Morphine

Acetaminophen (APAP, 4-Acetamidophenol; Sigma-Aldrich, UK) and morphine (Sigma-Aldrich, UK) were prepared in 0.9% saline (with warming for APAP). WIN55,212-2 (Tocris, UK) was made up in 5% DMSO/5% Tween 80/0.9% saline, RTX (Sigma-Aldrich, UK) in 10% ethanol/10% Tween 80/saline, indomethacin (Sigma-Aldrich, UK) in 5% sodium hydrogen carbonate (pH to 7.4), cinnamaldehyde (Fluka) in 0.5% Tween 80/saline and brewer’s yeast; (Acros Organics) in 0.9% saline. BCTP (7-tert-butyl-6-(4-chloro-phenyl)-2-thioxo-2,3-dihydro-1H-pyrido[2,3-d]pyrimidin-4-one) was suspended in 0.5% methyl cellulose. CHEM5861528 (Chembridge Corporation, USA), capsaicin (Sigma-Aldrich, UK) was administered in 10% DMSO/saline. Mice were exposed to isoflurane (Abbott Industries Ltd. UK) at 2.5% in 95%O₂/5% CO₂ for 5 minutes.

Morphine was obtained from Sigma Chemicals (Catalog# M8777, St. Louis, MO). Human and bovine forms of BCM-7 were custom synthesized by Neopeptide (Cambridge, MA).

Pure standards for GC-MS analysis were purchased from Sigma-Aldrich (St. Louis, MO) and Extrasynthese (Lyon, France). Glutamate, morphine, Tween 80 (polyoxyethylene sorbitan monooleate), and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO). All salts used in physiological solution, sucrose, glucose and NaOH, were purchased from Vetec (Rio de Janeiro, Brazil). HEPES [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid] was acquired from Amresco (Solon, OH).

Human SH-SY5Y neuroblastoma cells purchased from ICLC-IST (Genoa, Italy) were cultured in Dulbecco’s modified Eagle medium (DMEM), supplemented with 10% (v/v) fetal bovine serum (FBS), 100 units/mL penicillin, 100 μg/mL streptomycin and 2 mM glutamine. Cells were incubated at 37 °C in a humidified atmosphere containing 5% CO2 and were allowed to reach 80% confluence before starting any treatment. All reagents employed for cell culture were purchased from Lonza (Milan, Italy). SH-SY5Y cells were exposed to 10 μM morphine (Carlo Erba, Cornaredo, Italy), 0.25 μM buprenorphine (Sigma-Aldrich, Milan, Italy) or 10 μM tapentadol (Grünenthal GmbH, Aachen, Germany) in accordance with previous studies regarding morphine and tapentadol [37 (link),61 (link),62 (link)]. As regards buprenorphine, we referred to the drug potency of buprenorphine vs. morphine (25–100% more potent than morphine); therefore, we decided to use 0.25 μM [63 (link)].
Moreover, to test the influence of MOR activation on ROS generation, cells were pretreated with 10 μM naloxone (non-selective opioid antagonist, Tocris Biotechne, Milan, Italy) or with 10 μM β-funaltrexamine, β-FNA (MOR selective antagonist, Sigma-Aldrich, Milan, Italy), 30 min before morphine. Four/six biological replicates per treatment were utilized in each experiment.

Morphine (Sigma; Cat No: 25-27-2) and 5-aza-2′-deoxycytidine (5-aza-dC; Sigma; Cat No: 2353-33-5) were dissolved in saline; saline was set as the control. Suberoylanilide hydroxamic acid (SAHA; Selleck Chemicals, Houston, TX, United States; Cat No: S1047) was dissolved in 5% dimethyl sulfoxide (DMSO; Sigma; Cat No: D2650-100 mL); 5% DMSO was set as the control. Morphine was subcutaneously given with a dose of 1.5 mg per kg of body weight. The use of 5-aza-dC was continued for three consecutive days at 5 μg/5 μL daily, 30 min before surgery. SAHA was continued for 7 consecutive days, at a dose of 50 mg/kg in a volume of 5 μL, 30 min before surgery. All drug doses are based on preliminary experiments (Zhou et al., 2014 (link); Cao et al., 2015 (link); Garriga et al., 2018 (link)), and did not alter the nociceptive response per se. Intrathecal injections were performed manually between L5 and L6 spinal segments, through the theca of the spinal cord into the subarachnoid space (Hylden and Wilcox, 1980 (link)). On entry of the needle, a quick flicking motion of the mouse’s tail or leg was noticed. For anesthesia, sodium pentobarbital and isoflurane (Sigma; Cat No: 26675-46-7) were used.

The solvents hexane, ethyl acetate, methanol, butanol and acetic acid were purchased from Trinity College Dublin HMF facilities (Dublin, Ireland). High-performance liquid chromatography (HPLC) grade water was obtained from a deionized water treatment system from PureLab Option, -Evoqua (Pittsburgh, PA, USA). Morphine, acetylsalycilic acid (ASA), formalin and carrageenan were purchased from Sigma-Aldrich, Dublin, Ireland.