BACKGROUND: Intrathecal neostigmine produces antinociceptive effects in many pain tests. Comparisons of pre- versus postinjury drug interventions have been made using the formalin test as a model for central sensitization. The purpose of this study was to compare behavioral antinociceptive responses with spinal fos-like immunoreactivity (FLI) in an intrathecal neostigmine injection between pre- versus post-formalin administration in a rat pain model. METHODS: Sprague-Dawley rats (250 300 g) were prepared with a PE 10 indwelling intrathecal catheter to receive either saline or neostigmine. The formalin test was performed on the second day after surgery with 5% 100microl. All rats were randomly divided into one of five groups; Sham, basal, control, pretreatment or posttreatment group. The Sham group (n = 4) received a dry needle at the hindpaw and the basal group (n = 6) received saline 100microl at the hindpaw. Saline 20microl was injected intrathecally after the formalin test in the control group (n = 8). Neostigmine (7.5microgram/10microl in volume) was administered intrathecally 7 minutes before the formalin test in the pretreatment group (n = 8) and 7 minutes after the formalin injection in the posttreatment group (n = 8). Pain related behavior was quantified by counting the incidence of flinches of the injected hindpaw for 60 minutes. Two hours after injection of formalin, animals were perfused with PBS and the spinal cords were removed immediately. The effect of pre- versus posttreatment with the neostigmine on c-Fos protein in the cord was examined by immunohistochemistry using specific antibodies. Immuno-positive cells in laminae I-II and III-V in the lumbar enlargement were calculated. RESULTS: Intraplantar formalin injection produced a biphasic appearance of the flinch behavior (phase 1 = 0 5 minutes, phase 2 = 20 60 minutes) in the control, pretreatment and postreatment groups. In the pretreatment group, rats showed less remarkable phase 1 and 2 responses than other groups. Flinches of phase 1 were 2.3 2.1, 33.2 7.6 (P< 0.05 vs. basal), 10.0 6.6 and 33.0 13.4(P< 0.05 vs. basal) in basal, control, pre- and posttreatment groups respectively. Flinches of phase 2 were 0.3 0.5, 224.5 66.4 (P< 0.05 vs. basal), 120.6 151.3 and 187.5 76.8 (P< 0.05 vs. basal) in basal, control, pre- and posttreatment groups respectively. Similarly, the pretreatment group virtually inhibited formalin-induced expression of FLI (P< 0.05 vs. control, posttreatment) in the laminae I-II ipsilateral to injection. The posttreatment group inhibited formalin-indeced expression of FLI (P< 0.05 vs. control) in the laminae III-V ipsilateral to injection. CONCLUSIONS: Pretreatment of intrathecal neostigmine inhibits nociceptive behaviors and FLI in the laminae I-II following formalin injection in rats, suggesting that pretreatment plays an important role in the development of plasticity in this model.