Culturing Murine Pelvic Ganglia

MPG tissue was harvested and maintained as described previously.15 (link) Briefly, MPG tissue was isolated from male mice under a dissecting microscope and transferred into sterile vials containing Hank's Balanced Salt Solution (HBSS; Gibco). MPG tissue was then cut into small pieces and plated on 8-well Nunc Lab-Tek Chamber Slides coated with 0.1 mg/ml poly-D-lysine hydrobromide (Sigma-Aldrich). Tissue was covered with Matrigel (Ca# 354234, Becton Dickinson, Mountain View, CA, USA) and then plates were incubated for 5-10 minutes at 37°C in a humidified 5% CO₂ atmosphere, after which 200 µl of complete Neurobasal medium (Ca# 21103-049, Gibco) supplemented with 2% serum-free B-27 (Ca# 17504-044, Gibco) and 0.5 nM GlutaMAX-I (Ca# 35050-061, Gibco) was added. To mimic the in vivo neuroinflammatory conditions of CNI-induced ED, we treated MPG tissue with 2.5 μg/ml LPS (Sigma-Aldrich) immediately after adding medium, as described previously.30 (link) The medium was changed every 2 days, and 5 days later, tissue was fixed in 4% paraformaldehyde for at least 30 minutes and neurite outgrowths were immunostained with an anti-neurofilament antibody (Ca# N5389, 1:50; Sigma-Aldrich).