinical disease progression compared to electrophys-iological parameters and histopathological analysis of the CNS tissue.

3.1.MOG-induced electrophysiological dysfunction in DA

rat model

EAE induction resulted in a prolongation of somatosensory evoked potential(SEP)and VEP latencies,as well as a reduction in SEP amplitude(Fig.1).VEP amplitudes were more vari-able compared to the other cortical EP responses,in agreement with observations in the guinea pig[4],Lewis rat[15]and rhe-sus monkey[19];signi?cant alterations in the mean P1to N2 peaks were rarely seen(data not shown).However,SEP and VEP latency delays based on the P1peak positively correlated with increasing disease severity(Fig.1A and C)and temporal development of EAE(Fig.1B and D).A peak-to-peak decrease in SEP amplitude from P1to N2was inversely related to the appearance and severity of clinical symptoms(Fig.1E).MOG concentrations≥60?g elicited the most consistent and robust EP responses in the DA rat,regardless of whether a transversal or longitudinal analysis was performed.

3.2.FTY720prophylaxis prevents evoked potential disturbances in MOG-EAE

Oral prophylactic dosing with FTY720(0.4mg/kg),start-ing at immunization and continuing for2weeks,signi?cantly protected against disease-related mortalities and weight loss even in a very severe form of EAE where rats were injected with75?g MOG(Fig.2A).FTY720completely prevented the emergence of neurological de?cits during treatment,quanti?ed by the cumulative disease score±S.E.M.for FTY720(0±0) compared to vehicle(14.3±1.2)from day0to13.

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

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Fig.1.Electrophysiological alterations correlate with EAE onset and disease severity.(A–E)Nerve conductance pattern in DA rats injected on day 0with adjuvant alone ( ;n =8)or MOG at about 60?g ( ;n =6).Mean EP values ±standard error mean (S.E.M.)are plotted as log 10on the y -axis against a linear scale for clinical scores and logit for days after immunization on the x -axis;latencies are shown in millisecond (ms)and amplitudes in microvolt (?V).Signi?cant prolongation of EP latencies,based on the P1peak,occurred soon after EAE onset as depicted on day 15for SEP (B)and VEP (D).Latency delays for SEP (A)and VEP (C)positively correlated with increasing paralysis in MOG-injected animals,indicated by R values (p =0.004and 0.025,respectively).Reduction of the SEP amplitude (E)from P1to N2was inversely related to disease progression in MOG controls (p =0.03for R value),with a maximum decrease already occurring by about day 15when animals exhibit hindlimb weakness or ataxia.EP latencies and amplitudes were not signi?cantly altered in animals injected with adjuvant alone (A–E).p -Values are expressed as the comparison of latency values in MOG controls vs.adjuvant (B and D)using one-way ANOV A,followed by a post hoc Tukey test.

quanti?ed by a delay in the P1peak compared to longitudi-nal measurements in adjuvant-injected animals (Fig.2B)and to baseline recordings on day 0.Signi?cant prolongation of SEP and VEP latencies in the MOG/vehicle controls positively correlated with EAE onset and high disease severity around day 10(Fig.2C and D,respectively).Reduction of the SEP amplitude from P1to N2(Fig.2B)also showed a tight tem-poral relationship with the emergence of clinical symptoms in the vehicle group (R =?0.97,data not shown);the VEP amplitude did not signi?cantly differ between the experimental groups.

FTY720signi?cantly prevented electrophysiological distur-bances in both the SEP and VEP responses,demonstrated by latency measurements taken during and after the 2-week treat-ment (Fig.2C and D).As illustrated by tracings on day 24(Fig.2B),the EP latency and amplitude under FTY720were almost identical to those of the negative (adjuvant)controls,which were injected with CFA and vehicle-treated.3.3.Maintenance of nerve conduction correlates with preservation of CNS structural integrity

Preventive therapy with FTY7200.4mg/kg,starting at 50?g MOG immunization and continuing for 3weeks,fully blocked the development of EAE symptoms (Fig.3A).The animals were still protected against wasting and the emergence of paralysis after FTY720discontinuation,in contrast to sustained disease in the vehicle group.To evaluate the nerve conductance velocity in a transversal study utilizing 50?g MOG,EP recordings were taken at weekly intervals throughout the disease course.Vehicle-treated animals exhibited a signi?cant delay in SEP and VEP latencies by day 33(Fig.3B and C,respectively),in addition to a reduction in the SEP amplitude (Fig.3D).Consistent with its effectiveness on clinical symptoms,FTY720signi?cantly pre-vented EP disturbances even up to 2weeks after drug cessation (Fig.3B–D).To compare the clinical and electrophysiological

pro?le with CNS histology,the animals were sacri?ced on day 33following the EP analysis (Fig.4).

MOG-immunized animals that received vehicle from day 0to 21developed chronic-protracted EAE (Fig.3A),accompanied by extensive perivascular in?ltrates throughout the CNS and con?uent demyelination in the optic chiasms,optic nerves and spinal cords (Fig.4A–C and I and J).All vehicle-treated animals showed marked axonal loss,as indicated by a clear reduction of Bielschowsky silver impregnation in the optic nerves and spinal cord (Fig.4D and K);some animals exhibited additional lesions at other predilection sites of MS.In sharp contrast,we found no detectable pathological changes in the CNS under FTY720pro-phylactic treatment,based on the light microscopic evaluation of in?ammation,demyelination and axonal integrity (Fig.4E–H and L–N).

3.4.FTY720therapeutic treatment restores neuronal function in fully established EAE

To explore the therapeutic potential of FTY720under more clinically relevant and stringent conditions,we administered the drug as rescue therapy in fully established EAE.Three-week treatment with FTY720at 0.4mg/kg,starting 25days after 60?g MOG-immunization and after the second bout of com-plete paralysis,rapidly reversed symptoms to a residual de?cit in tail tonus (Fig.5A).This was accompanied by a signi?cant increase in body weight under FTY720therapy (Fig.5B).In contrast,the positive controls continued to exhibit sustained paraplegia with severe wasting.

EAE induction with MOG at 60?g provides an optimal experimental condition for evaluating therapeutic treatment modalities since disease symptoms are sustained at near max-imum levels by day 25without extensive mortality (Fig.5).Moreover,alterations in nerve conductance have reached peak levels by day 25,as demonstrated in Fig.1using a comparable immunization protocol.For these reasons we sought to deter-

B.Balatoni et al./Brain Research Bulletin74(2007)307–316311

Fig.2.FTY720prophylaxis 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 with75?g MOG.Animals received daily treatment per os with vehicle( ;n=7)or FTY7200.4mg/kg( ;n=7)for 2weeks,starting from immunization on day0.Negative controls were injected with CFA alone and vehicle-treated(*;n=6).FTY720signi?cantly prevented clinical symptoms,disease-related mortalities and weight loss(A)during treatment and up to almost2weeks after drug cessation.Level of signi?cance was determined by one-way ANOV A of area under the curve(AUC)values from day0to24,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 day0.(B)Representative SEP(left)and VEP (right)tracings from individual animals on day24.FTY720protected against peak latency(P1)delays and amplitude(P1to N2)reductions compared to the vehicle control,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 of SEP(C)and VEP(D)latencies coincided with EAE onset and high disease severity around day10in the vehicle group( ;R=0.99and0.97,respectively).FTY720 ( )signi?cantly prevented MOG-induced latency delays for both SEP(C)and VEP(D),as shown on days12–24.EP responses did not differ between FTY720and the 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 ANOV A,followed by a post hoc Tukey test.

Fig.3.Transversal electrophysiological analysis of MOG-EAE using FTY720prophylactic treatment.(A)Mean disease score±S.E.M.during a5week study where DA rats were immunized with50?g MOG on day0and given vehicle( )or FTY720( )at0.4mg/kg for3weeks.Negative controls were immunized with CFA alone and given vehicle;n=12for each group.Vehicle-treated animals exhibited sustained neurological de?cits throughout the study(A).In contrast, FTY720totally prevented clinical symptoms,even after drug cessation.Level of signi?cance was determined by one-way ANOV A of AUC values from day0to33, 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 were taken at weekly intervals during the disease course.From day18onwards,mean SEP(B)and VEP(C)latencies±S.E.M.tended to be progressively delayed in the vehicle group( ),with signi?cant prolongation by day33(data not shown for days12,18and24).A signi?cant reduction in mean SEP amplitude±S.E.M.also occurred by day33in vehicle-treated animals(D).FTY720( ),on the other hand,effectively protected against alterations in the EP responses(B–D)for at least 2weeks after treatment ended,showing a similar pattern to na¨?ve(×)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 na¨?ve rats,negative(adjuvant)and positive(MOG)controls vs.FTY720-treated(B–D) using one-way ANOV A,followed by a post hoc Tukey test.

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Fig.4.Representative optic nerve and spinal cord histology from above study (Fig.3A)where MOG-immunized rats received vehicle or FTY720from day 0to 21.Following EP recordings and ?xation on day 33,serial cross-sections of the brain and spinal cord were stained with hematoxylin and eosin (HE),anti-ED1,luxol fast blue (LFB),and Bielschowsky (BIEL)silver impregnation.(A–D,I–K)EAE controls exhibited widespread in?ammation (A)of the optic nerves,both of which were mainly in?ltrated by activated ED1+macrophages (B)and were completely demyelinated (C),coupled with a reduction of axonal density (D).In the spinal cord,in?ammation (I)was associated with con?uent demyelination (J)and reduction of axonal density (K)in the dorsal,lateral and ventral column;arrows denote lesions in serial spinal cord-sections from EAE controls.(E–H,L–N)FTY720-treated animals,in contrast,showed no pathological alterations in the brain,optic tract,or spinal cord.Scale bar =400?m (A–H,depicted in A)and 800?m (I–N,depicted in I).

mine if FTY720could also rescue the animals from ongoing neurophysiological disturbances,based on EP recordings taken on day 53post-immunization.

Therapeutic intervention with FTY720,starting on day 25,not only reversed established clinical symptoms within the 3-week treatment period (Fig.5)but also restored the SEP nerve conductance rate to that measured in age-matched na¨?ve rats (Fig.6).Even 1week after drug cessation,the FTY720-treated animals exhibited a signi?cant normalization of SEP latencies (Fig.6A),as well as a marked increase in the SEP amplitude (Fig.6B).The positive controls continued to exhibit clear latency prolongation and amplitude reduction,compared to na¨?ve (Fig.6)and adjuvant-injected animals (Fig.1).Sub-sequent to the EP recordings,both experimental groups were sacri?ced for histopathological analysis (Table 1).3.5.FTY720rescue therapy reduces neuropathological damage in the CNS

In agreement with our previous observations of MOG-induced EAE in the DA rat [49,50,58],clinical disability is associated with extensive CNS lesions and large plaques of demyelination by day 25when FTY720rescue therapy was ini-tiated,as shown in the cerebellum (Fig.7);neuropathological damage is also depicted on day 33in the optic nerves and spinal cord (Fig.4A–D and I–K).Histological evaluation of brain and spinal cord samples taken on day 53,following transversal EP

Table 1

Summary of CNS demyelination and in?ammation from the FTY720rescue therapy study (Figs.5and 6)where DA rats were immunized with 60?g MOG and treated with 0.4mg/kg FTY720from day 25to 45Animal

Demyelination index In?ammatory index a Brain

Spinal cord Spinal cord Control-1 3.0 3.50Control-2 4.0 3.50.53Control-30 3.00Control-4

4.0 2.502.8±1.9*

3.1±0.5*0.13±0.3FTY720-10 2.00.17FTY720-20 2.00FTY720-300.50FTY720-40 2.00FTY720-5

00.000±0*

1.3±1.0*

0.03±0.1

Brain and spinal cord samples were taken on day 53post-immunization after the ?nal EP recordings.Aggregate results for both groups are expressed as mean ±S.D.

a Score 0signi?es the presence of macrophages but no perivascular in?am-matory in?ltrate.

*p <0.05by Mann–Whitney U test between demyelination index for controls and FTY720-treated in both brain and spinal cord samples.

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Fig.5.FTY720rescue therapy reverses established EAE symptoms.(A and B)Mean clinical scores and body weights ±S.E.M.during a 7.5week EAE study where DA rats were immunized with 60?g MOG on day 0.Daily oral treatment with vehicle (?;n =4)or 0.4mg/kg FTY720( ;n =5)started on day 25after the second disease bout and continued for 3weeks.Negative controls (*;n =7)were immunized with CFA alone and given vehicle.Clinical symptoms under FTY720therapy were signi?cantly reversed to background levels (A),concomitant with a progressive gain in body weight (B)compared to the positive controls.One-way ANOV A was performed on AUC values from day 25to 53,followed by a post hoc Tukey test.No deaths occurred in any experimental group.

recordings and 1week after drug cessation (Fig.5),clearly demonstrated that therapeutic treatment with FTY720signif-icantly reduced the extent of demyelination.As summarized in Table 1,75%of the positive control animals continued to exhibit severe demyelination which was especially evident in the medulla oblongata and cerebellar white matter;the patho-logical damage was indistinquishable from that seen on day 25in the cerebellum (Fig.7A).Demyelinated areas were accom-panied by in?ammation throughout the entire lesion and mainly consisted of activated ED1+macrophages,the extent of

which

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 and treated with FTY720from day 25to 45.SEP values are depicted as mean latency and amplitude ±S.E.M.for age-matched na¨?ve rats (;n =5),positive controls ( ;n =4)and FTY720-treated animals ( ;n =5).The control group exhibited pronounced latency prolongation (A)and amplitude reduction (B).Therapeutic treatment with FTY720signi?cantly reversed the latency response to levels approximating those seen in na¨?ve animals (A)and likewise normalized the SEP amplitude (B).p -Values are expressed as the multiple comparison of EP values in na¨?ve rats,positive (MOG)controls and FTY720using one-way ANOV A,followed by a post hoc Tukey test.

was similar to that observed on day 25(Fig.7B and C).In actively demyelinating lesions,macrophages contained degra-dation products positive for CNPase and PLP (M.K.S.,data not shown).

All FTY720-treated animals were strikingly devoid of lesions in the brain and showed signi?cantly less demyelination in all levels of the spinal cord (Table 1).As expected,almost no resid-ual T cells were found within the spinal cord of either group,taking into account that perivascular in?ammation is known to largely resolve by this late stage of MOG-EAE in the DA rat and predominately comprises ED1+macrophages [50,58].Acute axonal damage,as assessed by immunocytochemistry for amyloid precursor protein (APP),was pronounced in the brain lesions of positive control animals and was primarily localized to demyelinated areas in the cerebellar white matter and medulla oblongata.

In contrast,there was no evidence for APP positive axons in the brain under FTY720treatment.Animals in both

Fig.7.(A–C)Representative brain histology on day 25post-immunization in DA rats diseased with EAE.The cerebellar white matter shows extensive demyelination,which is exempli?ed by the absence of MBP-positive axons (A).Widespread in?ammation occurred throughout the entire lesion and this coincided with areas of demyelination (arrows),shown in a serial cross-section stained by HE (B).Perivascular in?ltrates and demyelinated areas mainly contained activated ED1+macrophages (C).Scale bar =120?m for A and B and 50?m for C.

314 B.Balatoni et al./Brain Research Bulletin74(2007)307–316

groups showed equivalent signs of early remyelination,as evi-dent by?bers positive for myelin proteins within the lesion and alongside areas of demyelination.

4.Discussion

To more ef?ciently manage progressive disability in MS patients,it will be necessary to identify therapeutic strategies that prevent and curtail ongoing pathogenesis as well as repair the residual damage that has already occurred.While considerable progress has been made with the former through the develop-ment of anti-in?ammatory and immunomodulatory agents,there are currently no effective repair therapies that are routinely used in MS patients[13,34].Various MOG-EAE studies have high-lighted the striking similarities of this experimental disease in rats to the clinical course and pathology in MS[29,39,40,50,58], reinforcing the usefulness of preclinical models in drug discov-ery and in the ongoing search for novel neuroprotective agents.

By applying electrophysiology techniques to the DA rat model,we have now provided further insight into the pathogenic processes underlying EAE development.Our?ndings demon-strate that MOG immunization elicits marked prolongation of SEP and VEP latencies,assessed by the P1peak,and reduc-tion of SEP amplitude from P1to N2.These changes in nerve conductance velocity were signi?cantly associated with the development and severity of clinical symptoms.Concor-dance with previous studies was most consistently seen in the prolongation of SEP latency,as observed in Lewis rats after adoptive-transfer of EAE[20]or upon active immunization [14].A delay in VEP latency also was seen in the guinea pig[4],monkey[19]and Lewis rat[15].Some EAE studies, however,reported no strict relationship between electrophysio-logical parameters and the clinical state[18]while others only noted a change in SEP latency but not in VEP[11].MOG-EAE in the DA rat is regarded as one of the most robust MS models.Therefore,it is likely that some disparity in the EP response using different EAE-induction protocols is related to strain susceptibility and disease severity.

Our results,along with previous studies in other MS models [7,17,27,44,57],clearly demonstrate that prophylactic treat-ment with FTY720at clinically relevant concentrations prevents the onset and development of neurological disability.We have extended these?ndings and show for the?rst time that functional electrophysiological parameters,namely SEP and VEP,are pre-dictive for the ef?cacy of FTY720in DA rats immunized with MOG and that this correlates with the CNS histopathological pro?le.Notwithstanding,the more critical mechanistic stud-ies that will impact future management of MS revolve around therapeutic treatment modalities.To identify and develop more effective drugs for this indication,it will be necessary to bet-ter understand the pathogenesis of acute versus progressive MS relative to the respective roles of in?ammation,demyelination, axonal injury and restoration of neuronal function[1,28,48].

Here we show that delayed initiation of FTY720therapy during established MOG-EAE markedly attenuated clinical symptoms,which was con?rmed by a concomitant gain in body weight.Reversal of severe paralysis coincided with almost nor-

mal somatosensory responses by day53,corresponding to about 1week after drug cessation.Histopathological analysis of brain and spinal cord revealed several key features that distinguish these FTY720-treated animals from the positive controls and that would account,at least in part,for improvements seen in both the SEP recordings and clinical scores.The most obvi-ous amelioration under FTY720treatment included signi?cantly less demyelination in the spinal cord and no detectable lesions in the brain parenchyma.In contrast,75%of the positive controls showed a high level of demyelination in association with acute impairment of axonal transport in the cerebellum and medulla oblongata,the latter assessed by the extent of APP+axons.

As suggested100years ago[36],remyelination of primary demyelinated lesions is now considered a normal response dur-ing the early stages of MS[13,16],even in some patients with progressive disease[41],and a consistent feature in experimental models[29,39,40].In the present study,we detected a similar extent of early remyelination in both the positive control and FTY720groups by day53.The absence of residual demyeli-nation in brain samples from all animals therapeutically treated with FTY720,coupled with about50%reduction of demyeli-nating lesions in the spinal cord,may provide a mechanistic explanation for the observed reversal of clinical de?cits and nor-malization of somatosensory responses.Since both the FTY720 and positive control animals displayed a comparable level of remyelination,we cannot exclude the possibility that FTY720-mediated ef?cacy is simply due to a profound anti-in?ammatory effect which results in a restoration of nerve conductance and

a reversal of clinical symptoms.Nonetheless,it is tempting to

speculate that FTY720-P additionally interacts with its cognate S1P receptors on glial cells to exert a centralized effect in the CNS.The molecular events leading to functional restoration of nerve conduction are complex and not fully understood,yet it is becoming increasingly clear that cross talk between neu-rons and glia plays an essential role.For example,signals from oligodendrocytes and astrocytes are required for axonal survival and profoundly in?uence neuronal excitability,axonal transport, clustering of ion channels and synaptic transmission[43,47].

Reciprocal signals provided by the axon in turn help to regulate the proliferation,survival and differentiation of glial cells.

We favor the hypothesis that FTY720is able to sustain and restore nerve function in MOG-induced EAE by complementary actions involving S1P receptor modulation in the periphery and within the CNS,based on the following paradigms:(a)inter-nalization of S1P1on lymphocytes[10]prevents the entry of encephalogenic T cells into the brain and spinal cord,(b)stabi-lization of vascular endothelium by translocation of cadherins and catenins[6,31,45]preserves integrity of the blood-brain-barrier,and(c)signaling of glial and/or neuronal cells promotes endogenous repair mechanisms.Regarding the latter,it is note-worthy that the nervous system is a major locus for constitutive S1P receptor expression in glial cells and neurons[22,23,53].

Four of the?ve known S1P receptor subtypes display a dis-tinct distribution pattern within speci?c brain regions and cell https://www.doczj.com/doc/fb7654793.html,S expression of S1P5,for example,is restricted to oligodendrocytes[52]and expressed throughout development to the mature myelin-forming cell[24].Subsequent to the discov-Edited by Foxit Reader

Copyright(C) by Foxit Software Company,2005-2007 For Evaluation Only.

B.Balatoni et al./Brain Research Bulletin74(2007)307–316315

ery that S1P acts as an important regulator of cell growth[59], it has become increasingly clear that this sphingolipid mediator may induce the survival of such cells in the CNS upon receptor ligation[22,23,53].Indeed,recent studies have demonstrated that FTY720-P promotes the survival of oligodendroglial lin-eage cells[25].A key consideration behind these in vitro results is the?nding that FTY720can distribute to the CNS[38],which contains sphingosine kinase-2[5]for ef?cient phosphorylation of FTY720in the DA rat brain(C.A.F.and Andreas Billich, submitted for publication).

Additional studies are necessary to further elucidate the molecular events that culminate in the remarkable ef?cacy observed with FTY720therapy,not only in experimental models of demyelination but more importantly in MS patients[26],who continue to bene?t from this novel oral agent.Key questions that remain to be addressed would include the effect of S1P receptor modulation on neuronal plasticity and regeneration,particularly involving remodeling of axonal connections,neurotrophin pro-?les,ion channel rearrangement,mitochondrial dysfunction and impaired electron transport[28,56].

Acknowledgements

We thank Hans Lassmann for reviewing this manuscript, Chris Linington for kindly providing the MOG plasmid,Marijke Nefzger for statistical consultation,Sam C.Wilkerson III for IT assistance,and Franz Hammerschmid for protein analysis.All experiments were supported by Novartis Pharma AG. References

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Sphingosine-1-phosphate,a novel lipid,involved in cellular proliferation, J.Cell Biol.114(1991)155–167.

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员会),负责对脐带血造血干细胞库设置的申请、验收和考评提出论证意见。专家委员会负责制订脐带血 造血干细胞库建设、操作、运行等技术标准。 第八条脐带血造血干细胞库设置的申请者除符合国家规划和布局要求,具备设置一般血站基本条件之外, 还需具备下列条件: (一)具有基本的血液学研究基础和造血干细胞研究能力; (二)具有符合储存不低于1 万份脐带血的高清洁度的空间和冷冻设备的设计规划; (三)具有血细胞生物学、HLA 配型、相关病原体检测、遗传学和冷冻生物学、专供脐带血处理等符合GMP、 GLP 标准的实验室、资料保存室; (四)具有流式细胞仪、程控冷冻仪、PCR 仪和细胞冷冻及相关检测及计算机网络管理等仪器设备; (五)具有独立开展实验血液学、免疫学、造血细胞培养、检测、HLA 配型、病原体检测、冷冻生物学、 管理、质量控制和监测、仪器操作、资料保管和共享等方面的技术、管理和服务人员; (六)具有安全可靠的脐带血来源保证; (七)具备多渠道筹集建设资金运转经费的能力。 第九条设置脐带血造血干细胞库应向所在地省级卫生行政部门提交设置可行性研究报告,内容包括:

艾灸之精髓

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艾灸的原理

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精神分裂症的发病原因是什么

精神分裂症的发病原因是什么 精神分裂症是一种精神病,对于我们的影响是很大的,如果不幸患上就要及时做好治疗,不然后果会很严重,无法进行正常的工作和生活,是一件很尴尬的事情。因此为了避免患上这样的疾病,我们就要做好预防,今天我们就请广州协佳的专家张可斌来介绍一下精神分裂症的发病原因。 精神分裂症是严重影响人们身体健康的一种疾病,这种疾病会让我们整体看起来不正常,会出现胡言乱语的情况,甚至还会出现幻想幻听,可见精神分裂症这种病的危害程度。 (1)精神刺激:人的心理与社会因素密切相关,个人与社会环境不相适应,就产生了精神刺激,精神刺激导致大脑功能紊乱,出现精神障碍。不管是令人愉快的良性刺激,还是使人痛苦的恶性刺激,超过一定的限度都会对人的心理造成影响。 (2)遗传因素:精神病中如精神分裂症、情感性精神障碍,家族中精神病的患病率明显高于一般普通人群,而且血缘关系愈近,发病机会愈高。此外,精神发育迟滞、癫痫性精神障碍的遗传性在发病因素中也占相当的比重。这也是精神病的病因之一。 (3)自身:在同样的环境中,承受同样的精神刺激,那些心理素质差、对精神刺激耐受力低的人易发病。通常情况下,性格内向、心胸狭窄、过分自尊的人,不与人交往、孤僻懒散的人受挫折后容易出现精神异常。 (4)躯体因素:感染、中毒、颅脑外伤、肿瘤、内分泌、代谢及营养障碍等均可导致精神障碍,。但应注意,精神障碍伴有的躯体因素,并不完全与精神症状直接相关,有些是由躯体因素直接引起的,有些则是以躯体因素只作为一种诱因而存在。 孕期感染。如果在怀孕期间,孕妇感染了某种病毒,病毒也传染给了胎儿的话,那么,胎儿出生长大后患上精神分裂症的可能性是极其的大。所以怀孕中的女性朋友要注意卫生,尽量不要接触病毒源。 上述就是关于精神分裂症的发病原因,想必大家都已经知道了吧。患上精神分裂症之后,大家也不必过于伤心,现在我国的医疗水平是足以让大家快速恢复过来的,所以说一定要保持良好的情绪。

基于单片机的自动存包系统设计

基于单片机的自动存包系统设计 摘要 近年来,随着生活水平的提高,人们对于社会消费品的质量和数量的要求也在逐渐增加。为了更好的为广大顾客服务,在一些商场、影院、超市等公共场合通常设置有自动存包柜,本次便是针对这一现象进行设计。 本文详细介绍了国内自动存包控制系统的发展现状,发展中所面临的问题。并详细介绍了本系统采用的AT89S52单片机做控制器,可以同时管理四个存包柜。柜门锁是由继电器控制,当顾客需要存包的时候,可以自行到存包柜前按“开门”键,需要顾客向光学指纹识别系统输入个指纹,然后通过继电器进行开门(用亮灯表示),顾客即可存包,并需将柜门关上。当顾客需要取包时,要将只要将之前输入的指纹放置于指纹识别器前方,指纹识别器采集到指纹信息输出相应的高低电平信号传给单片机,系统比较密码一致后,发出开箱信号至继电器将柜门打开,顾客即可将包取出。它具有功能实用、操作简便、安全可靠、抗干扰性强等特点。 关键词:自动存包柜,单片机,指纹识别器

李少鹏:基于单片机的自动存包系统设计 Based on single chip microcomputer automatic package design Abstract In recent years, with the improvement of living standards, people for social consumer goo ds quality and quantity requirements are to increase gradually. In order to better service for the g eneral customers, in some stores, movie theaters, supermarkets public Settings are to be put auto matically usually bag ark, it is functional practical, simple operation, safe and reliable, anti-jamm ing strong sexual characteristics. Domestic deposit automatic control system are introduced in detail in this paper the development of the status quo, problems faced in the development of. And introduces in detail the system adopts single chip microcomputer controller, can simultaneously manage multiple pack ark. Cupboard door lock controlled by relay, when customers need to save package, will be allowed to save package before the ark according to the "open" button, need customer to the system input fingerprint, and then through the relay to open the door (with lighting), customers can save package, and the cupboard door must be closed. When customers need to pick up package, as long as before the input fingerprint should be placed on the fingerprint recognizer, fingerprint recognizer collecting to the fingerprint information and output the corresponding high and low level signal to the microcontroller, the system is password consistent, signal out of the box to the relay Key words: Automatic Storage Bag, Microcontroller, Fingerprint recognizer。

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