Effect of glibenclamide on antinociceptive effects of antidepressants Hajhashemi V and Amin B CLINICS 2011;66(2):321-325 Modulation of microglia activation is another mechanism counteracting with excitotoxicity induced by glutamate that has an important role in pain signaling.18 In a recent study it has been indicated that amitriptyline increases reuptake of glutamate through its transporters resulting in prevention of N-methyl-D-aspartate (NMDA) induced excitotoxicity.19-20 Neural K+ channels are typically classified to four classes according to their structure, and specific agonists and antagonists that consist of voltage-gated (Kv), calcium- activated (KCa), inward rectifier (Kir) and two-pore (K2P) K+ channels. It has been established that central K+ channels especially ATP-sensitive K+ channels (KATP) belonging to the Kir family are involved in the perception of pain.21 Central administration of K+channel openers, such as diazoxide, minoxidil, lemakalim and cromakalim, produced antinoci- ception and potentiated analgesic effect produced by opioid and a2-adrenoceptor agonists.22,23 Furthermore, central administration of ATP dependent K+channel blocker, gliqui- done, a sulfonylurea, reversed the analgesic action of amitriptyline and clomipramine in a hot plate test.15 In another study intracerebro-ventricular injection of antisense oligonucleotide produced a dose-dependent inhibition of clomipramine and amitriptyline antinociception.24 It seems that activation of G-protein coupled receptors by agonists such as a2-adrenoceptors, opioids, GABAB, muscarinic M2, adenosine A1, serotonin 5-HT1A and cannabinoid receptors and some nonsteroidal anti-inflammatory drugs (NSAIDs) can activate these K+ channels.21 The purpose of this work was to examine if intraperitoneal administration of a KATP blocker, glibenclamide, one of the most important drugs in treatment of diabetic patients could also affect antinociptive effects of antidepressants with different mechanisms of action with respect to reuptake inhibition of 5-HT or NA. For this purpose, the effect of pretreatment with glibencla- mide on the analgesic effect of amitriptyline as a dual action, non selective inhibitor of noradrenaline and serotonin reuptake, fluvoxamine as a selective serotonin reuptake inhibitor (SSRI) and maprotiline as a selective noradrenaline reuptake inhibitor (SNRI) was assessed in mice. MATHERIAL AND METHODS Animals Male NMRI mice (Pasteur Institute, Tehran, Iran), weight range between 25-30 g were used. They were housed in cages in groups of six at 21 ¡ 2˚C in a 12 h light-dark cycle. Tap water and standard food pellets were available ad libitum. In order to minimize circadian rhythm influence, all experiments were conducted between 08:00 and 13:00 h, in a noise-free room with controlled lighting. Minimum of six mice were used for each treatment group. All procedures were approved by the Ethical Committee of the Isfahan University of Medical Sciences, and conducted in accor- dance with the internationally accepted principles for laboratory animal use and care. At the end of the experi- ments animals sacrificed in an ether chamber. Thirty minutes before testing, animals were placed in observation plexiglas box for accommodation. Drugs The following drugs were used: Amitriptyline hydro- chloride (Iran Daru Pharmaceutical Co., Tehran, Iran), maprotiline (Razk Pharmaceutical Co., Tehran, Iran), flu- voxamine (Abidi Pharmaceutical Co., Tehran, Iran) and 322 glibenclamide (Sigma, St Louis, Mo, USA). All drugs except glibenclamide were dissolved in normal saline 0.9% (w/v) and glibenclamide was suspended in water using 0.05% (w/ v) carboxymethylcellulose. Glibenclamide was injected 15 min before antidepressant drugs and antidepressants administered 30 min prior to foot pad injection of formalin. Measurement of antinociceptive activity For evaluating antinociceptive effect of drugs we used formalin test in mice. This is a widely used assay for analgesic agents, which allow explaining the effects of analgesic agents used in clinical practice and is consumed a method for clinical inflammatory pain.25 This method consists of two phase, phase I (acute phase, 0-5 min) and phase II (tonic phase, 20-40 min). Irritation in phase I produced by stimulation of nociceptors during the first 5 min after intra-plantar formalin injection and phase II is due to central sensitization inflammatory pathways. Twenty micro-liter of diluted formalin (2.5%) was injected sub- cutaneously into the dorsal surface of the right hind paw. Animals were then returned to the chambers, and measure- ment of the time spent for paw licking was performed during 0-5 (phase I) and 20-40 min (phase II) periods after formalin injection by a skilled observer blinded to drug treatments. All drugs were administered via intraperitoneal injections (i.p.). Statistical analysis The data were expressed as mean ¡ SEM and analyzed by one-way analysis of variance (ANOVA) followed by Scheffe post-hoc test, using SPSS 15.0 software. P value , 0.05 was considered significant. RESULTS Amitriptyline, fluvoxamine and maprotiline did not produce any considerable antinociception in the first phase of formalin test (data not showed). As shown in fig. 1 amitriptyline at doses of 5, 10 mg/kg significantly (P,0.001) inhibited paw licking behavior of second phase of formalin test and glibenclamide (15 mg/kg) partially reversed this effect. Antinociceptive effect of fluvoxamine on phase 2 has been shown in fig. 2. This drug showed significant analgesia in phase two, so that the percentage inhibition of paw licking behavior at doses of 20 and 30 mg/kg were 97% and 99% respectively. Glibenclamide alone had not any effect on formalin induced pain response, but at doses of 10 mg/kg and 15 mg/kg could partially reserve analgesic effect of 20 mg/kg fluvoxamine. Fig. 3 shows the effect of maproti- line on chronic phase of formalin. Maprotiline produced a considerable analgesic effect in phase two of formalin test at doses of 10 and 20 mg/kg. However, glibenclamide could not produce any significant inhibition of maprotiline- induced analgesia. DISCUSSION In this work, analgesic effects of three antidepressants belonging to different classes was examined in formalin test which is one of the most common animal tests for evaluation of analgesic drugs. In this study none of the drugs could significantly alter licking induced by formalin in phase one compared to control group, but all three antidepressants effectively attenuated pain behavior in