Culturing adult canine sensory neurons to optimise neural repair

Adult dorsal root ganglion (DRG) neurons as a versatile tool to study neuron-glia interactions ( Päiväläinen and others 2008 ) were initially isolated from embryonic chick and neonatal rodents ( Barbin and others 1984 ) and later on from adult rats ( Grothe and Unsicker 1987 ). Since protocols suitable for isolation of adult canine DRG neurons have not been made available so far, chimeric assays were established combining canine cells with fetal rat neurons ( Kamishina and others 2009 ). Briefly, ganglia were minced using scalpel blades and incubated in a mixture of type I trypsin, type IV-S hyaluronidase and type XI collagenase (each 0.5 per cent, Sigma-Aldrich) in Dulbecco's modified Eagle medium (DMEM; Gibco, Invitrogen) for 45 minutes at 37°C. Tissue was mechanically dissociated in DMEM with DNase I (0.05 per cent, Roche Diagnostics Mannheim) using successively narrowed flame-constricted Pasteur pipettes, pelleted by centrifugation (10 minutes, 200 xg), counted in a Neubauer chamber and seeded in DME medium at a density of 200 neurons per well on microtiterplates (CLS 3696, Corning, Sigma-Aldrich) coated with poly-l-lysin (100 μg/ml, Sigma-Aldrich) and poly-l-lysin/laminin (Becton Dickinson).