All experiments and procedures were carried out according to the German guidelines and regulations of the care and treatment of laboratory animals and the European Communities Council Directive of November 24, 1986 (86/609/EEC), amended September 22, 2010 (2010/63/EU).
For CGRP release experiments from the dura mater and trigeminal ganglia, home bred adult Wistar rats of both sexes (260–410 g) as well as adult C57 BL/6 mice of both sexes and different genetic configuration were used: C57 BL/6 wild type mice (18–31 g), C57 BL/6 knock out mice with functionally deleted TRPA1 (18–30 g) or deleted TRPV1 (19–31 g) receptor channels. Animals were euthanized by CO2 inhalation and decapitated. Rat skulls were freed from skin, muscles, eyes and mandibles, and sagittally divided into two halves. The brain was carefully removed taking care not to damage the dura mater lining the skull. Trigeminal ganglia (TGs) were dissected together with their dura sheath by cutting the trigeminal branches, the ophthalmic, maxillary and mandibular nerves at their utmost distal sites accessible, and kept in wells of a microplate filled with 120 µL of synthetic interstitial fluid (SIF). The microplate was placed on the surface of a pre-warmed water bath (37°C). Mice skulls were flayed and sagittally divided into two halves. Mice brains were carefully removed from the skull. One of the mice skull halves of each animal was used for dura release experiments, while from the other half the TG was dissected and processed like rat TGs. Skull halves for dura experiments were washed in gently flowing SIF for 30 min at room temperature and subsequently mounted upon the surface of a water bath of 37°C with the opening upside. Mice skull halves were sealed with a rim of Vaseline® on their outer surface in order to avoid spillage due to hairline cracks in the filigree mouse skull, and mounted accordingly. Skull cavities were filled with 500 µL (rat) or 200 µL (mouse) of SIF. The same volumes were used for pre-incubating solutions for 1 hour as well as for the respective incubating steps of 5 minutes each during CGRP release experiments. Evaporation during pre-incubation was prevented by a self-adhesive plastic film attached on top of the water bath containers.
All tissue samples were incubated with either butterbur root extract solved in ethanol containing different concentrations of petasins (3 µg/mL, 10 µg/mL, 30 µg/mL, 100 µg/mL), isopetasin solved in ethanol (3 µg/mL, 10 µg/mL, 30 µg/mL) or ethanol equivalent to the respective concentration of petasins or isopetasin (0.0316%, 0.105%, 0.316%, 1.055% ethanol) for 1 hour and subsequently washed with SIF three times, before fluid samples were sampled for measurements of CGRP concentration using a pipette without touching the tissues. The first two samples were taken after incubation with SIF for 5 minutes each in order to measure the basal CGRP release from the dura mater and the trigeminal ganglia. Then the TRPA1 receptor agonist allylisothiocyanate (mustard oil, MO) 5 x 10− 4 M (Wistar, C57 BL/6 wild type, C57 BL/6 TRPA1 knock-out) or the TRPV1 receptor agonist capsaicin (Caps) 5 x 10− 7 M in SIF (C57 BL/6 TRPV1 knock-out) was applied and incubated for 5 minutes, followed by one 5 min application of SIF. Finally, caps 5 x 10− 7 M in SIF (Wistar, C57 BL/6 wild type, C57 BL/6 TRPA1 knock-out), or MO 5 x 10− 4 M (C57 BL/6 TRPV1 knock-out) was applied for incubation of 5 minutes. After each incubation step the fluid was carefully removed and SIF was added for washing the tissues, before the next solution was added. From each sample 100 µl were collected in Eppendorf cups, diluted with 25 µL EIA buffer (Bertin Pharma, France) containing peptidase inhibitors, and stored on ice or deep-frozen until they were processed for CGRP measurement.
CGRP was measured using a CGRP enzyme immunoassay (EIA) kit (Bertin Pharma/SPIbio, Montigny Le Bretonneux, France) according to the manufacturer’s instruction. The sandwich technique of this assay consists of immune reactions of capture and tracer antibodies recognizing different CGRP epitopes, and the enzymatic activity of acetylcholinesterase (AChE, Ellman’s reagent). The emitted light is subsequently spectrophotometrically detected as a measure for the CGRP concentration in the samples, displayed as pg/mL. The detection limit of the assay is 5 pg/mL according to the manufacturer’s information.
Statistical analysis was performed with STATISTICA (StatSoft 7.0, Tulsa, OK). The consecutive measurements at intervals of 5 minutes were analysed with repeated measures ANOVA and least square difference (LSD) post-hoc test on the basis of the raw data. Comparisons between different concentrations and groups were analysed by univariate ANOVA and least square difference (LSD) post-hoc test on the basis of data normalized to the mean of the two initial CGRP measurements after SIF application in each experiment to compensate for inter-assay variations. Data are presented as means ± SEM, p < 0.05 was considered statistically significant. Panels were created with Origin® 2019 (OriginLab, Massachusetts).
Chemicals: Synthetic interstitial fluid (SIF) is composed of (in mM): 107.8 NaCl, 26.2 NaHCO3, 9.64 Na-gluconate, 7.6 sucrose, 5.55 glucose, 3.5 KCl, 1.67 NaH2PO4 x 2 H2O 1.53 CaCl2 x 2 H2O and 0.69 MgSO4 x 7 H2O, adjusted to pH 7.4. Butterbur root extract (Petadolex®, Weber & Weber, Inning/Ammersee, Germany) was dissolved in ethanol at a concentration of 9.483 mg/mL and step-wise diluted with SIF to the applied concentrations of 100, 30, 10 and 3 µg/mL. Isopetasin (HWI Group, Rülzheim, Germany) was correspondingly dissolved in ethanol and diluted with SIF to the respective concentrations. Accordingly, ethanol concentrations in the vehicle solutions tested were 10.55, 3.16, 1.05 and 0.315 µL/mL in SIF. Mustard oil (MO) (Merck, Darmstadt, Germany) and capsaicin (Caps) (Sigma-Aldrich, Taufkirchen, Germany) were dissolved in ethanol 1% and diluted with SIF to the final concentrations of 5 x 10− 4 M (MO) and 5 x 10− 7 M (Caps).