Serum free b 27

The mouse major pelvic ganglion tissues were prepared and maintained as described previously^{43 (link)} with minor modifications. The MPG tissues were isolated from male mice using a microscope, transferred into sterile vials containing Hank’s balanced salt solution (Gibco), and then rinsed and washed twice in PBS. The MPG tissues were cut into small pieces and the samples plated on poly-D-lysine hydrobromide-coated (Sigma-Aldrich) 12-well plate. The whole MPG tissue was covered with matrigel and the culture plate placed on ice for 5 minutes prior to incubation at 37 °C for 10–15 minutes in a 5% CO₂ atmosphere. We added 1 mL of complete Neurobasal medium (Gibco) supplemented with 2% serum-free B-27 (Gibco) and 0.5 nM GlutaMAX™-I (Gibco). The dishes were then incubated at 37 °C in a 5% CO₂ atmosphere. Three days after incubation, we evaluated neurite outgrowth.

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).