Collagenase a

Mice were anesthetized with isoflurane and sacrificed by decapitation and the TG or dura mater were removed and prepared for culture as previously described (24 (link)). For cultures with back-labelled TG neurons, TGs were taken 7 days following supra-dural application of the retrograde tracer, wheat germ agglutinin conjugated Alexa-Fluor 555 nm. After removal, tissue was placed in ice-cold Hanks balanced-salt solution (divalent free). Trigeminal tissue was dissociated enzymatically with collagenase A (1 mg/ml, 25 min, Roche, Indianapolis, IN) and collagenase D (1 mg/ml, Roche, Indianapolis, IN) with papain (30 units/ml) for 20 min at 37°C. Dural tissue was dissociated enzymatically with collagenase A (1 mg/ml, 30 min, Roche, Indianapolis, IN) and collagenase D (1 mg/ml, Roche, Indianapolis, IN) with papain (30 units/ml) for 25 min at 37°C. The tissues were then triturated through fire-polished Pasteur pipettes, and trigeminal cells were plated on poly-D-lysine (Becton Dickinson) and dural cells plated on untreated plates. After several hours at room temperature to allow adhesion, cells were placed in a room-temperature, humidified chamber in Liebovitz L-15 medium supplemented with 10% FBS, 10 mM glucose, 10 mM HEPES and 50 U/ml penicillin/streptomycin. Trigeminal cells were tested within 24 hours post plating and dural cell cultures were tested 2–4 days post plating.

Control and infected mice were sacrificed at 14 and 42 days post-infection, and one rear ankle joint and one half heart were stored immediately in dry ice and transferred to −80 °C until the time of DNA extraction. Each tissue was pulverized with liquid nitrogen pre-chilled mortar and pestle and transferred to 2.5 mL of a 1 mg/mL collagenase A (Boehringer Mannheim) solution in phosphate-buffered saline (pH 7.4). Digestions were carried out for 4 h at 37 °C. An equal volume of proteinase K solution (0.2 mg of proteinase K per mL, 200 mM NaCl, 20 mM Tris–HCl [pH 8.0], 50 mM EDTA, 1% sodium dodecyl sulfate) was added to collagenase digested tissues, and the mixture was incubated overnight at 55 °C. DNA was recovered by extraction of the digested sample with phenol-chloroform and subsequent ethanol precipitation. Resuspended samples were incubated with 0.1 mg/mL of DNase-free RNase for 1 h at 37 °C. Extractions and precipitations were repeated, and DNA was resuspended in 0.5 mL of TE. DNA concentration was determined by A260 and samples were used for quantitative PCR assays.