Many effects of TNF are mediated by synergistic interactions with other cytokines. of ion channels that mediate the transduction of thermal and mechanical stimuli or regulate excitability and action potential propagation (Bhave and Gereau, 2004). For example, the noxious heat transduction channel transient receptor potential vanilloid AMG 900 receptor 1 (TRPV1) can be sensitized by chronic TNF treatment (Nicol et al., 1997), a possible mechanism for the development of heat hypersensitivity, but it is not obvious how TRPV1 modulation would mediate enhanced sensitivity to mechanical stimuli. Among other ion channels critical in the genesis of inflammatory and neuropathic pain are the TTX-resistant (TTX-R) sodium channels (Bhave and Gereau, 2004; Wood et al., 2004a,b). In the present study, we examined the modulation of TTX-R Na+ channels in mouse DRG neurons by TNF. We show that acute application of TNF to cultured AMG 900 mouse DRG neurons rapidly enhances TTX-R currents via a TNF receptor 1 (TNFR1)- and p38-dependent pathway. These studies provide the first evidence AMG 900 of rapid receptor-mediated modulation of nociceptor excitability by TNF and may provide an explanation for the rapid sensitization to mechanical stimuli induced by TNF. Materials and Methods Adult male mice, 6C8 weeks old, of the following strains, ICR (Taconic Farms, Germantown, NY), Mice were allowed to acclimate for 1d before baseline testing. Mechanical sensitivity was assessed using von Frey hairs (North Coast Medical, San Jose, CA). Mice were placed on elevated wire mesh and allowed to acclimate to the testing environment for 2 h before testing. The plantar surface of the hindpaw was stimulated with a series of von Frey hairs. Each filament was applied five times, and threshold was determined as the lowest force that induced hindpaw withdrawal on at least three Rabbit polyclonal to HISPPD1 of five trials. Baseline values were defined as the mean of three measurements before injection. To test the effect of TNF on the basal mechanical sensitivity, 1 ng of TNF (in 10 l) was AMG 900 injected into the hindpaw plantar surface, and the paw-withdrawal thresholds of the ipsilateral hindpaw were measured at 30, 45, 60, and 90 min after injection. For the inhibitor experiments, vehicle (0.12% DMSO, 10 l) or SB202190 [4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)1Himidazole] (12 m in 10 l) was injected in the paw 20 min before the injection of TNF. Thermal level of sensitivity was measured using radiant warmth applied to the plantar surface of the hindpaw (IITC Existence Sciences, Woodland Hills, CA). Paw-withdrawal latency was measured. The heat stimulus was terminated having a withdrawal response or cutoff at 20 s to avoid cells damage. Before injections, three withdrawal latencies were recorded and averaged as the baseline for each animal. In < 0.01, ANOVA). TNF (1 ng in 10 l) was injected intradermally in the planter surface of the hindpaw, and withdrawal latencies were measured at 30, 45, 60, and 90 min after injection. DRG neuronal ethnicities were prepared using methods much like those described in our earlier publications (Hu et al., 2002; Yang and Gereau, 2004). DRGs were removed and collected in chilly (4C) PBS without Ca2+ or Mg2+ (Mediatech, Herndon, VA). Ganglia were incubated in 15 U/ml papain in HBSS (Mediatech) for 18 min at 37C. After this initial enzyme treatment, the ganglia were rinsed three times in HBSS and then incubated for 18 min with 1.5 mg/ml collagenase (Sigma, St. Louis, MO) in HBSS at 37C. After washing three times with HBSS, ganglia were softly triturated having a flame-polished Pasteur pipette. The cells fragments were centrifuged at 1000 rpm for 5 min, and the pellet was resuspended in neurobasal tradition press (Invitrogen, Grand island, NY) with 5% FBS, AMG 900 1% B27, 100 U/ml penicillin/streptomycin, and 2 mm glutamax. Cells were plated onto poly-d-lysine-coated 12 mm glass coverslips and managed at 37C inside a 95% airC5% CO2 incubator over night. Electrophysiological recordings were.