Primary tDRG cultures

Bilateral thoracic (T1–T4) DRG neurons were harvested and cultured from neonatal (P0–P5) mice as previously described³⁹. Briefly, ganglia were removed under sterile conditions and enzymatically dissociated at 37 °C in Hank’s balanced salt solution (HBSS) containing trypsin (180–200 U/ml; Worthington, Freehold, NJ, USA) and buffered with HEPES (pH 7.4). The resulting cell suspension was washed twice in serum-containing Leibovitz's L-15 medium to inactivate the trypsin and plated on laminin-coated glass-bottom Petri dishes (35 mm) made in-house. The neurons were grown in L-15 medium supplemented with vitamins, cofactors, penicillin–streptomycin, 5 mM glucose, 5% rat serum and NGF (10 ng/ml; Alomone Labs, Jerusalem, Israel). Cultures were maintained at 37 °C in a humidified atmosphere of 95% air-5% CO₂ and fed every 4 days with growth media. To eliminate non-neuronal cells, cultures were treated with cytosine arabinoside (10 μM; Sigma, St. Louis, MO, USA) from days 2 to 4, which prevents the proliferation of non-neuronal cells resulting in a neuron-enriched primary culture. Established DRG cultures were incubated for 24 h at 37 °C; (5% CO₂; humidified) in growth media alone (control) or with 1 µg/ml lipopolysaccharide (LPS, Sigma-Aldrich, MO, USA)-supplemented growth media. To inhibit RAGE function, some cultures were incubated with either the RAGE antagonist FPS-ZM1 (10 μM), or the vehicle (0.01% dymethyl sulfoxide), for 48 hr before electrophysiological recording.