Oral Communications - Contribution of non-neuronal cells to chronic pain of peripheral origin 35P C58 The inducible isoform of nitric oxide synthase (iNOS) has an important peripheral role in the development of inflammatory and neuropathic pain

We have investigated the role of the inducible isoform (iNOS) in models of inflammatory and neuropathic hypersensitivity using immunohistochemistry, chemiluminescence and the selective iNOS inhibitor GW274150 (Alderton et al, 2005). Male random hooded rats (200-250g) were used. Freund’s complete adjuvant (FCA): animals received 0.1ml of FCA (1mg/ml) or saline intraplantar into the left hind paw. The hypersensitivity was assessed at different time points (6h, 24h, 48h, 1 week and 3 weeks) after the FCA. The hypersensitivity was determined using the dual channel weight averager. At each time point animals were humanely killed, the paws, dorsal root ganglion (DRG), spinal cord and brain were removed and snap frozen. In the chronic constriction injury (CCI) model, under anaesthesia (isofluorane) the common left sciatic nerve was exposed and four loose ligatures of chromic gut (4.0) tied around it. The rats were allowed a period of 3 days to recover from the surgery before the hypersensitivity was determined using the algesymeter (Randall & Selitto, 1957) from day 3 to days 23 post surgery. Again after testing, different groups of animals at each time point were humanely killed and the sciatic nerves, DRG, spinal cords and brains were taken. In the FCA model GW274150 (1-30mg/kg p.o.) was dosed 24h post FCA and the effect on the hypersensitivity was determined at 1 and 6h post dose. In the CCI model GW274150 (3-30mg/kg s.c.) was dosed 23h post surgery and the effect on the hypersensitivity was determined 1h post dose. Nitrate/nitrite levels were determined using chemiluminescence, iNOS expression was determined using immunohistochemistry. Results are expressed as mean ±S.E.M. (n=7-10) and statistical analysis was carried out using one-way ANOVA followed by Dunnett’s test where p<0.05 is considered significant. iNOS was detected locally in the paw 6h after FCA injection and was associated with macrophages. The expression plateaued 24-72h post FCA and then slowly declined. This was associated with the development of the hypersensitivity. GW274150 (130mg/kg p.o.) suppressed the accumulation of nitrite (ED50=1.9±0.74mg/kg.) and partially reversed the hypersensitivity (max effect:52±5% at 30mg/kg). In the CCI model iNOS was only detected around the sciatic nerve after 3 days, appeared to concentrate proximal to the ligatures and was associated with inflammatory cells (macrophages). Between 7 and 26 days iNOS expression had spread away from the ligatures and surrounded the nerve fibres. GW274150 (3-30mg/kg s.c.) on day 23 produced a dose-related inhibition of the CCI-induced hypersensitivity 1h post dose (max effect: 105±29% at 30mg/kg s.c.; ED50=3.02 (0.5-17)mg/kg). In conclusion, the study supports an important role for peripherally expressed iNOS in both inflammatory and neuropathic pain, and therefore GW274150 may have clinically utility in the treatment of these pain states. Alderton WK et al. (2000). In Biology of Nitric Oxide, ed. Moncada S, Gustaffson LE, Wiklund NP & Higgs E, p. 22. Portland Press.