Flupirtine as Neuroprotective Add-On Therapy in Autoimmune Optic Neuritis
2008; Elsevier BV; Volume: 173; Issue: 5 Linguagem: Inglês
10.2353/ajpath.2008.080491
ISSN1525-2191
AutoresMuriel B. Sättler, Sarah K. Williams, Clemens Neusch, Markus Otto, Jens R. Pehlke, Mathias Bähr, Ricarda Diem,
Tópico(s)Tryptophan and brain disorders
ResumoMultiple 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-β, 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. 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-β, 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. Flupirtine is a nonopioid analgesic, approved for long-term use in patients.1Herrmann WM Hiersemenzel R Aigner M Lobisch M Riethmüller-Winzen H Michel I Long-term tolerance of flupirtine. Open multicenter study over one year.Fortschr Med. 1993; 111: 266-270PubMed Google Scholar Neuroprotective properties of flupirtine have been previously demonstrated in models of cerebral ischemia.2Rupalla K Cao W Krieglstein J Flupirtine protects neurons against excitotoxic or ischemic damage and inhibits the increase in cytosolic Ca2+ concentration.Eur J Pharmacol. 1995; 294: 469-473Crossref PubMed Scopus (53) Google Scholar, 3Block F Pergande G Schwarz M Flupirtine reduces functional deficits and neuronal damage after global ischemia in rats.Brain Res. 1997; 754: 279-284Crossref PubMed Scopus (40) Google Scholar In human brain slices a reduction of tumor necrosis factor-related apoptosis-inducing-ligand-induced neurodegeneration was described for this analgesic compound.4Dörr J Roth K Zurbuchen U Deisz R Bechmann I Lehmann TN Meier S Nitsch R Zipp F Tumor-necrosis-factor-related apoptosis-inducing-ligand (TRAIL)-mediated death of neurons in living human brain tissue is inhibited by flupirtine-maleate.J Neuroimmunol. 2005; 167: 204-209Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar As one of the mechanisms underlying its neuroprotective properties, an up-regulation of the anti-apoptotic protein Bcl-2 was identified.5Perovic S Pialoglou P Schroder HC Pergande G Muller WE Flupirtine increases the levels of glutathione and Bcl-2 in hNT (human Ntera/D1) neurons: mode of action of the drug-mediated anti-apoptotic effect.Eur J Pharmacol. 1996; 317: 157-164Crossref PubMed Scopus (36) Google Scholar In an animal model of myelin oligodendrocyte glycoprotein (MOG)-induced optic neuritis we previously observed a down-regulation of Bcl-2 to be one of the signaling events involved in the degeneration of retinal ganglion cells (RGCs), the neurons that form the axons of the optic nerve (ON).6Hobom M Storch MK Weissert R Maier K Radhakrishnan A Kramer B Bähr M Diem R Mechanisms and time course of neuronal degeneration in experimental autoimmune encephalomyelitis.Brain Pathol. 2004; 14: 148-157Crossref PubMed Scopus (137) Google Scholar, 7Sättler MB Merkler D Maier K Stadelmann C Ehrenreich H Bähr M Diem R Neuroprotective effects and intracellular signalling pathways of erythropoietin in a rat model of multiple sclerosis.Cell Death Differ. 2004; 11: S181-S192Crossref PubMed Scopus (154) Google Scholar In contrast to other models of experimental autoimmune encephalomyelitis (EAE), MOG-induced EAE in rats produces an encephalitogenic T-cell activation in parallel with a demyelinating autoantibody response.8Stefferl A Brehm U Storch M Lambracht-Washington D Bourquin C Wonigeit K Lassmann H Linington C Myelin oligodendrocyte glycoprotein induces experimental autoimmune encephalomyelitis in the “resistant” Brown Norway rat: disease susceptibility is determined by MHC and MHC-linked effects on the B cell response.J Immunol. 1999; 163: 40-49PubMed Google Scholar, 9Gold R Linington C Lassmann H Understanding pathogenesis and therapy of multiple sclerosis via animal models: 70 years of merits and culprits in experimental autoimmune encephalomyelitis research.Brain. 2006; 129: 1953-1971Crossref PubMed Scopus (778) Google Scholar Additionally, the extent of axonal and neuronal injury is similar to that of the human disease and begins shortly after immunization.6Hobom M Storch MK Weissert R Maier K Radhakrishnan A Kramer B Bähr M Diem R Mechanisms and time course of neuronal degeneration in experimental autoimmune encephalomyelitis.Brain Pathol. 2004; 14: 148-157Crossref PubMed Scopus (137) Google Scholar, 10Kornek B Storch MK Weissert R Wallstroem E Stefferl A Olsson T Linington C Schmidbauer M Lassmann H Multiple sclerosis and chronic autoimmune encephalomyelitis: a comparative quantitative study of axonal injury in active, inactive, and remyelinated lesions.Am J Pathol. 2000; 157: 267-276Abstract Full Text Full Text PDF PubMed Scopus (774) Google Scholar Current therapies for multiple sclerosis (MS) mainly target the inflammatory infiltration.11Rieckmann P Multiple Sklerose Therapie Konsensus Gruppe (MSTKG) Escalating immunomodulatory therapy of multiple sclerosis.Nervenarzt. 2006; 77: 1506-1518Crossref PubMed Scopus (48) Google Scholar However, in chronic progressive disease stages the present treatment strategies are insufficient, as neuronal and axonal degeneration continues to progress, finally leading to persisting neurological impairments.12Bjartmar C Kidd G Mork S Rudick R Trapp BD Neurological disability correlates with spinal cord axonal loss and reduced N-acetyl aspartate in chronic multiple sclerosis patients.Ann Neurol. 2000; 48: 893-901Crossref PubMed Scopus (483) Google Scholar, 13Wujek JR Bjartmar C Richer E Ransohoff RM Yu M Tuohy VK Trapp BD Axon loss in the spinal cord determines permanent neurological disability in an animal model of multiple sclerosis.J Neuropathol Exp Neurol. 2002; 61: 23-32Crossref PubMed Scopus (223) Google Scholar As of yet, no approved therapy targeting the neurodegenerative aspect of this disease is available. The possibility of an oral application and the involvement of Bcl-2 in the degeneration of RGCs in MOG-induced optic neuritis, led us to investigate the neuroprotective properties of flupirtine in our animal model. All animal protocols were approved by the local authorities in Braunschweig, Germany. Female BN rats 8 to 10 weeks of age were obtained from Charles River (Sulzfeld, Germany). Recombinant rat MOGIgd, corresponding to the N-terminal sequence of rat MOG (amino acids 1 to 125) was used to induce MOG-EAE. The rats were anesthetized by inhalation of diethyl ether and were then injected intradermally at the base of the tail with a total volume of 200 μl of inoculum, containing 50 μg of MOG kindly provided by Doron Merkler (Department of Neuropathology, University of Göttingen, Göttingen, Germany) in saline emulsified (1:1) with complete Freund's adjuvant (Sigma, St. Louis, MO) containing 200 μg of heat-inactivated Mycobacterium tuberculosis (strain H 37 RA; Difco Laboratories, Detroit, MI). The rats were scored for clinical signs of EAE either until day 8 after clinical manifestation or, if they developed no clinical signs of EAE, until day 25 after immunization. The signs were scored as follows: grade 0.5, distal paresis of the tail; grade 1, complete tail paralysis; grade 1.5, paresis of the tail and mild hind leg paresis; grade 2.0, unilateral severe hind leg paresis; grade 2.5, bilateral severe hind limb paresis; grade 3.0, complete bilateral hind limb paralysis; grade 3.5, complete bilateral hind limb paralysis and paresis of one front limb; grade 4, complete paralysis (tetraplegia), moribund state, or death. This score reflects the amount of spinal cord lesions and does not include visual symptoms. Recordings of visual evoked potentials (VEPs) were performed as described previously.14Meyer R Weissert R Diem R Storch MK de Graaf KL Kramer B Bähr M Acute neuronal apoptosis in a rat model of multiple sclerosis.J Neurosci. 2001; 21: 6214-6220Crossref PubMed Google Scholar In six animals of each group, the mean visual acuity was calculated from the smallest size of alternating bars for which specific VEP potentials were recordable at day 1 of the clinically apparent disease or, if the animal developed no clinical signs of EAE, at day 19 after immunization with MOG.6Hobom M Storch MK Weissert R Maier K Radhakrishnan A Kramer B Bähr M Diem R Mechanisms and time course of neuronal degeneration in experimental autoimmune encephalomyelitis.Brain Pathol. 2004; 14: 148-157Crossref PubMed Scopus (137) Google Scholar A second session of VEP recordings was performed in the same animals at day 8 after clinical manifestation of EAE or, if the animal developed no clinical signs of EAE, at day 25 after immunization. At the end of the experiment, the rats received an overdose of chloral hydrate and were perfused via the aorta with 4% paraformaldehyde. Data analysis of the VEP recordings was performed by an investigator blinded to the treatment applied. The rats were randomly allocated to four different groups: interferon (IFN)-β1a monotherapy, flupirtine monotherapy, combination of flupirtine and IFN-β1a, and vehicle treatment. To avoid direct interference with the immunization, all treatments were started on day 2 after immunization with MOG. In both the IFN-β1a monotherapy group (n = 6) and the combination therapy group (n = 6), animals were treated three times per week with subcutaneous applications of 300,000 U (1.1 μg) IFN-β1a (Serono, Unterschleiβheim, Germany) in 150 μl of 0.9% sodium chloride. The control group received vehicle (n = 12). In the flupirtine monotherapy group (n = 12) and the combination therapy group, pelleted rat chow incorporating 0.6125 g/kg flupirtine (Astra-Zeneca, Frankfurt, Germany) was fed to the rats. Considering a daily food uptake of 20 g per rat, this concentration in the food leads to a daily flupirtine uptake of 2.45 mg/kg body weight. This dosage is in a similar range as the daily uptake of a standard dosage in patients, in which 300 mg are routinely applied, resulting in 3.75 mg/kg body weight. The vehicle-treated rat groups were fed the identical standard rat chow not containing flupirtine. For intraocular injection of 2 mmol/L barium (Ba), animals were anesthetized with diethyl ether. By means of a glass microelectrode, 2 μl of the solution were injected into the vitreous space of each eye, puncturing the eye at the cornea-sclera junction. The injections were performed at days 5, 10, 15, and 20 after immunization. To determine the plasma level of flupirtine in rats treated with pelleted rat chow incorporating 0.6125 g/kg flupirtine, blood was collected 48 hours after initiating flupirtine application by sublingual puncture. The plasma sample was processed using a liquid/liquid extraction method. To 250 μl of the plasma test samples, QC samples, and calibration standards, 20 μl NaOH (1 mol/L) and 2 ml diethyl ether were added. After centrifuging at 2500 × g for 5 minutes the samples were kept for 20 minutes at −40°C. The organic phase was then decanted from the frozen aqueous phase and transferred to new polypropylene vials that contained 500 μl of n-hexane and 500 μl of HCl (0.01 mol/L). After further centrifugation, the upper organic phase was discarded and the vials were placed into a vacuum centrifuge for 10 minutes at room temperature. Five hundred μl of the residual solution was transferred into high performance liquid chromatography vials and 50 μl of each sample were then injected directly into the high performance liquid chromatography system for measuring the flupirtine concentration in the individual plasma samples. One week before immunization, retrograde prelabeling of RGCs was performed after anesthetizing the rats with 10% ketamine (0.75 ml/kg; Atarost GmbH and Co., Twistringen, Germany) together with 2% xylazine (0.35 ml/kg; Albrecht, Aulendorf, Germany). The skin was incised mediosagitally, and holes were drilled into the skull above each superior colliculus (6.8 mm dorsal and 2 mm lateral from bregma). Two μl of the fluorescent dye Fluorogold (FG) (5% in normal saline; Fluorochrome Inc., Englewood, CO) were injected stereotactically into both superior colliculi. Axonal transport of FG with consecutive labeling of RGCs takes place within the first 24 hours after FG injection so that RGCs are fully labeled at the time of EAE induction (our own previous observations). At the end of the experiment, retinas were dissected, flat-mounted on glass-slides, and examined by fluorescence microscopy (Axioplan 2; Zeiss, Göttingen, Germany) using a 4,6-diamidino-2-phenylindole filter (315/395 nm). RGC densities were determined by counting FG-labeled cells in three areas (62,500 μm2) per retinal quadrant at three different eccentricities of 1/6, 3/6, and 5/6 of the retinal radius in blinded samples. In sham-immunized controls we previously detected 2730 ± 145 RGCs per mm2 (mean ± SEM).15Diem R Hobom M Maier K Weissert R Storch MK Meyer R Bähr M Methylprednisolone increases neuronal apoptosis during autoimmune CNS inflammation by inhibition of an endogenous neuroprotective pathway.J Neurosci. 2003; 23: 6993-7000Crossref PubMed Google Scholar Rats were anesthetized by an intraperitoneal injection of ketamine and xylazine as described above. A skin incision close to the superior orbital rim was performed, and the right orbita was opened. The lachrymal gland was resected subtotally. After spreading of the superior extraocular muscles, the ON was exposed by longitudinal incision of the perineurium. ON transection was performed 2 mm from the posterior pole of the eye without damaging retinal blood supply. Retrograde labeling of RGCs was achieved by placing a small sponge soaked in 5% FG at the ocular stump of the transected ON. RGC counts were evaluated as described above at day 14 after ON transection. After perfusion of the rat with 4% paraformaldehyde, ONs were taken for histopathological evaluation and were paraffin-embedded. Histological evaluation was performed on 4-μm-thick slices. Luxol-fast blue staining was used to assess demyelination. Photos of vertical sections were taken using an Axiocam MR (Zeiss). The images were processed using Axiovision 4.2 software (Zeiss) to evaluate the demyelinated area as a percentage of the whole ON cross section. Additionally, immunohistochemistry was performed on ON cross-sections. ED-1-positive macrophages/activated microglia (MCA341R, diluted 1:500; Serotec, Oxford, UK), CD-3-positive T-cells (BZL03543, diluted 1:500; Biozol, Eching, Germany), and β-amyloid precursor protein (APP)-positive axons (MAB348, diluted 1:3000; Chemicon, Ford, UK) were detected using biotin-avidin detection. Spleen sections served as a control for ED1 and CD3 stainings. The evaluation of ED-1- or CD-3-positive cells was performed according to the following score: 0, no labeled cells; 1, a few positive cells in at least one of three different ON levels; 2, 10 to 50% of at least one ON cross section infiltrated with labeled cells; and 3, more than 50% of the ON cross section infiltrated with labeled cells in at least one ON level. The number of β-APP-positive axons was counted per cross section. For each histopathological parameter three different levels of each ON were evaluated. The investigators who performed neuropathological examinations were blinded to the treatment applied. Immunocytochemistry was performed on cultured RGCs maintained for 48 hours in normal conditions and in media lacking growth factors, with and without the presence of 200 μmol/L flupirtine. Cells were fixed in 4% paraformaldehyde and permeabilized with 0.3% Triton X-100. After blocking with 5% normal goat serum, cells were incubated overnight at 4°C in mouse anti-Bcl-2 (sc-7382, 1:100 in blocking solution; Santa Cruz Biotechnology, Inc., Santa Cruz, CA). After washing, cells were incubated with an Alexa 488-conjugated secondary antibody (Molecular Probes, Eugene, OR), washed, and mounted (Vectashield; Vector Laboratories, Burlingame, CA). Primary RGCs were obtained from 6- to 8-day-old Wistar rats as described previously.16Sättler MB Demmer I Williams SK Maier K Merkler D Gadjanski I Stadelmann C Bähr M Diem R Effects of interferon-beta1a on neuronal survival under autoimmune inflammatory conditions.Exp Neurol. 2006; 201: 172-181Crossref PubMed Scopus (33) Google Scholar After 24 hours, media containing forskolin, brain-derived neurotrophic factor, ciliary neurotrophic factor, and insulin was removed and replaced with media lacking these neurotrophic factors and containing flupirtine dissolved in 100% dimethyl sulfoxide (Astra Medica, Bad Homburg, Germany), at a concentration of 1 to 200 μmol/L. Cells were maintained in media lacking neurotrophic factors, and containing equivalent dimethyl sulfoxide levels, as controls. Previous studies demonstrated the most prominent effect of flupirtine in neuronal cell cultures at concentrations of 10 to 200 μmol/L.2Rupalla K Cao W Krieglstein J Flupirtine protects neurons against excitotoxic or ischemic damage and inhibits the increase in cytosolic Ca2+ concentration.Eur J Pharmacol. 1995; 294: 469-473Crossref PubMed Scopus (53) Google Scholar, 17Wood JP Pergande G Osborne NN Prevention of glutathione depletion-induced apoptosis in cultured human RPE cells by flupirtine.Restor Neurol Neurosci. 1998; 12: 119-125PubMed Google Scholar, 18Nash MS Wood JP Melena J Osborne NN Flupirtine ameliorates ischaemic-like death of rat retinal ganglion cells by preventing calcium influx.Brain Res. 2000; 856: 236-239Crossref PubMed Scopus (15) Google Scholar We assessed cell viability using a (3,4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction assay 48 hours after removal of neurotrophins. Viability was assessed by counting the number of surviving cells in six fields of view in each of three wells per concentration and was performed on three separate cell preparations to ensure reliability of the data. Results are expressed as a percentage of controls. After 24 hours in culture, primary RGCs were treated with increasing concentrations of buthionine sulfoximine (100 μmol/L to 10 mmol/L) both with and without the presence of 200 μmol/L flupirtine. Forty-eight hours later, cell viability was assessed using a MTT assay as before, and again repeated on three separate cell preparations. For Western blot analysis, animals received an overdose of chloral hydrate 6 hours after the last application of IFN-β1a or vehicle. The dissected retinas were homogenized and lysed (150 mmol/L NaCl, 50 mmol/L Tris, pH 8.0, 2 mmol/L ethylenediaminetetraacetic acid, and 1% Triton, containing 0.1 mmol/L phenylmethyl sulfonyl difluoride and 2 mg/ml pepstatin, leupeptin, and aprotinin) for 20 minutes on ice. Cell debris were then pelleted at 13,000 × g for 15 minutes. The protein concentration of the supernatant was determined using the BCA reagent (Pierce, Rockford, IL). After separation by reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the lysates (20 mg protein per lane), proteins were transferred to a polyvinylidene difluoride membrane and blocked with 5% skim milk in 0.1% Tween 20 in PBS-T. The membranes were incubated with the primary antibody against phospho-Akt (9271, 1:1000 in 1% skim milk in PBS-T; New England Biolabs GmbH, Schwalbach, Germany), Akt (9272, 1:1000 in 5% skim milk; New England Biolabs GmbH), phospho-MAPK 1 and 2 (9106, 1:200 in 1% skim milk in PBS-T; New England Biolabs GmbH), or Bax (sc-526, 1:1000 in 5% skimmed milk in PBS-T; Santa Cruz Biotechnology, Inc.). Membranes were washed in PBS-T and then incubated with horseradish peroxidase-conjugated secondary antibodies against rabbit IgG (1:2500 in 1% skim milk, Santa Cruz Biotechnology). For Western blot analysis of Bcl-2 levels (sc-7382, 1:200; 5% skim milk; Santa Cruz Biotechnology), a horseradish peroxidase-conjugated secondary antibody against mouse IgG was used (1:2000 in 1% skim milk in PBS-T, Santa Cruz Biotechnology, Inc.). MAPK 1 and 2 protein levels were detected using a primary antibody (sc-93-G, Santa Cruz Biotechnology Inc.) diluted 1:500 in 1% skim milk in PBS-T, and a horseradish peroxidase-conjugated secondary antibody against goat IgG (1:10,000 in PBS-T, Santa Cruz Biotechnology Inc.). Labeled proteins were detected using the ECL-plus reagent (Amersham, Arlington Heights, IL). To estimate the relative expression levels of the different proteins, the expression patterns were analyzed in the same retinal protein lysate. At least four different retinal protein lysates were used to study each effect. In addition, lysates were prepared from primary RGC cultures grown for 48 hours in either normal conditions, without growth factors, or a combination of growth factor withdrawal and increasing concentrations of flupirtine (10 to 200 μmol/L). After cellular lysis, lysates and Western blots were prepared as described above. The whole-cell patch-clamp technique was used to measure membrane currents in primary RGCs.19Hamill OP Marty A Neher E Sakmann B Sigworth FJ Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches.Pflugers Arch. 1981; 391: 85-100Crossref PubMed Scopus (15119) Google Scholar Cells were subjected to electrophysiological recordings after 3 to 8 days in culture. The culture dishes were placed on the stage of an inverted microscope (Axiovert 135; Zeiss, Oberkochen, Germany). RGCs were identified by their size, typical morphology, and current profile.20Lipton SA Tauck DL Voltage-dependent conductances of solitary ganglion cells dissociated from the rat retina.J Physiol. 1987; 385: 361-391PubMed Google Scholar Single-cell recording was then performed at room temperature (20 to 25°C). All indicated solutions were applied by continuous perfusion of the culture dishes. The following drugs were applied to the extracellular solution by a perfusion system: flupirtine (200 μmol/L, dissolved in 100% dimethyl sulfoxide), Ba2+ (1 mmol/L), and rTertiapin-Q (50 nmol/L; Alomone Labs Ltd., Jerusalem, Israel), a blocker of Kir1.1 and Kir3 channels.21Jin W Lu Z A novel high-affinity inhibitor for inward-rectifier K+ channels.Biochemistry. 1998; 37: 13291-13299Crossref PubMed Scopus (207) Google Scholar, 22Kitamura H Yokoyama M Akita H Matsushita K Kurachi Y Yamada M Tertiapin potently and selectively blocks muscarinic K(+) channels in rabbit cardiac myocytes.J Pharmacol Exp Ther. 2000; 293: 196-206PubMed Google Scholar An EPC-9 amplifier and the Pulse software (Heka, Lambrecht, Germany) were used to generate voltage jumps, inject constant currents and acquire data. A routine correction for leak currents and capacitive transients was performed using a P/n method. Only experiments with series resistances below 30 MΩ were used for evaluation. Series resistance errors were compensated in the range of 30 to 60% with a routine of the Pulse software. Data analysis was performed with the program PulseFit (Heka). Micropipettes were pulled from borosilicate glass capillaries (Harvard Apparatus Ltd., Edenbridge, UK) on a horizontal puller (Zeitz Instumente, Augsburg, Germany). When filled with internal solution the pipette resistance ranged from 2 to 6 MΩ. Patch pipettes were filled with an intracellular solution containing (in mmol/L): 130 KCl, 10 NaCl, 2 MgCl2, 10 EGTA, and 10 Hepes. The external solution contained (mmol/L): 130 NaCl, 5 KCl, 2 MgCl2, 2 CaCl2, 10 Hepes, and 10 d-glucose. The external solution for perfusion experiments contained (mmol/L): 104 NaCl, 50 KCl, 2 CaCl2, 2 MgCl2, 10 Hepes, and 10 d-glucose. Solutions were adjusted to pH 7.4. To measure Kir currents, we changed the external solution to one containing 50 mmol/L KCl in place of 50 mmol/L NaCl. Inwardly rectifying potassium (Kir) currents were distinguished from other potassium currents by their sensitivity to the application of Ba2+ and tertiapin.21Jin W Lu Z A novel high-affinity inhibitor for inward-rectifier K+ channels.Biochemistry. 1998; 37: 13291-13299Crossref PubMed Scopus (207) Google Scholar, 22Kitamura H Yokoyama M Akita H Matsushita K Kurachi Y Yamada M Tertiapin potently and selectively blocks muscarinic K(+) channels in rabbit cardiac myocytes.J Pharmacol Exp Ther. 2000; 293: 196-206PubMed Google Scholar, 23Scroggs RS Todorovic SM Anderson EG Fox AP Variation in IH, IIR, and ILEAK between acutely isolated adult rat dorsal root ganglion neurons of different size.J Neurophysiol. 1994; 71: 271-279PubMed Google Scholar Inward currents were elicited by applying hyper- and depolarizing voltage steps from −150 mV to 0 mV in 10 mV steps starting from a holding potential of −20 mV. Data are presented as mean ± SEM. For multiple group comparisons, statistical significance was assessed using a Bonferroni-corrected one-way analysis of variance. Student's t-test was used to asses RGC densities in vitro as well as the results from the patch clamp experiments. A P value less than 0.05 was considered to be statistically significant. In rats with MOG-induced optic neuritis we analyzed the effects of flupirtine as an add-on therapy to IFN-β1a, one of the immunomodulatory therapies commonly used in MS patients. Three hundred thousand units of IFN-β1a were applied subcutaneously three times per week. Twenty-four hours after being fed with pelleted rat chow incorporating 0.6125 g/kg flupirtine, the rat plasma levels of flupirtine were within the therapeutic range achieved in patients taking a standard dosage of flupirtine (Figure 1A).24Niebch G Borbe HO Hummel T Kobal G Dose-proportional plasma levels of the analgesic flupirtine maleate in man. Application of a new HPLC assay.Arzneimittelforschung. 1992; 42: 1343-1345PubMed Google Scholar In comparison to vehicle-treated controls, the application of flupirtine had no effect on the clinical disease course of MOG-induced EAE. In contrast, application of IFN-β1a with and without combining it with flupirtine significantly improved the functional deficits at each time point analyzed (Figure 1B). The general clinical score in our animal model is mainly determined by spinal cord lesions, visual functions are not included.25Weissert R Wallstrom E Storch MK Stefferl A Lorentzen J Lassmann H Linington C Olsson T MHC haplotype-dependent regulation of MOG-induced EAE in rats.J Clin Invest. 1998; 102: 1265-1273Crossref PubMed Scopus (220) Google Scholar We performed electrophysiological recordings of VEPs to examine the effects of flupirtine and IFN-β1a treatment on visual functions. In an individual rat, an intact function of both, the neuronal cell bodies in the retina and their associated axons in the ON is essential to generate VEPs. VEPs represent the electrical response of the visual association cortex to a light stimulus presented to one eye. Pattern VEPs with different sizes of alternating black and white bars were used to estimate the animal's visual acuity. We have previously shown that healthy sham-immunized rats have visual acuity values of 1.31 ± 0.16 cycles per degree.14Meyer R Weissert R Diem R Storch MK de Graaf KL Kramer B Bähr M Acute neuronal apoptosis in a rat model of multiple sclerosis.J Neurosci. 2001; 21: 6214-6220Crossref PubMed Google Scholar A severe decline of specific cortical potentials in response to pattern stimulation occurred at the day of clinical manifestation of the disease. At that time point none of the vehicle-treated controls had detectable visual acuity values (Figure 2, A–E). Rats treated with flupirtine together with IFN-β1a showed significantly higher visual ac
Referência(s)