Protocol and consent forms were approved by the Committee for Ethics of the University of Munich and ethical approval to study drug effects in healthy volunteers was provided by the German Federal Institute for Drugs and Medical Devices. In accord with the declaration of Helsinki (Seoul 2008) all participants provided their written informed consent prior to participation in the study. ClinicalTrials registration is NCT01450865.
In vitro studies
Procurement and preparation of human sural nerve Segments of sural and peroneal nerve were obtained from patients previously scheduled for biopsy (n=7; 1 female, 6 male, age range: 48–80). The diagnosis precipitating biopsy was typically polyneuropathy of unknown aetiology. Patients were informed about the procedure by an anaesthesiologist prior to surgery. Upon surgical removal nerve segments were immediately placed in physiological solution containing (in mM): 117 NaCl, 3.6 KCl, 2.5 CaCl2, 1.2 MgCl2, 1.2 NaH2PO4, 25 NaHCO3, 11 D-glucose (bubbled to pH 7.4 with 95 % O2 – 5 % CO2).
Immunohistochemistry Individual fascicles were isolated from nerve segments and teased apart with fine forceps. After incubating in PBS containing 0.3% collagenase (30 min, room temp.) and washing twice in PBS, preparations were mounted on gelatine-coated slides, dried overnight and fixed in 4% paraformaldehyde for 30 minutes. For immunostaining, preparations were blocked in PBS containing 0.2% Triton 100 and 10% natural goat serum at room temperature for 40 minutes, followed by overnight incubation at 4°C with rabbit anti-KCNQ2-N antibodies diluted 1:200  raised and affinity-purified as previously described [19, 20]. KCNQ2 antibodies were directed against residues 13–37 of the conserved intracellular N-terminal region [21, 22]. Monoclonal antibodies against peripherin (diluted 1:200) and the alpha subunits of voltage-gated (NaV) sodium channels (panNaV, diluted 1:200) were used. After incubation with the primary antibody, slides were washed three times in PBS and incubated with fluorescein- and rhodamine-conjugated donkey cross-affinity-purified secondary antibodies at room temperature for two hours (all diluted 1:400). Slides were then washed three times with PBS and covered with Aqua-Poly/Mount Coverslipping medium. Digital images were taken with a laser-scanning confocal microscope (Bio-Rad MRC-1024, Bio-Rad Laboratories GmbH, Munich, Germany). No evidence of antibody cross-reactivity was observed in multilabeling experiments with single label and secondary-only controls.
Threshold tracking Nerve fascicles were mounted between stimulating and recording electrodes in an organ bath perfused continuously with physiological solution tempered to 32°C. Constant current was used for stimulation (1 ms, A395, WPI, Sarasota, USA) and extracellular signals were amplified (NPI, Tamm, Germany), filtered (30 Hz - 1.3 kHz), sampled (BNC 2120, National Instruments, USA) and stored to disk.
Electrical excitability of myelinated axons was assessed using the TRONDXS4 protocol in QTRAC (© Prof. Bostock, Institute of Neurology, London, UK).
Study design Volunteers were recruited for a randomised, double-blind, placebo-controlled, two-way, cross-over clinical phase I trial (RCT) to investigate the effect of oral flupirtine on the electrical excitability of peripheral myelinated axons. Exclusion criteria were: ongoing medication, prevailing organic disease, previous forearm trauma, primary organ failure, pregnancy or breast-feeding. All female participants were subject to a pregnancy test (Cyclotest, UEBE, Wertheim, Germany) prior to inclusion.
Subjects were assigned randomly to a study arm in accordance with an algorithm developed by the Institute of Medical Information Technology, Biometry and Epidemiology, University of Munich, Germany (IBE), stratifying for gender. Subjects received a set of sealed sequentially numbered pill-boxes each containing either flupirtine or placebo tablets to be taken prior to the recording sessions. Both the subjects and study physicians were blind to the group allocation.
Preliminary results indicated that a sample size of 12 would be needed to detect a decrease in the primary outcome measure (RRP) with 80% power at a significance level of 5%. With provision for drop-outs, 20 subjects were recruited, with an allocation ratio of 1:1.
Participants were evaluated on seven occasions in which motor axon excitability parameters and ischemic EMG signals were recorded (Additional file 1: Figure S1). The first 3 recording sessions, scheduled not less than 48 hours apart, were used to establish baseline values. The 4th recording session comprised 2 sequential recordings of axonal excitability, one immediately prior to pharmacologic intervention and a second two hours after. EMG during ischemia was only recorded after medication. The 5th session was a control recording performed 48 hours after the 4th. The 6th recording session was scheduled 7 ± 1 days after the 5th and comprised 2 sequential recordings immediately before and 2 hours after medication. The final (7th) recording session was a control recording performed 48 hours after the 6th. A pain questionnaire was assessed after the 3rd, the 4th and the 6th session.
At the time of examination, subjects lay comfortably in a temperature controlled room.
Outcome measures The primary outcome measure was the relative refractory period (RRP) as determined with threshold tracking. All other indices were considered secondary outcome measures.
Threshold tracking All experiments were performed by the same examiner. Skin temperature was determined at the wrist at the end of each recording session using a thermocouple (Voltcraft, Hirschau, Germany).
The median nerve of the right hand was used for nerve excitability studies. A gel disc electrode (H92SG, Kendall-Arbo, Neustadt, Germany) positioned at the wrist between palmaris longus and flexor carpi radialis tendons served as the cathode. The anode was placed over the course of the median nerve approximately 2 cm proximal to the cathode. EMG from abductor pollicis brevis (APB) was recorded from one electrode situated over the motor point and a second electrode over the proximal interphalangeal joint.
Constant current pulses (DS5, Digitimer, UK) were used to determine electrical excitability in QTRAC using the TRONDXM4 protocol with same measures as for in vitro experiments (see above). The EMG signal was amplified (gain 200-500x), filtered (bandpass 0.3 Hz to 10 kHz) and sampled at 50 kHz (BNC-2120, National Instruments, Austin, USA).
Ischemia A cuff around the upper arm inflated to >200mmHg was used to examine flupirtine and placebo effects on ischemic and post-ischemic EMG from APB. Muscle fasciculations are known to appear in some subjects upon recovery from a 10 minute period of ischemia of the lower arm with the intensity and incidence increasing with the period of ischemia . EMG signals were filtered, amplified and sampled at 20 kHz (ADInstruments GmbH, Spechbach, Germany). EMG was recorded continuously over a 21 minute period comprising one minute baseline followed by 10 minutes of ischemia and a further 10 minutes post-ischemia.
Volunteers rated the intensity of sensations before, during and after ischemia on a visual analogue scale (0–100 mm with 0 being no pain and 100 being maximal pain) and completed the Short Form McGill Pain Questionnaire (SF-MPQ) for sensations experienced during the first 5 minutes following release of the pressure cuff, i.e. post-ischemia. The SF-MPQ comprises eleven sensory and four affective pain descriptors that can be ranked in intensity from 0 = none to 3 = severe. The sum of ranked values provides a sensory (SPRI; 0–33), an affective (APRI; 0–12) and a total pain score (TPRI; 0–45).
Axonal excitability Electrical excitability of myelinated axons determined both in vitro and in vivo were analyzed off-line with the MEM routine in QTRAC. Six parameters were taken directly from the QTRAC analysis, namely the strength-duration time constant, rheobase current, refractoriness at 2 and 2.5 ms, superexcitability at 5 and 7 ms and during the 90-100 ms hyperpolarizing and depolarizing current pulses of the threshold electrotonus. The relative refractory period was determined using custom written software in Igor Pro (WaveMetrics, Lake Oswego, USA) by determining the first zero crossing of an exponential fit to recovery cycle data points less than 5 ms.
Electromyography Rectified and integrated EMG was used to quantify continuous signals. EMG power was calculated by discrete fast Fourier transform (FFT) of EMG recorded in the time domain.
Mean and standard deviation are used for population descriptors while mean and standard error of the mean are indicated for comparisons between groups. Normality was tested using Kolmogorov-Smirnov analysis. For parametric datasets Student’s paired t-test was used for pairwise comparisons. Unpaired datasets were compared using one-way ANOVA with post hoc Tukey-Kramer. Non-parametric datasets were analysed using Wilcoxon ranked sign test for paired data, and Kruskal-Wallis ANOVA with post hoc Mann–Whitney-U tests for non-paired data. The level of statistical significance is indicated throughout with * for p < 0.05 and ** for p < 0.01.
Chemicals and drugs
Triton 100, natural goat serum and the monoclonal antibody against panNaV were purchased from Sigma-Aldrich (Munich, Germany), the monoclonal antibody against peripherin was obtained from Chemicon (EMD Millipore Corporation, Billerica, USA). Anti-KCNQ2/Kv7.2 was kindly provided by M. Schwake (Kiel, Germany). Fluorescein-and rhodamine-conjugated donkey cross-affinity-purified secondary antibodies were purchased from Invitrogen (Paisley, UK). Aqua-Poly/Mount Coverslipping Medium was purchased from Polysciences Europe (Eppelheim, Germany). Flupirtine (in vitro) was purchased from Sigma (Taufkirchen, Germany) and XE991 from Biotrend (Wangen, Switzerland). The final concentration of test substances used in vitro was achieved by diluting stock solutions in the perfusing solution on the day of the experiment.
Blinded oral preparations of flupirtine 100 mg (AWD.pharma GmbH & Co.KG, Radebeul, Germany) and placebo tablets (7 mm capsules (Winthrop Arzneimittel GmbH, Frankfurt, Germany) were performed by the Pharmaceutical Department, University of Munich encapsulating both in gelatine (size 00, white opaque; Shionogl Qualicaps S.A., Alcobendas / Madrid, Spain) containing lactose, ferric oxide and food colouring E172 (all Fagron GmbH, Barsbüttel, Germany).