Disease Modification in Multiple Sclerosis by Flupirtine-Results of a Randomized Placebo Controlled Phase II Trial.

Central nervous system inflammation and neurodegeneration are the pathophysiological hallmarks of multiple sclerosis (MS). While inflammation can readily be targeted by current disease modifying drugs, neurodegeneration is by far less accessible to treatment. Based on suggested additional neuroprotective capacities of the orally available non-opioid and centrally acting analgesic drug flupirtine maleate we hypothesized that treatment with flupirtine maleate might be beneficial in MS patients. The flupirtine as oral treatment in multiple sclerosis (FLORIMS) study was a multi-center, randomized and stratified, placebo-controlled double-blind phase II trial to investigate safety and efficacy in terms of clinical and radiographical activity of flupirtine maleate (300 mg per day) given orally for 12 months, add-on to interferon beta 1b subcutaneously in patients with relapsing remitting MS. Due to a substantial delay in recruitment, enrolment of patients was prematurely terminated after randomization of only 30 of the originally planned 80 patients. Of these, 24 regularly terminated study after 12 months of treatment. Data were analyzed as originally planned. Treatment with flupirtine maleate was overall well tolerated. We observed moderate and asymptomatic elevations of liver enzymes in several cases but no overt hepatotoxicity. Neither the intention to treat nor the per protocol analysis revealed any significant treatment effects of flupirtine maleate with respect to occurrence of MS relapses, disability progression, or development of new lesions on cranial MRI. However, substantial methodological limitations need to be considered when interpreting these results. In conclusion, the results of the FLORIMS study neither add further evidence to nor argue against the hypothesized neuroprotective or disease modifying effects of flupirtine maleate in MS.

Brain Metabolite Changes in Patients with Relapsing-Remitting and Secondary Progressive Multiple Sclerosis: A Two-Year Follow-Up Study.

Magnetic resonance spectroscopy (MRS) provides the unique ability to monitor several disease-related pathological processes via their characteristic metabolic markers in vivo. In the present study metabolic compositions were assessed every six months over the period of two years in 36 patients with Multiple Sclerosis (MS) including 21 relapsing-remitting (RR), 15 secondary progressive (SP) patients and 12 normal subjects. The concentrations of the main MRS-detectable metabolites N-acetylaspartate and N-acetylaspartylglutamate (tNAA), creatine and phosphocreatine (tCr), choline containing compounds (Cho), myo-Inositol (Ins), glutamine and glutamate (Glx) and their ratios were calculated in the normal appearing white matter (NAWM) and in selected non-enhancing white matter (WM) lesions. Association between metabolic concentrations in the NAWM and disability were investigated. Concentration of tNAA, a marker for neuroaxonal integrity, did not show any difference between the investigated groups. However, the patients with SPMS showed significant reduction of tNAA in the NAWM over the investigation period of two years indicating diffuse neuroaxonal loss during the disease course. Furthermore, we found a significant increase of Ins, Ins/tCr and Ins/tNAA in WM lesions independently from the course of the disease suggesting ongoing astrogliosis in silent-appearing WM lesions. Analyzing correlations between MRS metabolites in the NAWM and patients clinical status we found the positive correlation of Ins/tNAA with disability in patients with RRMS. In SPMS positive correlation of Cho with disability was found.

Treatment with atacicept enhances neuronal cell death in a rat model of optic neuritis.

We investigated the effect of atacicept, a recombinant fusion protein blocking BLyS and APRIL and acting on B cells, on degeneration of retinal ganglion cells (RGCs) during experimental autoimmune encephalomyelitis (EAE). We used myelin oligodendrocyte glycoprotein in Brown Norway rats to induce a variant of EAE which involves B cells and leads to severe optic neuritis. Intraperitoneal treatment with atacicept at some of the studied dose levels (100 or 200 µg) resulted in increased apoptosis of retinal ganglion cells whereas at a tenfold lower dose or in vehicle-treated animals no such effect became apparent. Also the extent of inflammation, demyelination, and axonal loss of the optic nerve was more pronounced in rats treated with the higher atacicept dose level. The present study describes observational evidence for adverse effects of atacicept on neuronal survival during EAE.

Functional and structural evaluation of lamotrigine treatment in rat models of acute and chronic ocular hypertension.

Voltage gated sodium channels (Nav), are proposed mediators of neuronal damage in ischemic and excitotoxicity disease models. We evaluated the neuroprotective effects of lamotrigine, a Nav blocker, in the acute and chronic rat ocular hypertension models. Additionally, expression of the main Nav subtypes in the optic nerve (ON) was assessed to test whether their upregulation plays a role in the pathogenesis of ocular hypertension induced optic neuropathy. Unilateral intraocular pressure (IOP) elevation was induced for 60 min (80 mmHg) and 14-21 days (670-859 mmHg*day) in the acute and chronic models, respectively. Lamotrigine was administered at dosages of 10 mg/kg twice daily and 12.5 mg/kg once daily in the acute (n = 9) and chronic (n = 11) trials, respectively. Treatment began 2 days prior to IOP elevation until sacrifice. Outer and inner retinal function was evaluated with dark- and light-adapted flash electroretinography and pattern electroretinography, respectively, 6 and 14 days post acute IOP elevation and 13, 28 and 48 days post chronic IOP elevation. Retinal ganglion cell and axon densities and inflammatory reaction were evaluated through Fluorogold, Bielschowsky's silver impregnation and ED1 labeling respectively. Immunohistochemistry for Nav1.1, 1.2 and 1.6 was performed in ONs of untreated rats 7 and 15 days post IOP elevation in the acute model and after 7, 28 and 50 days in the chronic model. In the acute model, no differences were found in the a-wave amplitudes between lamotrigine-treated and vehicle-treated rats. B-wave amplitudes decreased by 40-66% in both treatment groups 6 days post IOP elevation, with no significant difference between groups (p = 0.38). However, a partial recovery of b-wave amplitudes was found in lamotrigine-treated rats between day 6 and day 14 post procedure (p < 0.05). No differences were found in any other parameter tested in this model. Similarly, lamotrigine treatment did not result in any beneficial effect in structural parameters of the chronic model. Functional evaluation of this model was inconclusive due to super-normal values in the hypertensive eyes. Up-regulation of Nav1.1 and 1.2 expression was found in both models, beginning by day 7; an increase of the former continued in a time-dependent manner in the chronic model. Nav1.6 labeling was inconclusive. In conclusion we found lamotrigine treatment to be mostly ineffective in both acute and chronic ocular hypertension models.

A randomized, double-blind, phase 2 study of erythropoietin in optic neuritis.

OBJECTIVE: Based on findings in animal models of autoimmune optic nerve inflammation, we have assessed the safety and efficacy of erythropoietin in patients presenting with a first episode of optic neuritis. METHODS: Patients with optic neuritis who attended the University Hospitals of Homburg/Saar, Göttingen, or Hamburg (Germany) were included in this double-blind, placebo-controlled, phase 2 study (ClinicalTrials.gov, NCT00355095). They were randomly assigned to groups receiving either 33,000IU recombinant human erythropoietin intravenously daily for 3 days or placebo as an add-on therapy to methylprednisolone. The primary outcome parameter was change in retinal nerve fiber layer (RNFL) thickness after 16 weeks. Secondary outcome parameters included optic nerve atrophy as assessed by magnetic resonance imaging, and changes in visual acuity, visual field, and visual evoked potentials (VEPs). RESULTS: Forty patients were assigned to the treatment groups (21/19 erythropoietin/placebo). Safety monitoring revealed no relevant issues. Thirty-seven patients (20/17 erythropoietin/placebo) were analyzed for the primary endpoint according to the intention-to-treat protocol. RNFL thinning was less apparent after erythropoietin treatment. Thickness of the RNFL decreased by a median of 7.5µm by week 16 (mean ± standard deviation, 10.55 ± 17.54µm) compared to a median of 16.0µm (22.65 ± 29.18µm) in the placebo group (p = 0.0357). Decrease in retrobulbar diameter of the optic nerve was smaller in the erythropoietin group (p = 0.0112). VEP latencies at week 16 were shorter in erythropoietin-treated patients than in the placebo group (p = 0.0011). Testing of visual functions revealed trends toward an improved outcome after erythropoietin treatment. INTERPRETATION: These results give the first indications that erythropoietin might be neuroprotective in optic neuritis.

An optical coherence tomography study on degeneration of retinal nerve fiber layer in rats with autoimmune optic neuritis.

PURPOSE: The aim of the present study was to evaluate the ability and accuracy of spectral domain optical coherence tomography (OCT) for in vivo monitoring of retinal ganglion cell degeneration in a rat model of myelin oligodendrocyte glycoprotein-induced optic neuritis. METHODS: First, OCT imaging was established for imaging of all retinal layers in Brown Norway rats. Second, thickness measurements of retinal nerve fiber layer (RNFL) were performed by periodically imaging during the development and progression of autoimmune optic neuritis. Third, the reproducibility of OCT measurements was determined by comparing RNFL measurements of two independent investigators. Fourth, OCT data were correlated with histopathology obtained ex vivo after the final imaging session. RESULTS: Results showed that RNFL thickness declined significantly before clinical manifestation of the disease and decline progresses continuously during the disease course. RNFL thickness measured by OCT had good repeatability and also corresponded with histomorphometric measurements. The reproducibility was limited because of the post-processing analyses performed by manual measurements. CONCLUSIONS: In summary, it is shown here for the first time that OCT can reliably monitor neurodegeneration in an experimental model of autoimmune optic neuritis in rodents. Moreover, in comparing RNFL thickness decline with histopathological analyses of the optic nerve, these results suggest an early, and in part, inflammation-independent process of RNFL degeneration in autoimmune optic neuritis.

Correlation of optical coherence tomography with clinical and histopathological findings in experimental autoimmune uveoretinitis.

Optical coherence tomography (OCT) is becoming the state-of-the-art method for the non-invasive imaging of a variety of ocular diseases. The aim of this study was to assess the application of OCT for the in vivo monitoring and follow-up of pathological changes during experimental autoimmune uveoretinitis (EAU) in rats. Initially we established OCT imaging in healthy brown Norway rats and correlated it with retinal histology. Subsequently, we induced EAU and imaged animals by OCT throughout the pre-peak, peak, and post-peak phases of the disease. The sensitivity of OCT imaging was determined by comparison with clinical EAU and histopathology scores obtained ex vivo at several time points throughout the disease course. Our data demonstrate that OCT imaging of the healthy rat retina closely correlates with histological observations and allows the clear visualization of all retinal layers. After induction of EAU, the first pathological changes could be detected by OCT at day (d) 8 post-immunization (p.i.) which corresponded to the time point of clinical disease onset. An increase in retinal thickness (RT) was detected from d10 p.i. onwards which peaked at d16 p.i. and decreased again to near control levels by d20 p.i. We introduce a novel semi-quantitative OCT scoring which correlates with histopathological findings and complements the clinical scores. Therefore, we conclude that OCT is an easily accessible, non-invasive tool for detection and follow-up of histopathological changes during EAU in rats. Indeed, significant differences in RT between different stages of EAU suggest that this OCT parameter is a sensitive marker for distinguishing disease phases in vivo.

Anti-inflammatory effects of FTY720 do not prevent neuronal cell loss in a rat model of optic neuritis.

In multiple sclerosis, long-term disability is caused by axonal and neuronal damage. Established therapies target primarily the inflammatory component of the disease, but fail to prevent neurodegeneration. Fingolimod (codenamed FTY720) is an oral sphingosine 1-phosphate (S1P) receptor modulator with promising results in phase II trials in multiple sclerosis patients and is under further development as a novel treatment for multiple sclerosis. To evaluate whether FTY720 has neuroprotective properties, we tested this drug in a rat model of myelin oligodendrocyte glycoprotein-induced optic neuritis. FTY720 exerted significant anti-inflammatory effects during optic neuritis and reduced inflammation, demyelination, and axonal damage; however, FTY720 treatment did not prevent apoptosis of retinal ganglion cells (RGCs), the neurons that form the axons of the optic nerve. Consistent with this lack of effect on RGC survival, FTY720 treatment did not improve visual function, nor did it prevent apoptosis of RGCs in vitro. We observed a persistent activation of apoptotic signaling pathways in RGCs under FTY720 treatment, a possible underlying mechanism for the lack of neuroprotection in the presence of strong anti-inflammatory effects, Furthermore, FTY720 shifted the remaining inflammation in the optic nerve toward neurotoxicity by modest up-regulation of potential neurotoxic cytokines. We conclude that FTY720-induced anti-inflammation and axon protection did not of itself protect neurons from apoptotic cell death.

Future neuroprotective strategies.

Accumulating impairment in patients suffering from multiple sclerosis (MS) is the result of increasing axonal damage and neurodegeneration. Current therapeutic strategies only target the inflammatory side of this disease. Convincing effects of all established treatment options in chronic progressive disease stages are lacking. In recent years much progress has been made unraveling the molecular mechanisms involved in neuronal and axonal loss in this neuroinflammatory disease. Understanding the mechanisms of neurodegeneration in MS and its animal models is essential to select therapeutic targets which are most likely to reduce long-term disabilities in patients. In this article we review the current knowledge of possible strategies for future neuroprotective therapies for MS patients and point out which treatment options are currently being evaluated in clinical trials.

Role of n-type voltage-dependent calcium channels in autoimmune optic neuritis.

OBJECTIVE: The aim of this study was to investigate the role of voltage-dependent calcium channels (VDCCs) in axon degeneration during autoimmune optic neuritis. METHODS: Calcium ion (Ca(2+)) influx into the optic nerve (ON) through VDCCs was investigated in a rat model of optic neuritis using manganese-enhanced magnetic resonance imaging and in vivo calcium imaging. After having identified the most relevant channel subtype (N-type VDCCs), we correlated immunohistochemistry of channel expression with ON histopathology. In the confirmatory part of this work, we performed a treatment study using omega-conotoxin GVIA, an N-type specific blocker. RESULTS: We observed that pathological Ca(2+) influx into ONs during optic neuritis is mediated via N-type VDCCs. By analyzing the expression of VDCCs in the inflamed ONs, we detected an upregulation of alpha(1B), the pore-forming subunit of N-type VDCCs, in demyelinated axons. However, high expression levels were also found on macrophages/activated microglia, and lower levels were detected on astrocytes. The relevance of N-type VDCCs for inflammation-induced axonal degeneration and the severity of optic neuritis was corroborated by treatment with omega-conotoxin GVIA. This blocker led to decreased axon and myelin degeneration in the ONs together with a reduced number of macrophages/activated microglia. These protective effects were confirmed by analyzing the spinal cords of the same animals. INTERPRETATION: We conclude that N-type VDCCs play an important role in inflammation-induced axon degeneration via two mechanisms: First, they directly mediate toxic Ca(2+) influx into the axons; and second, they contribute to macrophage/microglia function, thereby promoting secondary axonal damage. Ann Neurol 2009;66:81-93.

Flupirtine as neuroprotective add-on therapy in autoimmune optic neuritis.

Multiple sclerosis (MS) is a common inflammatory disease of the central nervous system that results in persistent impairment in young adults. During chronic progressive disease stages, there is a strong correlation between neurodegeneration and disability. Current therapies fail to prevent progression of neurological impairment during these disease stages. Flupirtine, a drug approved for oral use in patients suffering from chronic pain, was used in a rat model of autoimmune optic neuritis and significantly increased the survival of retinal ganglion cells, the neurons that form the axons of the optic nerve. When flupirtine was combined with interferon-beta, an established immunomodulatory therapy for MS, visual functions of the animals were improved during the acute phase of optic neuritis. Furthermore, flupirtine protected retinal ganglion cells from degeneration in a noninflammatory animal model of optic nerve transection. Although flupirtine was shown previously to increase neuronal survival by Bcl-2 up-regulation, this mechanism does not appear to play a role in flupirtine-mediated protection of retinal ganglion cells either in vitro or in vivo. Instead, we showed through patch-clamp investigations that the activation of inwardly rectifying potassium channels is involved in flupirtine-mediated neuroprotection. Considering the few side effects reported in patients who receive long-term flupirtine treatment for chronic pain, our results indicate that this drug is an interesting candidate for further evaluation of its neuroprotective potential in MS.

Autoimmune optic neuritis in the common marmoset monkey: comparison of visual evoked potentials with MRI and histopathology.

PURPOSE: To assess the use of visual evoked potentials (VEPs) for the in vivo detection of impaired visual function in a marmoset model of multiple sclerosis. The sensitivity of the VEP recordings was determined by comparison with magnetic resonance imaging (MRI) and histopathology. METHODS: Baseline VEPs were recorded in six healthy marmoset monkeys in response to light-flash stimulation. Experimental autoimmune encephalomyelitis (EAE) was induced in four of the six monkeys. Clinical scores were assessed daily, and VEPs were recorded every second week. In vivo MRI and subsequent histopathology of the brains and optic nerves were performed at the end of the study. RESULTS: After induction of EAE, all four marmosets exhibited clinical signs between day 26 and 38 after immunization. VEPs were normal during the induction phase of the disease, but deteriorated in amplitude with the occurrence of clinical symptoms in all animals. MRI revealed bilateral optic neuritis and signal alterations in the optic tracts and occipital subcortical white matter in two of the animals. In the remaining two animals, MRI detected signal alterations in the occipital subcortical white matter. Histopathologic results were concordant with the MRI findings. CONCLUSIONS: VEPs are an easily accessible noninvasive tool for measuring visual function and diagnosing impairment of the visual pathway in a marmoset EAE model.

Biological markers for axonal degeneration in CSF and blood of patients with the first event indicative for multiple sclerosis.

Axonal degeneration is now recognized as an important pathological feature of multiple sclerosis (MS). Acute axonal damage happens early in the disease course, and therefore early changes might occur in markers in body fluids, such as cerebrospinal fluid (CSF) and blood. In our study we investigated the relevance of serum and CSF markers for axonal damage in patients with clinically isolated syndrome indicative for MS. We measured the concentration of tau, phospho-tau, S100B, Amyloid beta and neuron specific enolase (NSE) in CSF and serum. Interestingly, the NSE concentration in CSF and serum was decreased in clinically isolated syndrome (CIS)-patients in comparison to the control group indicating reduced neuronal metabolic activity in the early stage of the disease. Concerning other biomarkers, we did not observe any changes in the concentrations between groups. Moreover, we did not detect any correlation between Expanded Disability Status Scale (EDSS) and the concentration of investigated proteins.

Strain-specific susceptibility for neurodegeneration in a rat model of autoimmune optic neuritis.

Heterogeneity in clinical disease course and histopathology complicates the treatment of multiple sclerosis. We detected important differences in neurodegeneration in various subtypes of myelin oligodendrocyte glycoprotein (MOG)-induced optic neuritis. Dark Agouti (DA) rats showed a significantly higher survival of retinal ganglion cells in comparison to Brown Norway rats. After surgical transection of the optic nerve neuronal loss was similar in both rat strains. We identified an increased expression of interleukin 1beta and glial cell line-derived neurotrophic factor in DA rats as the possible mechanism of the observed endogenous neuroprotection in MOG-induced optic neuritis.

Neurodegeneration and -protection in autoimmune CNS inflammation.

Neurodegeneration in multiple sclerosis (MS) is the structural correlate of permanent neurological disability in patients. The histopathological features of neurodegeneration include destruction of axons as well as apoptotic cell death of neuronal cell bodies. Therapeutic efforts to control these clinically important aspects of MS pathology showed limited success so far. In this review article, we give an overview about the current knowledge concerning the molecular mechanisms of neurodegeneration in autoimmune inflammation that is mainly derived from animal models. Further, we critically discuss experimental neuroprotective strategies with respect to their functional relevance and differentiate between anti-apoptotic and axon protective treatment approaches.

Effects of glatiramer acetate and interferon-beta on neurodegeneration in a model of multiple sclerosis: a comparative study.

Axonal destruction and neuronal loss occur early during multiple sclerosis (MS), an autoimmune inflammatory central nervous system disease that frequently manifests with acute optic neuritis. Glatiramer acetate (GA) and interferon-beta-1b (IFN-beta-1b) are two immunomodulatory agents that have been shown to decrease the frequency of MS relapses. However, the question of whether these substances can slow neurodegeneration in MS patients is the subject of controversy. In a rat model of experimental autoimmune encephalomyelitis, we investigated the effects of GA and IFN-beta-1b on the survival of retinal ganglion cells (RGCs), the neurons that form the axons of the optic nerve. For each substance, therapy was started 14 days before immunization, on the day of immunization, or on the day of clinical disease onset. After myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis became clinically manifest, optic neuritis was monitored by recording visual evoked potentials. The function of RGCs was measured by electroretinograms. Although early GA or IFN-beta-1b treatment showed benefit on disease activity, only treatment with GA exerted protective effects on RGCs, as revealed by measuring neurodegeneration and neuronal function. Furthermore, we demonstrate that this GA-induced neuroprotection does not exclusively depend on the reduction of inflammatory infiltrates within the optic nerve.

Effects of interferon-beta-1a on neuronal survival under autoimmune inflammatory conditions.

Interferon-beta-1a (IFN-beta-1a) is an approved treatment for multiple sclerosis (MS). It improves the disease course by reducing the relapse rate as well as the persistent neurological deficits. Recent MRI and post-mortem studies revealed that neuronal and axonal damage are most relevant for chronic disability in MS patients. We have characterized previously time course and mechanisms of neuronal apoptosis in a rat model of myelin oligodendrocyte glycoprotein (MOG)-induced optic neuritis. In this animal model, application of IFN-beta-1a three times per week slightly decreases the loss of retinal ganglion cells (RGCs), the neurons that form the axons within the optic nerve. In contrast to neurotrophic factors, this cytokine does not directly protect cultured RGCs from apoptosis. We conclude that IFN-beta-1a is a suitable candidate to be combined with a directly neuroprotective agent in order to further decrease axonal and neuronal degeneration in MS patients.

HIV-Tat-mediated Bcl-XL delivery protects retinal ganglion cells during experimental autoimmune optic neuritis.

In multiple sclerosis (MS), post-mortem studies of human brain tissue as well as data from animal models have shown that apoptosis of neurons occurs to a significant extent during this disease. As neurodegeneration in MS correlates with permanent neurological deficits in patients, understanding the mechanisms would be an important pre-condition for designing appropriate neuroprotective therapies. Myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis often affects the optic nerve and leads to consecutive apoptosis of retinal ganglion cells (RGCs), the neurons that form its axons. In this study, we fused Bcl-XL to the protein transduction domain of the HIV-transactivator of transcription. Thereby, this anti-apoptotic member of the Bcl-2 family was delivered into RGCs of rats with electrophysiologically diagnosed optic neuritis. Transduction of Bcl-XL in our study led to significant rescue of RGCs indicating the relevance of this pathway for neuronal survival under autoimmune inflammatory conditions.

Simvastatin treatment does not protect retinal ganglion cells from degeneration in a rat model of autoimmune optic neuritis.

In patients with multiple sclerosis (MS), non-remitting deficits are mainly caused by axonal and neuronal damage. We demonstrated previously that myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis in rats provokes severe axonal and neuronal injury even before clinical manifestation of the disease. In our present study, we investigated effects of simvastatin treatment on degeneration of retinal ganglion cell (RGC) bodies as well as their axons during MOG-induced optic neuritis. Electrophysiological functions of optic nerves and RGCs were analyzed in vivo. Although neuroprotective effects of simvastatin have been demonstrated before in other experimental settings, we did not observe an increase in RGC survival nor an improvement of visual functions. As we could not reproduce the anti-inflammatory effects that were observed under statin therapy in other EAE models, we hypothesize that patients suffering from optic neuritis might not take advantage of simvastatin applications.

Combined therapy with methylprednisolone and erythropoietin in a model of multiple sclerosis.

Neurodegenerative processes determine the clinical disease course of multiple sclerosis, an inflammatory autoimmune CNS disease that frequently manifests with acute optic neuritis. None of the established multiple sclerosis therapies has been shown to clearly reduce neurodegeneration. In a rat model of experimental autoimmune encephalomyelitis, we recently demonstrated increased neuronal apoptosis under methylprednisolone therapy, although CNS inflammation was effectively controlled. In the present study, we combined steroid treatment with application of erythropoietin to target inflammatory as well as neurodegenerative aspects. After immunization with myelin oligodendrocyte glycoprotein (MOG), animals were randomly assigned to six treatment groups receiving different combinations of erythropoietin and methylprednisolone, or respective monotherapies. After MOG-induced experimental autoimmune encephalomyelitis became clinically manifest, optic neuritis was monitored by recording visual evoked potentials. The function of retinal ganglion cells, the neurons that form the axons of the optic nerve, was measured by electroretinograms. Functional and histo pathological data of retinal ganglion cells and optic nerves revealed that neuron and axon protection was most effective when erythropoietin treatment that was started at immunization was combined with high-dose methylprednisolone therapy given from days 1 to 3 of MOG-induced experimental autoimmune encephalomyelitis. In contrast, isolated neuronal or axonal protection without clinical benefit was achieved under monotherapy with erythropoietin or methylprednisolone, respectively.