Neuronal nitric oxide synthase expression in developing and adult human CNS. to the lack of spinal rather than peripheral iNOS. Two additional observations indicate that this antinociceptive effects of iNOS inhibition are dependent on a loss of stimulation of PG synthesis. First, intrathecal injection of the COX inhibitor indomethacin, which exerted pronounced antinociceptive effects in wt mice, was completely ineffective in iNOS?/? mice. Second, treatment with the NO donor RE-2047 not only completely restored spinal PG production and thermal sensitization in iNOS?/? mice but also its sensitivity to indomethacin. In both types of mice induction of thermal hyperalgesia was accompanied by similar increases in COX-1 and COX-2 mRNA expression. The stimulation of PG production by NO therefore involves an increase in enzymatic activity, rather than an alteration of COX gene expression. These results indicate that NO derived from spinal iNOS acts as a fast inductor of spinal thermal hyperalgesia. A modified Hargreaves plantar test (Hargreaves et al., 1988) was used to assess thermal hyperalgesia in mice. A metal grid bottom instead of a glass floor in the observation cage and 10.5 13.0 4.5 cm boxes to restrict animal movement were used. Zymosan A (Sigma, Deisenhofen, Germany) was injected subcutaneously into the plantar side of right hindpaws, and paw withdrawal latencies (PWL) were determined on exposure of the paws to a defined NS1 thermal stimulus were measured using a commercially available apparatus (Hargreaves Test Ugo Basile Biological Research Apparatus, Comerio, Italy). Mice were kept in 10-Undecenoic acid the test cages for 1 d to allow accommodation. On day 2, each mouse was tested several times to gain baseline PWL. On day 3 thermal hyperalgesia was assessed for 8 hr starting 15 min after subcutaneous zymosan injection (3.0 mg/ml in 20 l of PBS, containing NaCl 8 gm/l, Na2HPO4 2.9 gm/l, KCl 0.2 gm/l, KH2 PO4 0.24 gm/l). Experiments were performed in air conditioned rooms (22C) between 12 A.M. and 8 P.M. In some experiments the assessment of thermal hyperalgesia was continued for 7 d (one measurement per day). Right (injected) and left (noninjected) paws were measured alternately in intervals of 5C10 min. At 1 hr intervals, PWL were averaged. Under control conditions, PWL were identical in wt (10.40 0.35 sec; = 40) and iNOS?/?mice (10.25 0.20 sec;= 18). In an initial set of experiments, zymosan (20 l) was tested in concentrations of 12.0, 6.0, or 3.0 mg/ml. Zymosan injection caused a dose-dependent increase in areas [PWL observation interval [seconds hours]; calculated using the linear trapezoidal rule for each mouse] between right and left hindpaw PWL from 0.17 1.75 (PBS) to 10.10 1.81 (3.0 mg/ml) to 17.47 2.55 (6.0 mg/ml) and to 23.30 2.22 (12.0 mg/ml). Injection of vehicle did not affect nociceptive behavior in any of the experiments. For all subsequent assessments an intermediate zymosan concentration of 3.0 mg/ml was used for the detection of pro-nociceptive and anti-nociceptive effects. Male iNOS?/? mice weighing 26.7 (22.9C36.8) gm [mean (range)] with the genetic background of C57/Bl6 mice and male C57/Bl6 mice (wt) weighing 21.2 (19.6C26.1) gm were used for all experiments. Breeding pairs of iNOS ?/? mice (Laubach et al., 1995) were obtained from The Jackson Laboratory (Bar Harbor, ME). INOS ?/? mice show no major abnormalities (Laubach et al., 1995; MacMicking et al., 1995; Wei et al., 1995). Mice were housed under a 12 hr light/dark cycle and cared for according to the guidelines of the Institutional Animal Care and Use Committee. Water and food were given All drugs were dissolved in isotonic, physiological solvents. Indomethacin was dissolved 10-Undecenoic acid as described elsewhere (Shen and Winter, 1977). Briefly, for a 10 mmsolution, 17.9 mg of indomethacin, 15.3 mg of Na2CO3 10 H2O, and 5 ml of artificial CSF (ACSF) consisting of (in mm): 151.1 Na+, 2.6 K+, 0.9 Mg2+, 1.3 Ca2+, 122.7 Cl?, 21.0 10-Undecenoic acid mmHCO3?, 2.5 mmHPO4?, and 3.5 dextrose, pH 7.20, was used. Intraperitoneal drug or vehicle (PBS) injections (50 l) were given into the lower remaining abdominal quadrant. Intrathecal shots were performed relating to Hylden and Wilcox (1980). In short, mice had been anesthetized with isoflurane, and 5 l of medication including 10-Undecenoic acid solutions or 10-Undecenoic acid automobile (ACSF) had been injected in to the vertebral subarachnoid space between L5 and L6 30 min prior to the administration of zymosan utilizing a 26 measure needle mated to a 10 l Hamilton syringe. Mice displaying neurological abnormalities had been excluded. We added 1% dark printer ink (Pelikan, Hannover, Germany) to all or any solutions useful for intrathecal shots. Proper intrathecal shots were confirmed by inspection of pieces from the spinal-cord after lumbar laminectomy. After conclusion of the Hargreaves check, mice were killed under CO2 anesthesia by intracardial decapitation and puncture. Hindpaws as well as the thoracolumbar section from the spinal cord had been eliminated for morphological.