4.1. Isolation and Cultivation of Primary Canine Dorsal Root Ganglion Neurons

Dorsal root ganglia (DRG) were obtained from 3 female and 5 male, 6 months old, healthy Beagle dogs, which were control animals from an unrelated study (33.19-42502-05-16A043). No animals were killed for the purpose of this particular study. The experiments were approved by the Lower Saxony State Office for Consumer Protection and Food Safety (permission number: 16A 024). Isolation and dissociation of primary DRG neurons was performed as described previously [31] with slight modifications. Briefly, surrounding fat and connective tissue as well as blood vessels were removed from the DRG and they were minced and kept on ice in Hanks’s balanced salt solution (HBSS) 1x (Gibco, Life Technologies; Paisley, UK). Consequently, they were digested with type IV and type XI collagenase, type IV hyaluronidase and trypsin (Sigma-Aldrich Chemie GmbH; Taufkirchen, Germany) for 60 min at 37 °C. Afterwards, they were dissociated using successively narrowed, flame-constricted Pasteur pipettes and the addition of DNase I (Roche Diagnostics GmbH; Mannheim; Germany). Cells were centrifuged and resuspended in Dulbecco’s modified Eagle’s Medium (DMEM; Gibco, Life Technologies; Paisley, UK) supplemented with 10% fetal calf serum (FCS; PAA Laboratories GmbH; Pasching, Austria) and neurons were separated from the other cells using a two-step Percoll-gradient (25% and 27%) (GE HealthCare BioSciences, Fisher Scientific; Rockford, Tempe, AZ, USA). Neurons were resuspended in Sato’s medium [63], counted in a Neubauer chamber (Roth C. GmbH & CO KG; Karlsruhe, Germany), and plated at a density of 150 neurons per well in half-area 96 well microtiter plates (Nunc GmbH & CO KG; Wiesbaden, Germany) coated with poly-L-lysin (100 µg/mL; Sigma-Aldrich Chemie GmbH; Taufkirchen, Germany) and laminin (Becton, Dickinson and Company; Franklin Lakes, NJ, USA). Neurons were identified in the Neubauer chamber by their large, round, granular cell-body, which was surrounded by a phase-bright halo [46]. Neurons were maintained in Sato’s medium supplemented with 1% bovine serum albumin (BSA; Sigma-Aldrich Chemie GmbH; Taufkirchen, Germany) and 2.1 ng/100 µL nerve growth factor (NGF; Peprotec Inc., Tebu-Bio; Frankfurt, Germany). Cultures were kept in an incubator at 37 °C and 5% CO₂. Twice weekly 60% of the medium was exchanged with fresh medium. Non-cryo and cryo neurons were observed by phase contrast microscopy at 24 h in culture in order to study their morphology and assess the general presence of neurite outgrowth. Quantification of neurite outgrowth was carried out after fixation and immunofluorescence at 6 DiV (Figure 6).

Experimental design: while non-cryo neurons were directly seeded after isolation, dissociation and counting, cryo-neurons were cryopreserved an thawed after 3 months. The day the neurons were seeded represents day 0 of the experiment. Part of the cultures were fixed for comparative immunofluorescence and morphometric analysis at 6 DiV for non-cryo and cryo neurons and at an additional time-point (12 DiV) for cryo neurons. The remaining DRG neuronal cultures were infected with 2 different strains of CDV (CDV-5804 PeGFP, CDV R252) at 6 DiV and kept in culture until 12 DiV or 6 dpi, respectively, when they were processed for immunostaining and further analysis. Non-cryo = non-cryopreserved neurons, cryo = cryopreserved neurons, DiV = days in vitro, dpi = days post infection, CDV = canine distemper virus, eGFP = enhanced green fluorescent protein.