Bd1063

Spinal cord organotypic cultures (SCOCs) were prepared from lumbar sections of Sprague–Dawley pups (8–9 days-old) as previously described (Mòdol-Caballero et al., 2017 (link)). After harvesting, the spinal cord was cut in 350 μm thick transverse sections, that were transferred on Millicell-CM nets (0.4 μm, PICM03050, Millipore) and then into a six-well plate with the incubation medium [50% (v/v) minimal essential medium (MEM, M5775, Sigma), 2 mM glutamine, 25 mM HEPES, 25% (v/v) Hank’s Balanced Salt Solution (HBSS−/−, 14,175, Gibco) supplemented with 25.6 mg/ml glucose and 25% (v/v) heat-inactivated horse serum (26,050–088, Gibco), pH = 7.2). After 7 days in vitro (DIV), chronic excitotoxicity was induced by adding DL-threo-β-hydroxyaspartic acid (THA; 100 μM), a selective inhibitor of glutamate transport (Rothstein et al., 1993 (link)). The Sig-1R ligands were simultaneously co-added in the culture medium and renewed at each medium change twice per week. Sig-1R ligands PRE-084, BD1063 and SA4503 (Tocris) were tested at three different concentrations (30, 3 and 0.3 μM). Riluzole (5 μM) was also assayed as positive control. Slices were maintained for 28 DIV and then fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS). The in vitro experiments have been performed in three independent cultures and resulting in 12 slices for each experimental condition.

Cocaine HCl was obtained commercially from Sigma-Aldrich and the Hässleholm Hospital Pharmacy, Sweden, and dissolved in saline. BD 1063 was purchased from Tocris and dissolved in saline. An initial batch of Siramesine was provided by H. Lundbeck A/S and a second batch was purchased commercially from Sigma-Aldrich. Siramesine was dissolved in saline using 1% Tween80 and subsequent sonication for 10 min.

Dextromethorphan hydrobromide and quinidine sulfate were provided by Avanir Pharmaceuticals, Inc. (Aliso Viejo, CA; for the behavioral studies) or purchased from Sigma-Aldrich (St. Louis, MO; for the binding assays). Imipramine hydrochloride was purchased from Sigma-Aldrich (St. Louis, MO). Fluoxetine hydrochloride, BD1063 (1-[2-(3,4-dichlorophenyl)ethyl]-4-methylpiperazine dihydrochloride), and BD1047 (N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(dimethylamino)ethylamine dihydrobromide) were obtained from Tocris (Ellisville, MO). [³H](+)-Pentazocine (34.8 Ci/mmol) was procured from Perkin Elmer (Hopkington, MA). All other chemicals and reagents were purchased from standard commercial suppliers (Sigma-Aldrich, St. Louis, MO).

The drugs used were paclitaxel and the σ₁ receptor antagonist BD-1063 (both from Tocris Cookson Ltd., Bristol, United Kingdom). Paclitaxel was dissolved in a solution of 50% Cremophor EL and 50% absolute ethanol to obtain a concentration of 6 mg/ml. This paclitaxel solution was diluted in sterile physiological saline to a final concentration of 2 mg/10 ml just before its administration. For control treatments, the paclitaxel-vehicle solution was also diluted just before its administration in saline at the same proportion as the paclitaxel solution. Paclitaxel (2 mg/kg) was administered intraperitoneally (i.p.) in a volume of 10 ml/kg once per day for 5 consecutive days (cumulative dose of 10 mg/kg); a schedule of paclitaxel treatment that produces a painful neuropathy in mice [5 (link),28 (link),29 (link),40 (link)]. The control group was administered with the vehicle for paclitaxel according to the same schedule.
BD-1063 (32 mg/kg) was dissolved in saline just before the s.c. administration of a volume of 5 ml/kg in the interscapular area. This dose of BD-1063 produces a significant anti-allodynic effect in several models of pain [23 (link),28 (link),62 (link)]. The control animals received s.c. the same volume of saline. BD-1063 was s.c. administered to avoid any possibility of chemical interaction with paclitaxel solution (which was i.p. administered).