Boldine

Daily boldine (Millipore Sigma, Jaffrey, NH, USA) administration started at 3 dpi. boldine was added to cages at 2:00 PM. It was prepared by dissolving boldine in a mix of DMSO and peanut oil (Sigma). This mixture was then added to peanut butter (PB) so that 1.0 g of total bolus could be given once per day at a dose of 50 mg/kg. The final concentration of DMSO was less than 2.5%. Single-housed animals were familiarized with 1.0 gram of peanut butter for a week prior to surgery. All animals consumed 100% of their daily PB mix within 1 h and continued to do so throughout the remainder of the study. Vehicle-treated SCI and laminectomy-only animals (shams) received daily equal amount of the PB mix without boldine.

A total of 97 retinal explants obtained from P9 wild-type mice from 50 wild-type animals were cultured as previously described [32 (link),33 (link)] and divided among the experiments. Briefly, the retina was placed with the pigment epithelium facing down on cell culture inserts (Millicell, PIHA03050, Merck, Darmstadt, Germany) with 1 mL of culture containing 10% of fetal bovine serum (FBS), which was replaced every two days. The cultures were incubated at 37 °C in a 5% CO₂ water-jacketed incubator (Thermo Scientific, Waltham, MA, USA). Once retinal explants were cultured, an adaptation period of two days was performed before the start of the different treatments. Moderate glucose concentration (15 mM) was used as a control, and high glucose (30 mM) was used to simulate diabetic conditions. The reagents used in this study were boldine (100 µM) (Cat# B3916-5, Sigma Aldrich, St. Louis, MO, USA), L-NAME (1 mM) (Cat# 80210, Cayman, Ann Harbor, MI, USA), and SIN-1 (10 µM) (Cat# 0756, Tocris Bioscience, Bristol, UK).