Flupirtine plasma levels and clinical disease course. A: Twenty-four hours after application of 0.6125 g/kg flupirtine incorporated in pelleted rat chow, plasma concentrations of 624 ± 86 ng/ml flupirtine were observed in healthy rats, whereas none of the vehicle-treated (veh) plasma probes showed relevant flupirtine levels, only background activity was detected (mean ± SEM; n = 5 for each group). B: Comparison of the clinical disease course in the four different treatment groups. ▴The mean clinical score in the rat group receiving combination therapy of flupirtine and IFN-β1a, ▪IFN-β1a monotherapy, •flupirtine monotherapy, and □vehicle-treated controls. Data are demonstrated as mean ± SEM. In comparison to vehicle-treated controls, the mean clinical score was significantly reduced if IFN-β1a monotherapy or IFN-β1a together with flupirtine were applied (one-way analysis of variance followed by Bonferroni correction, P < 0.05).

Effects of flupirtine application on ON histopathology at day 8 after clinical manifestation of MOG-EAE. A and B: Luxol-fast blue staining of ON cross-sections was used to determine the degree of demyelination. A similar degree of demyelination was observed in ON cross-sections of rats treated with flupirtine (A) in comparison to vehicle-treated controls (B). C and D: Inflammatory infiltration with ED1⁺ macrophages/microglia was unchanged with (C) and without (D) flupirtine treatment. E and F: Acute axonal damage was analyzed in β-APP-stained ON cross-sections of a flupirtine (E) and a vehicle-treated animal (F), and demonstrated no effect of flupirtine on the presence of β-APP-positive profiles (arrows). Scale bars: 100 μm (A–D); 2 μm (E, F).

Activation of Kir by 200 μmol/L flupirtine in cultured RGCs. Currents were evoked under control conditions (A) and after application of 200 μmol/L flupirtine (B) by hyperpolarizing voltage steps throughout the range of −100 to 0 mV from a holding potential of −20 mV. Representative traces of one RGC are shown without flupirtine (A), 1 minute after application of flupirtine (B) and 2 minutes after application of 200 μmol/L flupirtine and 1 mmol/L Ba²⁺ (C). Blockade of flupirtine-induced currents by co-application with Ba²⁺ identified the currents as inwardly rectifying K⁺ currents. D: Number of RGCs per mm² in retinal flat mounts at day 8 after clinical manifestation of MOG-EAE in rats receiving flupirtine or vehicle (veh) monotherapy, or intravitreal applications of barium combined with flupirtine (flu + Ba) or vehicle (veh + Ba). Data are presented as mean ± SEM. Statistically significant when compared to the vehicle-treated group (*P < 0.05).

Visual function and RGC survival in rats suffering from MOG-induced optic neuritis. A: Visual acuity determined by pattern VEP recordings at the day of clinical manifestation of MOG-EAE in comparison to sham-immunized controls (ctrl).14 Electrophysiological responses to pattern stimulation were not detectable in any of the vehicle-treated controls (veh), whereas in the flupirtine and IFN-β1a monotherapy groups a response to pattern stimulation using broad alternating black and white bars was present. The combination therapy groups receiving flupirtine together with IFN-β1a showed a significantly better electrophysiological response in pattern VEPs. Statistically significant when compared with vehicle-treated controls (*P < 0.05). B: Visual acuity determined by pattern VEP recordings at day 8 after clinical manifestation of EAE. Only a tendency for improved visual acuity was observed in the combination therapy group in comparison to vehicle-treated controls (P = 0.0658). C: Representative example of VEP recordings using flash stimulation in an IFN-β1a-treated rat. D and E: Representative examples of VEP recordings at EAE onset using pattern stimulation with 12 alternating black and white bars in an animal receiving flupirtine together with IFN-β1a (D) and a vehicle-treated control (E). F: Number of RGCs per mm² in retinal flat mounts at day 8 after clinical manifestation of MOG-EAE. Statistically significant when compared to vehicle-treated controls (*P < 0.05). G: Examples of retinal flat mounts after FG prelabeling in a rat receiving flupirtine together with IFN (flupirtine + IFN), IFN monotherapy (IFN), flupirtine monotherapy (flu), and a vehicle-treated control (veh). H: Number of RGCs per mm² in retinal flat mounts 2 weeks after surgical ON transection. A significantly higher density of retrogradely labeled RGCs was present in rats that had received flupirtine in comparison to vehicle-treated controls (veh, *P < 0.05). Data are given as mean ± SEM. One-way analysis of variance followed by Bonferroni correction was used for multiple group comparison. Scale bar = 50 μm.

Neuroprotective effects of flupirtine on RGCs in vitro and in vivo. A: Survival of cultured RGCs after growth factor withdrawal (−GF) is significantly increased if flupirtine is applied at a 200 μmol/L concentration. Statistically significant if compared with control (*P < 0.05). B: The percentage of RGC survival under glutathione depletion induced by buthionine sulfoximine is similar with (gray bars) and without (black bars) application of 200 μmol/L flupirtine. Buthionine sulfoximine induced a dose-dependent loss of cultured primary RGCs. C: Western blot analyses of retinal protein lysates obtained at the day of clinical manifestation of MOG-EAE in rats treated with flupirtine or vehicle (veh). Similar protein levels of pAkt, Akt, pMAPK1/2, MAPK1/2, Bcl-2, and Bax were observed. D: Western blot analysis of Bcl-2 expression in cultured primary RGCs. Unchanged protein level of Bcl-2 under growth factor withdrawal (−GF) with and without application of 10, 100, or 200 μmol/L flupirtine.