BACKGROUND: It has been previously shown that intrathecal administration of either ouabain or neostigmine can produce antinociceptive effects. Moreover, ouabain and neostigmine are differently associated with acetylcholine. OBJECTIVE: It has been hypothesized that intrathecal administration of ouabain, in combination with neostigmine, can produce antinociceptive synergistic effects. Atropine, as a competitive antagonist, was pre-injected to verify the mechanisms of action. DESIGN, TIME AND SETTING: This study was a randomized, controlled, animal experiment, performed at the State Key Laboratory of Oncology in Southern China between May 2006 and February 2007. MATERIALS: A total of 102 healthy, adult, Sprague Dawley rats were included. Ouabain and neostigmine (Sigma, USA), as well as atropine (Tanabe Seiyaku, Japan), were also used. METHODS: Varied doses of ouabain, neostigmine, and a combination of the two were intrathecally injected into rats. Six rats were allotted for each dose group. Intrathecal pretreatment with atropine was tested 10 minutes prior to intrathecal administration of neostigmine or the combination of ouabain and neostigmine. MAIN OUTCOME MEASURES: Tail-flick tests were performed to measure tail-flick latency (seconds) prior to and after administration. The response in the tail-flick test was expressed as the percentage of maximum possible effect (% MPE), where % MPE = [tail-flick latency after administration (seconds) -mean baseline value for tail-flick latency]/[10 seconds - the mean baseline value for tail-flick latency (seconds)] × 100%. RESULTS: Rat spinal intrathecal administration of either ouabain or neostigmine alone produced antinociceptive effects in a dose-dependent manner. Intrathecally administration of neostigmine (0.05, 0.1, 0.3 μg ) in combination with ouabain (1 μg ) produced enhanced antinociceptive effects, with a % MPE of 29%, 78%, and 95%, respectively (P < 0.05). Intrathecally administration of 0.3 μg neostigmine (% MPE: 45%), in combination with 1 μg ouabain (% MPE: 27%) produced potent antinociceptive effects (% MPE: 95%). Intrathecally pre-injected atropine antagonized the antinociceptive effects of neostigmine (3 μg), or a combination of ouabain (1 μg) and neostigmine (0.3 μg) (P < 0.01). CONCLUSION: Rat spinal intrathecal administration of either ouabain or neostigmine alone produced dose-dependent antinociceptive effects. Ouabain enhanced the antinociceptive effects of neostigmine. Atropine antagonized the antinociceptive effects of neostigmine or the combination of ouabain and neostigmine. This occurs possibly due to the fact that atropine is a competitive antagonist of the muscarinic acetylcholine receptors. BACKGROUND: It has been previously shown that intrathecal administration of either ouabain or neostigmine can produce antinociceptive effects. Moreover, ouabain and neostigmine are differently associated with acetylcholine. OBJECTIVE: It has been hypothesized that intrathecal administration of ouabain, in combination with neostigmine, can produce Atropine, as a competitive antagonist, was pre-injected to verify the mechanisms of action. DESIGN, TIME AND SETTING: This study was a randomized, controlled, animal experiment, performed at the State Key Laboratory of Oncology in Southern China between May 2006 and February 2007. MATERIALS: A total of 102 healthy adults, Sprague Dawley rats were included. Ouabain and neostigmine (Sigma, USA), as well as atropine (Tanabe Seiyaku, Japan), also also used. METHODS: Varied doses of ouabain, neostigmine, and a combination of the two were intrathecally injected into rats. Six rats were allotted for each dose grou p. Intrathecal pretreatment with atropine was tested 10 minutes prior to intrathecal administration of neostigmine or the combination of ouabain and neostigmine. MAIN OUTCOME MEASURES: Tail-flick tests were performed to measure tail-flick latency (seconds) prior to and after administration. The response in the tail-flick test was expressed as the percentage of maximum possible effect (% MPE), where% MPE = [tail-flick latency after administration (seconds) -mean baseline value for tail- flick latency] / [10 seconds - the mean baseline value for tail-flick latency (seconds)] × 100%. RESULTS: Rat spinal intrathecal administration of either ouabain or neostigmine alone produced antinociceptive effects in a dose- dependent manner. Intrathecally administration of neostigmine (0.05, 0.1, 0.3 μg ) in combination with ouabain (1 μg) produced enhanced antinociceptive effects with a% MPE of 29%, 78%, and 95%, respectively (P <0.05) Intrathecally administration of 0.3 μg neostigmine (% MPE: 45%), in combination with 1 μg ouabain (% MPE: 27%) produced potent antinociceptive effects (% MPE: 95%). Intrathecally pre-injected atropine antagonized the antinociceptive effects of neostigmine (3 μg), or a combination of ouabain ) and neostigmine (0.3 μg) (P0.01). CONCLUSION: Rat spinal intrathecal administration of either ouabain or neostigmine alone produced dose-dependent antinociceptive effects. Alabine enhanced the antinociceptive effects of neostigmine. Atropine antagonized the antinociceptive effects of neostigmine or the combination of ouabain and neostigmine. This occurs possibly due to the fact that atropine is a competitive antagonist of the muscarinic acetylcholine receptors.