Multiple neuroprotective mechanisms of minocycline in autoimmune CNS inflammation

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Neurobiology of Disease 25 (2007) 514–525
Multiple neuroprotective mechanisms of minocycline inautoimmune CNS inflammation

Katharina Maier,a,⁎ Doron Merkler,b Joachim Gerber,a Naimeh Taheri,a Antje V. Kuhnert,a

Sarah K. Williams,a Clemens Neusch,a Mathias Bähr,a and Ricarda Diema

aNeurologische Universitätsklinik, Robert-Koch-Strasse 40, D-37075 Göttingen, GermanybInstitut für Neuropathologie, Göttingen, Germany

Received 15 August 2006; revised 8 October 2006; accepted 29 October 2006Available online 18 January 2007

Axonal destruction and neuronal loss occur early during multiplesclerosis, an autoimmune inflammatory CNS disease that frequentlymanifests with acute optic neuritis. Available therapies mainly targetthe inflammatory component of the disease but fail to prevent neuro-degeneration. To investigate the effect of minocycline on the survival ofretinal ganglion cells (RGCs), the neurons that form the axons of theoptic nerve, we used a rat model of myelin oligodendrocyte glyco-protein (MOG)-induced experimental autoimmune encephalomyelitis.Optic neuritis in this model was diagnosed by recording visual evokedpotentials and RGC function was monitored by measuring electro-retinograms. Functional and histopathological data of RGCs andoptic nerves revealed neuronal and axonal protection when minocy-cline treatment was started on the day of immunization. Further-more, we demonstrate that minocycline-induced neuroprotection isrelated to a direct antagonism of multiple mechanisms leading toneuronal cell death such as the induction of anti-apoptotic intracel-lular signalling pathways and a decrease in glutamate excitotoxicity.From these observations, we conclude that minocycline exertsneuroprotective effects independent of its anti-inflammatory proper-ties. This hypothesis was confirmed in a non-inflammatory diseasemodel leading to degeneration of RGCs, the surgical transection of theoptic nerve.© 2007 Published by Elsevier Inc.

Keywords: Minocycline; Experimental autoimmune encephalomyelitis;Neuroprotection; Visual evoked potentials; Glutamate excitotoxicity

Introduction

Multiple sclerosis (MS) is a chronic inflammatory disease ofthe CNS and the most common disabling neurological disease inyoung adults (Noseworthy et al., 2000). The pathological

⁎ Corresponding author. Fax: +49 551 398405.E-mail address: [email protected] (K. Maier).Available online on ScienceDirect (www.sciencedirect.com).

0969-9961/$ - see front matter © 2007 Published by Elsevier Inc.doi:10.1016/j.nbd.2006.10.022

hallmarks are the destruction of myelin, axonal damage andcomplete axonal transection, even in the early stage of the disease(Ferguson et al., 1997; Trapp et al., 1998; Kornek et al., 2000). Inaddition to severe axonal pathology, apoptotic neuronal cell deathhas been described in demyelinated cerebral cortex lesions of MSpatients (Peterson et al., 2001) that might contribute to clinicaldisability as well as the development of progressive MS. Thecurrent therapeutic strategies for MS mainly target the immuno-logical aspect of the disease. These immunotherapeutic ap-proaches have been shown to be helpful, especially in therelapsing form of the illness, but nevertheless, many patients whoresponded well in the beginning continue to worsen over time.The effect of one of the standard immunotherapies, IFN-beta, ondevelopment of brain atrophy causes controversial discussion(Rovaris and Filippi, 2003; Hardmeier et al., 2005). Treatmentwith methylprednisolone, the standard therapy for acute MSrelapses, actually resulted in negative effects on neuronal survivalin a model of experimental autoimmune encephalomyelitis (EAE)by inhibiting an endogenous neuroprotective pathway (Diem etal., 2003). These findings suggest an urgent need to explore newtherapeutic strategies that prevent axonal loss and apoptoticneuronal cell death.

In the present study, we have investigated the effects ofminocycline on neuronal survival in myelin oligodendrocyteglycoprotein (MOG)-induced EAE. MOG-EAE is an establishedanimal model of MS closely representing the pathogenic lesionformation of this disease (Storch et al., 1998; Weissert et al., 1998;Stefferl et al., 1999). In this model, we have demonstrated thatinflammation of the optic nerve leads to acute degeneration ofoptic nerve axon fibers and consecutive apoptosis of retinalganglion cells (RGCs) (Meyer et al., 2001).

In previous EAE studies, minocycline has been shown tosuppress disease activity and progression by influencing theimmune response and inhibiting inflammatory cascades (Brundulaet al., 2002; Popovic et al., 2002). On the other hand, in a non-autoimmune model of demyelination, it has been shown thatsuppression of microglia/macrophage activation by minocycline

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may impair the process of remyelination (Li et al., 2005). However,an inflammation-independent aspect of action makes minocyclinean interesting drug for testing it in our EAE model: directneuroprotective effects have been described in non-inflammatorydisease models such as models of cerebral ischemia, traumaticbrain injury, Huntington’s and Parkinson’s disease, and amyo-trophic lateral sclerosis (Yrjanheikki et al., 1999; Chen et al., 2000;Du et al., 2001; Wu et al., 2002; Zhu et al., 2002). On the basis ofthese studies, we investigated the neuroprotective potential ofminocycline in an EAE model that especially reflects theneurodegenerative aspects of MS. In order to elucidate the relevantmechanisms of action and to differentiate between primary andsecondary neuroprotection, we combined electrophysiological andhistopathological methods with the investigation of intraneuronalsignalling cascades. Furthermore, we investigated the effect ofminocycline on neuronal survival in a primarily degenerativedisease model, the surgical transection of the optic nerve.

Materials and methods

Rats

Experiments were performed on 8 to 10 week old femaleBrown Norway (BN) rats obtained from Charles River (Sulzfeld,Germany). During the observation period, rats were kept underenvironmentally controlled conditions without the presence ofpathogens.

All experiments that involve animal use were performed incompliance with the relevant laws and institutional guidelines.These experiments have been approved by the local authorities ofBraunschweig, Germany.

Immunogen

rrMOGIgd, corresponding to the N-terminal sequence of ratMOG (amino acids 1–125), was expressed in Escherichia coli andpurified to homogeneity by chelate chromatography (Weissert etal., 1998). The purified protein in 6 M urea was then dialyzedagainst PBS to obtain a preparation that was stored at −20 °C.

Induction and evaluation of EAE

The induction of EAEwas performed as described by Diem et al.(2005). The animals were weighted daily and scored for clinicalsigns of EAE according to a score from 0 to 4 as described (Maier etal., 2004). Rats were followed until day 8 of the disease (end ofstudy). For each group, the clinical data are presented as acumulative score: the sum of all daily disease scores of all animalsper group was divided by the number of animals.

Retrograde labelling of RGCs

Adult BN rats were anaesthetized with an intraperitonealinjection of 10% ketamine (0.75 ml/kg; Atarost GmbH and Co.,Twistringen, Germany) together with 2% xylazine (0.4 ml/kg;Albrecht, Aulendorf, Germany), the skin was incised mediosagi-tally, and holes were drilled into the skull above each superiorcolliculus (6.8 mm dorsal and 2 mm lateral from bregma). Weinjected stereotactically 2 μl of the fluorescent dye Fluorogold (FG,5% in normal saline) (Fluorochrome Inc., Englewood, CO, USA)into both superior colliculi.

Electrophysiological recordings

Rats were anaesthetized by intraperitoneal injection of 10%ketamine (0.75 ml/kg; Atarost GmbH and Co., Twistringen,Germany) together with 2% xylazine (0.4 ml/kg; Albrecht,Aulendorf, Germany) and mounted on a stereotaxic device.Recordings of visual evoked potentials (VEPs) and electroretino-grams (ERG) were performed as described earlier (Meyer et al.,2001). VEPs and ERGs were recorded on the day of disease onsetand at day 8 of the disease.

Treatment of animals

Minocycline hydrochloride (Sigma, St. Louis, MO) wasdissolved in phosphate buffered saline (PBS) and administereddaily by intraperitoneal injections at a dosage of 50 mg/kg bodyweight. In our study, we used two different treatment protocols:One animal group (n=8) received minocycline from the day ofimmunization onwards until day 8 of MOG-EAE (“earlyminocycline”). In the “late minocycline” group (n=8), therapywas initiated at the day of disease onset and continued until day 8of MOG-EAE. The respective control group of animals (n=8)received 0.5 ml PBS given intraperitoneally from the day ofimmunization onwards. Pre-experiments with application ofvehicle over the different treatment periods (from immunizationor disease manifestation onwards) showed no differences concern-ing clinical outcome, electrophysiological or histopathologicaldata.

Detection of minocycline concentrations in serum andcerebrospinal fluid

At clinical onset of MOG-EAE, cerebrospinal fluid (CSF) andblood samples were taken 4 h after the last minocyclineapplication. Concentrations of minocycline in the samples weredetermined by the agar well diffusion technique using Mueller–Hinton agar with Bacillus subtilis (ATCC 6633, Difco, Detroit)spores. For serum and CSF samples, different standard curveswere constructed by using undiluted and 1:100-diluted drug-freerat serum. To avoid interassay variation, serum and CSF sampleswere measured in one assay. The measurements were repeatedtwice.

Intravitreal drug administration

In order to inhibit Akt phosphorylation or the phosphorylation ofmitogen-activated protein kinases (MAPKs) in RGCs, animals wereanesthetized with diethylether and received intravitreal injections ofwortmannin (WM; 0.1 mM, dissolved in 15% dimethylsulfoxide;Sigma, St. Louis, USA), or PD 98059 (2Vamino-3V-methoxyfla-vone, 2 μl of a 20 mM solution) (Calbiochem, San Diego, CA,USA). By means of a glass microelectrode, 2 μl of each substancewas injected into the vitreous space of each eye, puncturing the eyeat the cornea–sclera junction.

Quantification of RGC density

At the end of the second recording session, the rats received anoverdose of chloral hydrate and were perfused via the aorta with4% paraformaldehyde in PBS. The optic nerves and both eyes wereremoved, and the retinas were dissected and flat-mounted on glass

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slides. They were examined by fluorescence microscopy (Axiophot2, Zeiss, Göttingen, Germany) using an UV filter (365/397 nm),and RGC densities were determined by counting labelled cells inthree areas (62,500 μm2) per retinal quadrant at three differenteccentricities of 1/6, 3/6 and 5/6 of the retinal radius. Cell countswere performed by two independent investigators following a blindprotocol. To be able to correlate ERG and VEP data with retinalganglion cell counts and optic nerve histopathology, respectively,all these investigations were done in the same animals.

Histopathology

Histological evaluation was performed on paraformaldehyde-fixed, paraffin-embedded sections of optic nerves at day 8 ofMOG-EAE. Paraffin sections were stained with hematoxylin–eosin, Luxol fast blue, and Bielschowsky silver impregnation toassess inflammation, demyelination, and axonal pathology, respec-tively, as previously described (Storch et al., 1998). The degree ofinflammation was evaluated using a histological score described by(Maier et al., 2006). The determination of axonal densities anddemeylinated areas was also described earlier (Diem et al., 2005).The investigators who processed tissue sections and diagnosedoptic neuritis were blinded to the electrophysiological andimmunohistochemical results of the study.

Immunohistochemistry

Immunostaining was performed on cryostat retinal sections(18 μm thick) at day 1 of MOG-EAE after vehicle or minocyclinetreatment. Processing and staining of the control- and theminocycline-treated sections was performed simultaneously toavoid differences in labelling intensity. Antibodies were used at adilution of 1:1000 to detect the following excitatory amino acidtransporters (EAATs): the L-glutamate/L-aspartate transporter(GLAST) and the excitatory amino acid carrier-1 (EAAC1). Adilution of 1:100 was used for the detection of the glutamatetransporter-1 (GLT-1) (Chemicon, Hofheim, Germany). Antibodieswere diluted in 1% normal goat serum.

Sections were preincubated with 10% normal goat serum for1 h at room temperature to block unspecific binding of the antisera.This was followed by application of the primary antibodies(overnight, 4 °C) and goat anti-rabbit or goat anti-mouse IgG-conjugated Alexa Fluor® 488 in 10% normal goat serum for 1 h atroom temperature (MoBiTec, Göttingen, Germany). Retinalsections which were incubated in absence of the primary antibodiesserved as negative controls.

Western blot analysis

The Western blot analysis on retinal lysates was performed asdescribed elsewhere (Diem et al., 2003). After incubation with theprimary antibody against phospho-p44-phospho-p42 MAPKs(Thr202/Tyr204, New England Biolabs GmbH, Schwalbach,Germany; 1:250 in 1% skimmed milk in 0.1% Tween 20 inphosphate-buffered saline (TBS-T)) or against p44-p42 MAPKs(sc-93-G; Santa Cruz Biotechnology; 1:500 in 1% skimmed milkin TBS-T), membranes were washed in TBS-T and incubated withhorseradish peroxidase (HRP)-conjugated secondary antibodiesagainst goat IgG (Santa Cruz Biotechnology; 1:3000 in TBS-T).Labelled proteins were detected using the ECL-plus reagent(Amersham, Arlington Heights, IL, USA).

For Western blot analysis of Bcl-2 and Bax levels, the primaryantibody (Santa Cruz Biotechnology, Inc., California, USA) wasdiluted 1:200 in 5% skimmed milk in TBS-T. For protein detection,a HRP-conjugated secondary antibody against mouse or rabbit IgGwas used (Santa Cruz Biotechnology Inc., Santa Cruz, CA; 1:2000in 1% skimmed milk in TBS-T).

After incubation with the primary antibody against phospho-Akt(pAkt) (New England Biolabs GmbH, Schwalbach, Germany;1:1000 in 5% BSA in TBS-T), or Akt (New England BiolabsGmbH, Schwalbach, Germany; 1:1000 in 1% skimmed milk inTBS-T), membranes were washed in TBS-T and incubated withHRP-conjugated secondary antibodies against rabbit IgG (SantaCruz Biotechnology Inc., Santa Cruz, CA, USA; 1:2500 in 1%skimmed milk).

For Western blot analysis of β-tubulin as a housekeepingprotein, the primary antibody (Sigma-Aldrich, Germany) wasdiluted 1:1000 in TBS-T. Secondary antibody against mouse IgG(Santa Cruz Biotechnology Inc., Santa Cruz, CA; 1:2000 in 5%skimmed milk) was applied.

For detection of glutamate transporters, membranes wereincubated with antibodies against GLT-1, GLAST, or EAAC1(Chemicon International Inc, Temecula, CA, USA) at a dilutionof 1:1000 in 5% skimmed milk. After the washing step,secondary antibodies against rabbit, guinea pig, or mouse IgG(Santa Cruz Biotechnology Inc., Santa Cruz, CA; 1:2000 in 1%skimmed milk) were applied. For Western blot analysis of β-tubulin as a housekeeping protein, the primary antibody(Sigma-Aldrich, Germany) was diluted 1:1000 in TBS-T.Secondary antibody against mouse IgG (Santa Cruz Biotech-nology Inc., Santa Cruz, CA; 1:2000 in 5% skimmed milk) wasapplied.

Glutamate analyses

Retinal glutamate concentrations were measured at day 7 postimmunization, at the day of disease onset as well as at day 8 ofMOG-EAE. For that purpose, animals were sacrificed 4 h after thelast injection of minocycline or PBS. Ten eyes were included ineach group. The tissue was processed as described previously(Lund, 1986). Spectrophotometric measurements of L-glutamatevia enzymatic dehydrogenation with conversion of NAD+ toNADH were performed using the glutamate determination kit(Sigma-Aldrich Corporation, St. Louis, Missouri, USA). Thesamples of the different animal groups were processed simulta-neously and a triplicate testing was performed.

Unilateral optic nerve transection

Surgical procedures have been described elsewhere (Klöcker etal., 1997; Kermer et al., 1998). Animals received daily intraper-itoneal injections of minocycline 50 mg/kg body weight or PBS for14 days following optic nerve transection. Both groups contained 6rats each. At the end of the experiment, retinas were processed asdescribed previously (Kermer et al., 1998) and RGCs were countedby two independent investigators.

Statistical analysis

Statistical comparisons were made by one-way ANOVAfollowed by Duncan’s test. The Mann–Whitney test was used forstatistical analysis of the electrophysiological findings.

Fig. 2. Minocycline sufficiently penetrates into the CSF. CSF and serumconcentrations given as mean±S.E.M. were measured in six differentanimals. Filled inside bars represent minocycline concentrations measured inthe CSF whereas the serum concentration in the respective animal is given asan open white bar.


Results

Minocycline treatment delays disease onset and suppresses activityof MOG-EAE

Daily treatment with minocycline started at the day ofimmunization delayed the disease onset and reduced the severityof symptoms. The rationale for this early treatment lies in ourvery recent observation that significant RGC death started at day7 after immunization corresponding to a time point at least1 week before clinical onset of the disease (Hobom et al., 2004).In this group, disease onset was at day 22.6±3.3 and at day15.9±4.3 in the respective control group (mean±S.E.M.;p<0.05). The mean clinical score at day 1 of MOG-EAE was0.5 under daily minocycline applications in the early treatedgroup whereas vehicle-treated animals showed a mean clinicalscore of 0.8±0.3 (Fig. 1). Further during the disease course,none of the early minocycline-treated animals progressed beyondgrade 0.5 of the disease. In the control group at day 8 of MOG-EAE in contrast, 3 out of 8 animals showed hindleg paresis(grade 2.5), 4 out of 8 animals developed hindleg paralyses(grade 3), and one animal had a lethal course of the disease(grade 4) (mean clinical score: 0.5 for early minocycline-treatment vs. 2.9±0.2 for the vehicle-treated controls; mean±S.E.M.; n=8; p<0.05; Fig. 1). With the second set of experiments,we investigated whether minocycline also has beneficial effectson the disease course if therapy was started at the day when thefirst neurological symptoms occurred (“late-treatment” group). Inthis group, disease onset was around day 14 post immunizationand comparable to that of the vehicle-treated animals. However,we observed that within 3 days of the start of minocyclinetreatment, the neurological deficits stabilized and the mean scoreat day 8 of MOG-EAE was significantly lower in comparison tothe vehicle-treated controls (1.4±0.4 vs. 2.9±0.2; mean±S.E.M.,n=8, p<0.05; Fig. 1).

Fig. 1. Daily intraperitoneal application of minocycline started atimmunization delays disease onset and suppresses disease activity ofMOG-EAE. Mean clinical score under treatment with minocycline from theday of immunization (early mino) or disease onset (late mino) onwardscompared with vehicle-treated animals (vehicle). Data represent mean±S.E.M. of the daily score. The “early minocycline”-treated group is indicated bysquares whereas the mean score of the “late minocycline”-treated animals isrepresented by triangles. Diamonds show the daily clinical score of thecontrol group.

Detection of minocycline in the serum and cerebrospinal fluid

To ensure that minocycline sufficiently crossed the blood–brainbarrier in our animals, we measured its CSF and serumconcentrations using agars containing B. subtilis. Due to thepossibility of a delayed absorption into the blood after intraper-itoneal application, serum and CSF samples were taken 4 h afterthe last injection of minocycline. At this time, the mean serumlevel was 8.5±0.2 μg/ml (mean±S.E.M.; n=6). In the CSF, amean minocycline concentration of 0.5±0.03 g/ml (n=6) wasdetected (Fig. 2).

Improved electrophysiological function of the optic system underminocycline treatment

In order to examine the effect of minocycline on visualfunctions in the different rat groups, we performed recordings ofVEPs and ERGs in response to flash and pattern stimulation. Todiagnose optic neuritis, the first measurement was done at theday of disease onset. The second, follow-up recording wasperformed on day 8 of MOG-EAE. VEP flash stimulation wasused to test the axonal signalling of the optic nervecorresponding to the animal’s ability to discriminate betweenlight and dark. Pattern VEP recordings were performed toestimate the animal’s visual acuity. ERG measurements inresponse to flash stimulation indicate an intact function of thewhole retina, whereas ERG potentials induced by patternstimulation are a specific electrophysiological marker for RGCs.A strong correlation between the numbers of surviving retinalganglion cells and the responses to ERG pattern stimulation wasshown previously (Meyer et al., 2001). Further, we havedemonstrated that healthy control rats were able to discriminatea pattern of at least 24 to 36 alternating bars corresponding tovisual acuity values of 1.31±0.16 cycles per degree determinedby VEP recordings and 1.10±0.13 cycles per degree in the ERGmeasurements (Meyer et al., 2001).

Whereas all animals in this study responded to VEP flashstimulation at day 1 of MOG-EAE, visual acuity determined bypattern stimulation was clearly decreased in the vehicle-treatedgroup of rats as well as in the “late” minocycline-treated group.

Table 1Results of VEP and ERG recordings obtained at days 1 and 8 of MOG-EAE in vehicle- and minocycline-treated animals

VEP day 1flash

VEP day 8flash

VEP day 1pattern

VEP day 8pattern

ERG day 1flash

ERG day 8flash

ERG day 1pattern

ERG day 8pattern

Vehicle 8/8 4/8 0.3±0.11 cyc/° 0.22±0.06 cyc/° 8/8 2/8 0.21±0.01 cyc/° –Early mino 8/8 7/8 0.63±0.11 cyc/° 0.6±0.11 cyc/° 8/8 7/8 0.6±0.12 cyc/° 0.51±0.1 cyc/°Late mino 8/8 7/8 0.36±0.06 cyc/° 0.33±0.08 cyc/° 8/8 4/8 0.18±0.06 cyc/° 0.13 cyc/°Control: CFA-immunized 8/8

(day 18 p.i.)1.31±0.16 cyc/°(day 18 p.i.)

8/8(day 18 p.i.)

1.10±0.13 cyc/°(day 18 p.i.)

Treatment was started either at the day of immunization (early mino) or at the day of disease onset (late mino). Each group contained eight tested eyes. Thenumber of eyes with detectable potentials to flash stimulation is given as x out of eight tested eyes (x/8). The VEP and ERG responses to pattern stimulation aregiven as visual acuity values calculated from VEP and ERG amplitudes and the spatial frequency of the pattern stimulation. Control rats immunized with CFAwere measured on day 18 after immunization (p.i.) as described by Meyer et al. (2001).

Fig. 3. Minocycline treatment started at day of immunization improves ERGresponses during autoimmune optic neuritis. (a) Example of ERG potentialsat day 8 of MOG-EAE evoked by stimulation with 6 alternating bars in ananimal treated with minocycline. (b) Under the same recording conditions,only background noise levels of electrical activity were seen in an animalfrom the vehicle-treated group. The stimulating pattern is indicated on top ofthe recording sequences.


At clinical onset of MOG-EAE, the mean of the discernablealternating bars with detectable VEP responses was located inthe range of 6–12 bars in both of these groups. Thiscorresponds to visual acuity values of 0.3±0.11 cycles perdegree (“vehicle”), and 0.36±0.06 cycles per degree (“lateminocycline”) (mean±S.E.M.; Table 1). In contrast, rats whichwere treated with minocycline according to the early treatmentprotocol performed significantly better and were able todiscriminate 12–24 bars at this time point (visual acuity of0.63±0.11 cycles per degree, p<0.05; Table 1). During thefurther disease course, visual acuity in both animal groupstreated with minocycline remained stable (early minocycline:0.6±0.11 cycles per degree at day 8 of MOG-EAE; p<0.05when compared to vehicle; late minocycline: 0.33±0.08 cyclesper degree; p<0.05 when compared to controls). In contrast,the vehicle-treated animals showed a further progression ofvisual loss until day 8 of the disease (0.22±0.06 cycles perdegree).

To investigate the effect of minocycline on the function ofRGCs, we performed ERG recordings in the same animals. PatternERG is a specific electrophysiological marker for RGCs whereasflash ERG represents the function of all electrically active cells inthe retina (Meyer et al., 2001). At both time points (days 1 and 8of manifest EAE), most of the animals (92 out of 96 tested eyes)showed clear responses to flash ERG stimulation, indicating intactfunction of the entire retina. However, at the day of disease onsetas well as at day 8 of MOG-EAE, rats treated according to the“early minocycline” protocol showed significantly better ERGresults following pattern stimulation when compared to thevehicle-treated control group (visual acuity of 0.6±0.12 cyclesper degree vs. 0.21± 0.01 cycles per degree at day 1 of EAE;p<0.05; Table 1). At day 8 of the disease, visual acuity in the“early minocycline”-treated group was still 0.51±0.1 cycles perdegree (Table 1) whereas in the control group, none of the animalswas able to produce specific potentials in response to any kind ofalternating bars. Fig. 3a gives an example of ERG potentials inresponse to 6 alternating bars in a rat treated according to the“early minocycline” protocol. Fig. 3b, in contrast, shows onlybackground noise levels of electrical activity in a control animal atday 8 of MOG-EAE. As expected, ERG results in the “lateminocycline”-treated group at day 1 of MOG-EAE (visual acuityof 0.18±0.06 cycles per degree) did not differ from those of thecontrol animals. At day 8 of the disease, 2 out of 8 measured eyesin that group still responded to pattern ERG stimulation incomparison to no detectable ERG response at this time-point in thecontrol group of rats (Table 1).

Minocycline rescues RGCs during acute optic neuritis

Based on our above given electrophysiological results showinga detectable functional benefit in the ERG response after earlyminocycline treatment, we here investigated the effect ofminocycline on the survival of RGCs during acute optic neuritis.On day 8 of MOG-EAE, we compared the numbers of RGCsretrogradely labelled with FG in rats treated with minocycline withthose of the vehicle-treated animals. Previously, we have shownthat in healthy BN rats sham-immunized with CFA, 2730±145RGCs/mm2 were detectable (mean±S.E.M.; n=8) (Meyer et al.,2001). In our present study, the mean cell density in vehicle-treatedanimals at day 8 of MOG-EAE dropped to 797.1±84.2 (mean±S.E.M.; Fig. 4e). Simultaneously with decreasing numbers ofsurviving RGCs, we observed a predominance of microglia cellsin these retinas (Fig. 4b). In contrast, RGC counts of the earlyminocycline-treated group were significantly increased to 1488±135.3 (p<0.005; Figs. 4a and e). If minocycline treatment wasstarted at disease onset, the mean cell density was higher incomparison with the vehicle-treated animals, however, thisdifference was not statistically significant (990.0±161.2 vs.797.1±84.2 RGCs/mm2; Fig. 4e). In general, RGC counts inthese different rat groups were in good agreement with their abovedescribed responses to ERG pattern stimulation.

Fig. 5. Intracellular signal transduction cascades influenced by earlyminocycline treatment. In each column, Western blot analyses of differentproteins using the same retinal protein lysates are shown. (a) Note theincrease of phospho-Akt (pAkt) and phospho-MAPK (pMAPK) levelsunder minocycline treatment. The unphosphorylated forms of these proteinsare similar in each group. (b) Bcl-2 levels are increased under minocyclinetherapy whereas the expression of Bax is decreased.

Fig. 4. Daily treatment with minocycline started at immunizationsignificantly increases survival of RGCs during acute optic neuritis. (a)Representative whole mount area at 3/6 retinal radius from a minocycline-treated animal obtained at day 8 of MOG-EAE. An example of a FG-labelledRGC is indicated by the arrow. (b) The number of FG-labelled RGCs in avehicle-treated animal at day 8 of MOG-EAE is decreased when comparedto minocycline treatment. Note the predominance of microglia cells in thisretina (arrows). (c) Combined treatment with minocycline and PD 98059reduces the neuroprotective effect. d) The neuroprotective effect of dailyminocycline application is partially blocked by additional treatment withwortmannin. Scale bar, 100 μm. e) Data are given as the mean±S.E.M. ofretrogradely labelled RGCs/mm2 at day 8 of MOG-EAE. veh, vehicletreatment; late mino, minocycline treatment started at day of disease onset;early mino, minocycline treatment started at immunization; early mino+PD,combined treatment of minocycline and PD; early mino+WM, combinedtreatment of minocycline and wortmannin. * Statistically significant whencompared with the early minocycline group (p<0.05); ** statisticallysignificant when compared with vehicle-treated controls (p<0.01; one-wayANOVA followed by Duncan's test).


Minocycline activates three distinct intracellular anti-apoptoticpathways in RGCs

Recently, we observed a down-regulation of phospho-Akt(pAkt), a downstream component of the phosphatidylinositol-3kinase (PI3-K) signalling pathway, as well as a shift of two Bcl-2family members, Bax and Bcl-2, towards a pro-apoptotic ratio inRGCs during MOG-EAE (Hobom et al., 2004). We have alsoshown that a rescue of RGCs could be achieved under therapies

that increase the phosphorylation of Akt (Diem et al., 2001; Sättleret al., 2004). Phosphorylation of MAPKs and an increase of theBcl-2 concentration in RGCs also led to a better survival of theseneurons in our MOG-EAE model (Maier et al., 2004; Diem et al.,2005). To determine whether minocycline influences neuroprotec-tive signal transduction steps in RGCs, we performed Westernblots of total and phosphorylated MAPKs as well as of the inactiveand active form of Akt. As shown in Fig. 5a, “early minocycline”increased the phosphorylation of MAPKs and Akt in RGCs, iftherapy was started at the day of immunization. Furthermore, weobserved an up-regulation of the anti-apoptotic protein Bcl-2 and asimultaneous decrease in the expression of the pro-apoptoticprotein Bax (Fig. 5b).

We tested the functional relevance of this MAPK phosphoryla-tion for RGC survival by combining “early minocycline” withapplication of PD 98059, an inhibitor of the single upstreamMAPK kinase (MEK) (Kultz and Burg, 1998). The functionalsignificance of the Akt phosphorylation in this context was testedby using a combination of minocycline and WM, a specificinhibitor of PI3-K. Under combined treatment of minocycline andPD 98059, the mean cell density at day 8 of the diseasesignificantly decreased to 994.2±104.2 (mean±S.E.M., n=12eyes; p<0.05; Figs. 4c, e) when compared to the application ofminocycline alone (1488±135.3 RGCs/mm2). Injection of WM inaddition to minocycline treatment (n=12 eyes) also led tosignificantly decreased RGC counts at day 8 of MOG-EAE:903.5 ± 61.5 vs. 1488 ± 135.3 RGCs/mm2 (mean ± S.E.M.;p<0.005; Figs. 4d, e). Treatment with PD 98059 or WM alone(n=12 eyes each) did not alter the numbers of RGCs at day 8 ofMOG-EAE (PD: 597.8±104.7; WM: 620.4±88.1 RGCs/mm2)when compared to control counts (797.1±84.19).

Fig. 7. Up-regulation of glutamate transporters under minocycline therapy.Immunohistochemical stainings of vertical cryostat sections for differentglutamate transporters. GCL, ganglion cell layer; IPL, inner plexiform layer;


Reduced retinal concentration of glutamate under minocyclinetherapy

In order to determine whether minocycline has any influenceon glutamate production or metabolism during autoimmuneoptic neuritis, we investigated retinal concentrations of glutamateafter “early minocycline” or vehicle treatment. In retinas ofhealthy, sham-immunized animals, the mean glutamate concen-tration was in the range of 56.5±10.8 μmol/L (mean±S.E.M.;n=10 eyes). During induction of optic neuritis, glutamateconcentrations increased over time. At day 7 after immunization,however, the mean glutamate level was significantly lowerunder minocycline treatment in comparison to vehicle-treatedanimals (92.5±11 μmol/L vs. 257.5±33 μmol/L; p<0.05; Fig.6). In both groups, the concentration peaked at the day ofdisease onset but still stayed significantly lower under mino-cycline treatment (177.5±12.5 μmol/L vs. 384±22.5 μmol/L;p<0.05; Fig. 6). At day 8 of MOG-EAE, the mean glutamateconcentration declined to 141±39.5 μmol/L in minocycline-treatedanimals and to 275±60.5 μmol/L in vehicle-treated controls.

Expression of glutamate transporters in the retina

Based on the above given results showing decreased retinalglutamate concentrations in the minocycline-treated rat group, weexamined the expression of glutamate transporters after minocy-cline and vehicle treatment. From the different excitatory aminoacid transporters (EAATs) known to be involved in glutamatemetabolism of which there are known to be at least five, GLAST,GLT-1 and EAAC1 have been identified as playing a major rolein transmitter clearance in the retina (Danbolt, 2001). Weperformed immunostainings to localize GLAST, GLT-1 andEAAC1 in the different retinal layers and Western blotting toquantify and compare the expression levels. In our study, GLASTstaining was seen in astroglial elements corresponding to Müllercells, particularly labelling the outer and inner limiting membraneof the retinas (Figs. 7a, b). GLT-1 immunoreactivity wasprominent in the inner plexiform layer (IPL) of all stained

Fig. 6. Minocycline treatment significantly decreases retinal glutamatelevels. Glutamate concentrations (mean±S.E.M.) were measured in the ratretina at day 7 after immunization (d7pi), at the day of disease onset(EAEd1), and at day 8 of clinically manifest EAE (EAEd8). * Significantwhen compared to the respective glutamate concentration of the vehicle-treated control group (p<0.05).

INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclearlayer; OLM, outer limiting membrane. a, b) GLAST-1 immunoreactivity isfound throughout the retina and is more prominent in the minocycline-treated animal (a) in comparison to the vehicle-treated control (b). c, d)Immunofluorescence for GLT-1 is concentrated in the IPL and appearsstronger in the minocycline-treated retina (c) when compared to the vehicle-treated one (d). e, f) EAAC1 immunofluorescence was found in the IPL.Under minocycline therapy, staining is more intense (e) in comparison to theapplication of vehicle (f). Scale bar 50 μm. g) Western blot analysis of thedifferent glutamate transporters. Note the up-regulation of all three proteinsunder minocycline treatment.

sections (Figs. 7c, d). EAAC1 expression was predominantlyfound in the inner plexiform layer (IPL) (Figs. 7e, f). Westernblot analysis of the different glutamate transporters revealed anincreased expression of GLAST, GLT-1 and EAAC1 in theminocycline-treated rat group in comparison to the vehicle-treatedanimals (Fig. 7g).

Minocycline decreases the severity of optic neuritis

To correlate the VEP results with the histopathological data ofthe optic nerves, we assessed axonal density, demyelination and


inflammation. Optic nerve cross sections were stained withBielschowsky silver impregnation, Luxol-fast blue and haematox-ylin–eosin. Representative optic nerve sections of the differenttreatment groups are given in Fig. 8. In accordance with ourelectrophysiological findings, Bielschowsky silver impregnationrevealed significantly higher axon counts on day 8 of MOG-EAEin the “early minocycline” group (54.2±5.9 axons/mm2; mean±S.E.M.) when compared with vehicle-treated animals (13.3±2.0axons/mm2; p<0.005) or with the rats treated according to the “lateminocycline” protocol (24.6±6.1 axons/mm2; p<0.05). Further-more, the extent of myelin damage determined as the percentage ofthe demyelinated area with respect to the whole optic nerve cross-section revealed a pronounced myelin preservation in the “earlyminocycline” group (9.8±5.0%; mean±S.E.M.; p<0.0001) incomparison to the vehicle-treated animals (75.7±9.4%). Accord-ingly, the extent of inflammation after “early minocycline”treatment was significantly decreased in comparison to that invehicle-treated animals (infiltration score of 0.9±0.6 vs. 2.6±0.4;mean±S.E.M.; p<0.05).

Fig. 8. Histopathological analysis of representative transverse optic nerve sectioapplication of minocycline started at the day of disease onset (late mino), (i–l) dailyHematoxylin–eosin staining shows extensive cellular infiltration in the vehicle- andreduced in the early minocycline group (i). The optic nerve stained with Luxol-fast(f). Pronounced myelin preservation (blue) was detected in the early minocycline grwithin the optic nerve of an early minocycline-treated rat (k) when compared to themagnification of Bielschowsky silver stained axons. Blue arrows indicate remainin

The animal group which received minocycline from the day ofdisease onset onwards (“late minocycline”) showed a trendtowards a better histopathological outcome which was, however,not statistically significant when compared with the vehicle-treated controls (24.6±6.1 axons/mm2; 50.5±12% demyelination;inflammatory infiltration score of 1.7±0.5; Fig. 8).

Minocycline increases RGC survival after surgical transection ofthe optic nerve

In order to investigate the effects of minocycline in a non-inflammatory disease model leading to apoptotic cell death ofRGCs, we performed optic nerve transections. Surgical axotomy ofthe optic nerve is a well established in vivo model to investigatesecondary neuronal cell loss. In this model, a delayed death of 80–90% of RGCs occurs within 14 days (Villegas-Perez et al., 1993;Klöcker et al., 1998). In the present study, RGC counts at day 14after axotomy were significantly increased after daily intraper-itoneal application of minocycline when compared to vehicle-

ns obtained at day 8 of MOG-EAE. (a–d) Vehicle treatment, (e–h) dailyapplication of minocycline started at the day of immunization (early mino).late minocycline-treated optic nerve (a, e), whereas cellular infiltrates wereblue appears demyelinated (purple) after vehicle (b) and late mino treatmentoup (j). Bielschowsky silver impregnation shows marked axonal preservationone of the vehicle- and late minocycline-treated animal (c, g). (d, h, l) Higherg axons. Scale bars: 100 μm (a, b, e, f, i, j); 50 μm (c, g, k); 20 μm (d, h, i).


treated animals (610.7±31.4 vs. 346.5±16.4 RGCs/mm2; mean±S.E.M.; p<0.0001).

Discussion

Current MS therapies are only partially effective. Despite anti-inflammatory, immunosuppressive and immunomodulatory ap-proaches, neurodegeneration and consecutive disease progressioncannot be prevented in many patients (Trapp et al., 1998, 1999;Noseworthy et al., 1999). In the present study, we used a modelof MOG-induced optic neuritis which is characterized by acuteaxonal damage within the optic nerve and secondary apoptosis ofRGCs, the neurons that form its axons. As revealed byelectrophysiological and histopathological data from retinas andoptic nerves, we demonstrate that early minocycline treatment isbeneficial in this autoimmune disease model. Recently, agentssuch as statins or erythropoietin, which are already approved forthe treatment of non-autoimmune diseases, have been tested inboth animal models of MS and in clinical MS trials (Aktas et al.,2003; Sättler et al., 2004; Vollmer et al., 2004; Sättler et al.,2005). These substances showed either anti-inflammatory orneuroprotective effects and, therefore, have to be combined withcomplementary treatment approaches (Diem et al., 2005). Wehere show that minocycline, in contrast, reduces both the severityof optic neuritis and exerts direct neuroprotective effects. Thisdirect neuroprotection was confirmed by the survival promotingeffect of minocyline on RGCs we additionally observed in a non-inflammatory lesion model of the optic nerve. As the underlyingmechanisms of neuroprotection during optic neuritis we identi-fied an activation of intraneuronal cascades described in contextwith neurotrophin-dependent signalling as well as an up-regulation of glutamate transporters with consecutive reductionof excitotoxicity.

Glutamate excitotoxicity is an important factor that contributesto acute neuronal damage under several pathophysiologicalconditions (Beal, 1995; Dirnagl et al., 1999; Lee et al., 1999;Smith et al., 2000; Darman et al., 2004). In animal models of MSand MS patient studies glutamate concentrations have beenshown to be increased which leads to demyelination and acuteaxonal damage (Pitt et al., 2000; Srinivasan et al., 2005).Glutamate-induced cell death of RGCs, the neurons investigatedin our study, has been described after crush injury of the opticnerve (Sautter and Sabel, 1993; Yoles and Schwartz, 1998) andshowed a correlation with detectable glutamate concentrations inthe vitreous body (Vorwerk et al., 2004). As revealed byglutamate assays, the retinal concentration of the excitatoryneurotransmitter in our model strongly increased during theinduction of EAE and reached a peak concentration at the day ofdisease onset. In accordance with the findings of activatedmacrophages and microglia as the sources of increased glutamatelevel (Piani et al., 1991), the time of the maximum glutamateconcentration in our study correlated well with the time of thehighest amount of inflammatory infiltration within the optic nerveand the time course of RGC apoptosis (Meyer et al., 2001;Hobom et al., 2004).

Minocycline significantly decreased glutamate concentrationsin the retina at day 7 after immunization as well as at day 1 ofMOG-EAE in our study. The reduced excitotoxicity afterapplication of minocycline under in vitro conditions was explainedby its ability to inhibit microglial activation and to enhance glialglutamate transport (Tikka et al., 2002; Darman et al., 2004). In

order to differentiate whether the minocycline-induced reduction ofglutamate in our model was only a secondary phenomenonresulting from less severe optic neuritis under this therapy or apartially inflammation-independent effect, we investigated theexpression of retinal glutamate transporters. In non-inflammatorydisease models, a significant role for EAATs in maintaining normalglutamate levels has been demonstrated (Rothstein et al., 1995;Scott et al., 1995; Vorwerk et al., 2000). As revealed by Westernblot analysis in our study, minocycline treatment induced an up-regulation of GLAST, GLT-1 and EAAC1 indicating drug effectson glutamate homeostasis which can not be directly explained byits anti-inflammatory properties but rather reflects direct action ofminocycline on astroglial cells.

In addition to the influence of minocycline on glutamatetoxicity, anti-apoptotic effects of minocycline mediated throughseveral mechanisms at the mitochondrial level such as inhibitionof cytochrome c release or up-regulation of Bcl-2, were reportedfrom studies in models of Huntington’s disease and amyotrophiclateral sclerosis (Zhu et al., 2002; Wang et al., 2003). In ourmodel of MOG-induced optic neuritis, it has been shown that Bcl-2 and Bax, an anti- as well as a pro-apoptotic member of the Bcl-2 family, are involved in the early and severe cell death of RGCs:simultaneous with a down-regulation of Bcl-2, an increase in theamount of Bax in RGCs precedes the apoptotic cell death of theseneurons (Hobom et al., 2004). Apoptosis of RGCs aftermechanical lesion of the optic nerve was accompanied by areciprocal regulation of Bax and Bcl-2 as well (Isenmann et al.,1997). As revealed by Western blot analysis in our present study,minocycline acts on these two crucial members of the Bcl-2family by inducing a shift towards the anti-apoptotic side: theexpression of Bax was decreased under minocycline treatmentwhereas the Bcl-2 expression in RGCs was up-regulated. Since ithas been demonstrated that glutamate-induced toxicity can resultin a down-regulation of Bcl-2 and an increased expression of Baxin the affected neurons (Zhong et al., 1993; Wang et al., 1997;Schelman et al., 2004), the minocycline-induced shift in the Bcl-2family observed in our study might be secondary to its loweringeffect on retinal glutamate concentration. In accordance with thishypothesis, subtoxic amounts of glutamate have been shown toprotect neurons by increasing the expression of Bcl-2 as well asactivating the MAPK pathway via stimulating the secretion ofbrain-derived neurotrophic factor (Zhu et al., 2005). On the otherhand, there are controversies concerning the relevance ofglutamate toxicity for RGC degeneration in the non-inflammatorydisease model used in our present study (Russelakis-Carneiro etal., 1996; Kermer et al., 2001). After surgical transection of theoptic nerve, excitotoxicity might not play a major role for RGCdegeneration indicating a minocycline-induced activation of anti-apoptotic pathways within RGCs through mechanisms that do notdepend on glutamate metabolism. Additionally, minocyclinemight protect RGCs by inhibition of microglia activation afteraxotomy of the optic nerve as suggested by a recent study(Baptiste et al., 2005).

The functional relevance of the activation of MAPKs underminocycline treatment in our optic neuritis model was shown by acombined application of minocycline together with PD98059, aninhibitor of MEK, which phosphorylates and thereby activatesMAPKs. Under this combined therapy, the protective effect ofminocycline on RGCs was clearly decreased. Similar to the MAPKactivation in RGCs, minocycline led to an increased phosphoryla-tion of Akt. This effect might also be a consequence of the reduced


excitotoxicity under minocycline treatment as glutamate wasreported to decrease the phosphorylation of Akt in cerebellargranule neurons (Chalecka-Franaszek and Chuang, 1999). Thecombined treatment of minocycline and WM, the naturallyoccurring inhibitor of PI3-K (Alessi et al., 1996), in our presentstudy revealed the functional significance of the regulation of thispathway for RGC survival.

For most antibacterials, lipophilicity, molecular weight andserum protein binding determine the drug entry into the CSF(Norrby and Jonsson, 1985; Nau et al., 1994). Minocycline is asmall, highly lipophilic molecule capable of crossing the blood–brain barrier (Brogden et al., 1975). Intravenous administration ofminocycline results in a more predictable plasma concentrationthan that achieved by intraperitoneal injection (Fagan et al.,2004). Nevertheless, CSF and serum concentrations measuredafter intraperitoneal application of the drug in our study showedonly a small variation in all tested animals. In our model, whichrequires chronic neuroprotection, we chose daily intraperitonealdrug application. Two different treatment protocols werecompared giving minocycline either from the day of immuniza-tion or from disease manifestation onwards. The treatment did nothave any significant effect on neuronal survival when it wasstarted at disease onset of EAE. This observation can beexplained by our previous findings showing “pre-clinical”apoptotic RGC death which starts already 1 week beforemanifestation of neurological symptoms (Hobom et al., 2004).Overall, because of the direct effect of minocycline on immunedeviation and its suppression of inflammatory cascades (Brundulaet al., 2002; Popovic et al., 2002), treatment with the druginitiated at the onset of clinical signs can still attenuate thedisease course and stabilize the visual function as shown in ourstudy. Transferring our results to the human disease where thestarting point of neurodegeneration cannot be determined, thesefindings indicate the importance of both an early neuroprotectivetreatment and its extension over the time period of acuteneurological deterioration.

In summary, we demonstrate that minocycline acts beneficiallyon neurodegenerative aspects of a rat model of autoimmune opticneuritis. This minocycline-induced neuroprotection is related to adirect antagonism of multiple mechanisms leading to neuronalapoptosis. Additionally, minocycline suppresses autoimmuneinflammatory processes and thereby might exert secondaryneuroprotective effects as well. This dual mode of action makesminocycline, an already approved, safe and well tolerated drug,interesting for the therapy of MS patients. The clinical relevance ofthis treatment is corroborated by the recent observation thatinflammatory activity within the brain of MS patients was reducedafter 2 months of the therapy (Metz et al., 2004). Asneurodegeneration in the early stages of MS is difficult to detect,animal studies are helpful to evaluate the neuroprotective potentialof drugs and to provide a pathophysiological basis for ongoingclinical trials.

Acknowledgments

This work was supported by the Medical Faculty of theUniversity of Göttingen, Germany (junior research group; D. M.,R. D.), the the Gemeinnützige Hertie-Stiftung and the 6thFramework Program of the European Union, NeuroproMiSe,LSHM-CT-2005-018637. We thank Inna Boger and Nadine Meyerfor expert technical assistance.

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