*4.6. Primary Cultures of Neurons from Dorsal Root Ganglia*

Animals were sacrificed after 8 weeks from the intraperitoneal injection (at 16 weeks of age) and DRG neurons were obtained from all spinal levels of adult male CD1 mice as previously described [32,33]. The animals were exposed to CO2 inhalation (1 min) followed by decapitation according to the European Guidelines on Laboratory Animal Care, with the approval of the institutional Ethics Committee of the 'Horia Hulubei' National Institute of Physics and Nuclear Engineering (approval number, 31/11.06.2015). DRGs were removed under sterile conditions and were immediately transferred into IncMix solution (in mM, NaCl 155, K2HPO4 1.5, HEPES 5.6, Na-HEPES 4.8, glucose 5). After cleaning the ganglia from the surrounding tissue and counting them, DRGs were incubated in a mixture of 1 mg/mL Collagenase from *Clostridium histolyticum,* type 1A (#C9891, Sigma-Aldrich, St. Louis, MO, USA) and 1 mg/mL Dispase from *Bacillus polymyxa* (#17105041, GIBCO, Invitrogen, Carlsbad, CA, USA) in IncMix solution for 1 h at 37 ◦C. Following enzyme treatment, the ganglia were washed once in Dulbecco's modified Eagle's medium Ham's F-12 (DMEM F-12, #D8900, Sigma-Aldrich, St. Louis, MO, USA) with 10% horse serum (#H1270, Sigma-Aldrich, St. Louis, MO, USA), before mechanical trituration in 0.5 mL DMEM F-12. The dissociated neurons were then washed by centrifugation (at 1000× *g* for 10 min, 25 ◦C) followed by resuspension in fresh medium. Following the final wash, the cell pellet was resuspended in DMEM F-12, containing 10% horse serum and 50 μg/mL gentamicin (#G1272, Sigma-Aldrich, St. Louis, MO, USA). Following a second trituration, neurons were seeded on 13-mm coverslips, previously coated with 1 mg/mL poly-D-lysine (#P0899, Sigma-Aldrich, St. Louis, MO, USA) for 1 h at 37 ◦C and after 24 h were further processed for the immunostaining protocol. In the case of qRT-PCR protocol, the extracted DRGs were directly subjected to the RNA extraction protocol.
