Inibição da migração de neutrófilos e da hipernocicepção pelo pré-condicionamento isquêmico remoto : participação da via L- arginina-NO-GMPc-CANAIS K + ATP

Abstract

The reperfusion injury (RI) occurs when there is de lay in restoring blood flow to organs and tissues. Among the strategies proposed to attenuate the RI i s the ischemic preconditioning (IPC), which consists of induction of brief ischemic periods fol lowed by reperfusion performed before the sustained ischemic insult. In the first part, this study aim ed to evaluate the role of nitric oxide (NO), cycli c guanosine monophosphate (cGMP) and ATP-sensitive po tassium channels (K + ATP ) in the inhibitory effect of hind limb IPC on the mice peritoneal cavi ty neutrophil migration (NM). In the second part, t he objective was to evaluate the potential systemic an tinociceptive effect of IPC in mechanical plantar hypernociception test (MPH; Von Frey) in rats and t o evaluate the involvement of NO, cGMP and K + ATP channels in this event. In the first part, the IPC was induced by hind limb ischemia for 10 min followed by 30 min of reperfusion in wild and knock out mice to inducible NO synthase (iNOS -/-). The leukocyte rolling (LR), leukocyte adhesion (LA) and NM were induced by ip administration of Carrageenan (Cg, 500 μ g/cavity) and results expressed in number of leukoc ytes/min, number of adherent leukocytes/100 μ m 2 cells and number of neutrophils x 10 6 /cavity, respectively. Different groups of animals were treated with saline (SAL), A minoguanidine (AG, sc, 100mg/kg), ODQ (ip, 8 μ mol/kg) or Glibenclamide (GBC, sc, 20 mg/kg) 30 min before IPC induction. Controls received the same treatment, but without IPC. In the second part , the IPC was induced by hind limb ischemia for 10 min followed by 30 min of reperfusion in male Wista r rats (180-200g). Cg (300 μ g, intraplantar) or Prostaglandin E 2 (PGE 2 - 400 ng, intraplantar) were used as hypernocicept ive stimulus on left paw immediately after induction of IPC in contralateral paw. Different groups of animals were treated 30 min before induction of IPC with AG (100 μ g/kg, intraplantar), LNMMA (50 μ g, intraplantar), ODQ (8 μ g, intraplantar) or GBC (160 μ g, intraplantar). Controls received the same treatm ents, but without IPC. The quantification of MPH was performed throug h subtraction strength/pressure (g) required to cause withdrawal of paw in contact with an apparatu s of eletronic von Frey mensuread before hypernociceptive stimulus, by measure obtained 3h a fter administration of Cg or PGE2. The LR, LA and NM induced by Cg in the peritoneal cavity were significantly (p <0,01) inhibited by hind limb IPC of wild animals (LR= 75.94%, LA= 56.51%, NM= 79.01 %), but these effects were not observed in animals iNOS -/-. The treatment of wild animals wit h AG and ODQ, but not with GBC, abrogated the inhibitory effect of IPC on NM induced by Cg. Treat ment of animals with AG, GBC or ODQ did not significantly alter the NM induced by Cg in the per itoneal cavity. We also observed that IPC did not alter the TNF- α , IL1- β and CXCL1 chemokine levels induced by Cg in the pe ritoneal cavity. The mechanical hypernociception induced by Cg or PGE2 i n left hind paw was significantly reduced (p <0,01) when IPC was performed in the right paw (55% and 68%). The treatment of animals with AG, LNMMA, ODQ or GBC, before IPC, abrogated the antino ciceptive effect of IPC. Treatment of animals with AG, LNMMA, ODQ or GBC did not significantly al ter the hypernociception induced by noxious stimulation. Our results show that IPC has an inhib itory effect on remote NM, with NO an important mediator involved in this process, probably through the cGMP pathway. This inhibitory effect of IPC does not depend on the opening of K + ATP channels, or inhibition of synthesis and/or releas e of pro- inflammatory cytokines. The elucidation of the mech anism by which IPC inhibits the NM may make important contributions to clinical situations wher e neutrophil infiltration is a complicating factor. The IPC also has a potent inhibitory effect on inflamma tory pain, with NO seems to be one important mediator involved in this protective effect of IPC, acting through the cGMP/K + ATP channel pathway. This is the first demonstration described in the li terature of systemic antinociceptive effect of IPC, and the elucidation of the mechanisms involved in this process is fundamental for pain management induced by several inflammatory stimuli.