FundaÃÃo de Amparo à Pesquisa do Estado do Cearà / CoordenaÃÃo de AperfeiÃoamento de NÃvel Superior / CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / A lesÃo de reperfusÃo (LR) ocorre em diversos orgÃos e tecidos durante o restabelecimento do fluxo sangÃÃneo apÃs episÃdios de isquemia prolongada. Em diversas Ãreas clÃnicas esta lesÃo à temida pelas complicaÃÃes decorrentes da demora no restabelecimento do fluxo vascular. Nos transplantes de ÃrgÃos, nos retalhos microcirÃrgicos e nas cirurgias de revascularizaÃÃo miocÃrdica, a LR, dependendo de sua intensidade, pode prejudicar o benefÃcio destes procedimentos. Dentre as estratÃgias propostas para atenuar a LR, o prÃ-condicionamento isquÃmico (PCI) consiste na induÃÃo de pequenos perÃodos de isquemia seguidos de reperfusÃo, realizados antes da isquemia prolongada. Este estudo foi avaliar o efeito do PCI da pata posterior na sobrevida de retalhos cutÃneos randomizados em dorso (RCRD) e o efeito antiinflamatÃrio local e sistÃmico deste fenÃmeno. Para tanto o PCI foi induzido atravÃs da isquemia da pata posterior de ratos Wistar por 10 minutos seguida de 30 minutos de reperfusÃo. Diferentes grupos de ratos foram submetidos à confecÃÃo de RCRD, à induÃÃo de edema de pata (EP), ipsilateral (IPS) e contralateral (CON), por carragenina (Cg) ou dextran (Dx), cistite hemorrÃgica (CH) por ifosfamida (IFO) ou lesÃo gÃstrica (LG) por indometacina (INDO). Os grupos controles (C) receberam o mesmo tratamento, porÃm sem PCI. A Ãrea de sobrevida (AS) do RCRD foi calculada pela superfÃcie de tecido viÃvel (cm2), o EP atravÃs da variaÃÃo do volume das patas (ml), o edema vesical (EV) pelo peso Ãmido da bexiga (mg), o aumento da permeabilidade vascular (APV) pelo extravasamento de azul de Evans (&#61549;g), o Ãndice de lesÃo gÃstrica (ILG) pela soma das extensÃes das lesÃes encontradas na mucosa (mm), e a infiltraÃÃo neutrofÃlica (IN) pela atividade da mieloperoxidade (MPO) (U/mg). O PCI foi capaz de aumentar a AS do RCRD (C=4,16Â0,29 e PCI=6,42Â0,14, p<0,01). O EP e o APV induzidos por Cg (100&#61549;g, sc) ou Dx (200&#61549;g, sc), tanto na pata IPS quanto CON, foram reduzidos pelo PCI (EP IPS Cg â C=0,63+0,05 e PCI=0,26+0,02; APV IPS Cg â C=10,03+0,68 e PCI=5,82+1,18; EP IPS Dx â C=1,08+0,11 e PCI=0,54+0,06; APV IPS Dx â C= 24,38+3,02 e PCI=13,51+1,70; EP CON Cg â C=0,63+0,10 e PCI=0,30+0,05; APV CON Cg â C=16,16+2,18 e PCI=9,19+1,12; EP CON Dx â C=0,95+0,10 e PCI=0,49+0,10; APV CON Dx â C=24,09+2,97 e PCI=13,15+1,62; p<0,01). O PCI inibiu o EV e o APV da CH induzida por IFO (200mg/kg, ip) (EV â C=206,6+10,95 e PCI=126+5,66; APV â C=38,55+2,19 e PCI=17,74+2,18; p<0,001). Tanto a LG quanto a IN induzidas por INDO (20mg/kg, vo) foram bloqueadas pelo PCI (ILG â C=20,54+2,05 e PCI=2,00+0,93; IN â C=9,67+2,05 e PCI=3,49+0,83; p<0,001). Os resultados indicam que o PCI aumenta a sobrevida de RCRD e apresenta um efeito antiinflamatÃrio local e sistÃmico em modelos de inflamaÃÃo aguda. O esclarecimento do mecanismo atravÃs do qual o PCI atenua a lesÃo inflamatÃria poderà trazer importantes contribuiÃÃes em situaÃÃes clÃnicas onde esta lesÃo constitui um fator complicador, podendo propiciar o desenvolvimento de novas abordagens profilÃticas e/ou terapÃuticas. / The reperfusion injury (RI) occurs when there is delay in restoring blood flow to organs and tissues. Among the strategies proposed to attenuate the RI is the ischemic preconditioning (IPC), which consists of induction of brief ischemic periods followed by reperfusion performed before the sustained ischemic insult. In the first part, this study aimed to evaluate the role of nitric oxide (NO), cyclic guanosine monophosphate (cGMP) and ATP-sensitive potassium channels (K+ATP) in the inhibitory effect of hind limb IPC on the mice peritoneal cavity neutrophil migration (NM). In the second part, the objective was to evaluate the potential systemic antinociceptive effect of IPC in mechanical plantar hypernociception test (MPH; Von Frey) in rats and to 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 knockout mice to inducible NO synthase (iNOS -/-). The leukocyte rolling (LR), leukocyte adhesion (LA) and NM were induced by ip administration of Carrageenan (Cg, 500 &#61549;g/cavity) and results expressed in number of leukocytes/min, number of adherent leukocytes/100 &#61549;m2 cells and number of neutrophils x 106/cavity, respectively. Different groups of animals were treated with saline (SAL), Aminoguanidine (AG, sc, 100mg/kg), ODQ (ip, 8 &#61549;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 Wistar rats (180-200g). Cg (300 &#61549;g, intraplantar) or Prostaglandin E2 (PGE2 - 400 ng, intraplantar) were used as hypernociceptive 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 &#61549;g/kg, intraplantar), LNMMA (50 &#61549;g, intraplantar), ODQ (8 &#61549;g, intraplantar) or GBC (160 &#61549;g, intraplantar). Controls received the same treatments, but without IPC. The quantification of MPH was performed through subtraction strength/pressure (g) required to cause withdrawal of paw in contact with an apparatus of eletronic von Frey mensuread before hypernociceptive stimulus, by measure obtained 3h after 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 with AG and ODQ, but not with GBC, abrogated the inhibitory effect of IPC on NM induced by Cg. Treatment of animals with AG, GBC or ODQ did not significantly alter the NM induced by Cg in the peritoneal cavity. We also observed that IPC did not alter the TNF-&#61537;, IL1-&#61538; and CXCL1 chemokine levels induced by Cg in the peritoneal cavity. The mechanical hypernociception induced by Cg or PGE2 in 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 antinociceptive effect of IPC. Treatment of animals with AG, LNMMA, ODQ or GBC did not significantly alter the hypernociception induced by noxious stimulation. Our results show that IPC has an inhibitory 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 release of pro-inflammatory cytokines. The elucidation of the mechanism by which IPC inhibits the NM may make important contributions to clinical situations where neutrophil infiltration is a complicating factor. The IPC also has a potent inhibitory effect on inflammatory 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 literature 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.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / IntroduÃÃo:O prÃ-condicionamento isquÃmico (PCI) vem sendo considerado como um potente mecanismo endÃgeno capaz de inibir a resposta inflamatÃria. A migraÃÃo de neutrÃfilos (mn) à um evento central no desenvolvimento da reaÃÃo inflamatÃria. Nosso grupo tem demonstrado que o PCI inibe a mn em modelos experimentais, entretanto os mecanismos e mediadores envolvidos ainda nÃo sÃo conhecidos.Objetivo:Estudar a participaÃÃo dos mediadores Ãxido nÃtrico e MonÃxido de carbono, proteÃnas de adesÃo (ICAM-1 e &#946;2-integrina) e da expressÃo de CXCR2 no efeito inibitÃrio do PCI a distÃncia sobre a mn. MÃtodos:O modelo de PCI a distÃncia foi realizado com um torniquete no membro posterior direito de camundongos durante 10 minutos seguidos de 30 de reperfusÃo. ParticipaÃÃo do NO e CO foi investigada atravÃs de inibidores de iNOS (1400 W, 3 mg/kg ou Aminoguanidina (Amg), 50 mg/kg) e HO-1 (ZnPPIX, 10 mg/kg) como prÃ-tratamento de 30 min. Posteriormente, induziu-se peritonite comCarragenina(Cg) (500 mg /cav). Quatro horas apÃs, a cavidade peritoneal (cp) era lavada e leucÃcitos eram contados. ApÃs aquele mesmo procedimento, os animais foram submetidos a microscopia intravital (miv) para avaliar os efeitos do NO e CO nas vÃnulas mesentÃricas de 3 ordem.NeutrÃfilos de animais prÃ-condicionados e prÃ-tratados ou nÃo, foram utilizados para o ensaio de quimiotaxiain vitro, usandocomo estÃmulo a quimiocina KC(30ng/ml). AexpressÃo de CXCR2 e GRK2 dos neutrÃfilos foi determinada por citometria de fluxo e imunofluorescÃncia, respectivamente.AsparticipaÃÃes de ICAM-1/CD54 e &#946;2-integrina/CD11b foram investigadas em camundongos nocautes para os genes dessas molÃculas. A mn nesses animais foi avaliada segundo protocolo jà descrito pelo lavado peritonela.Na investigaÃÃo do papel do NO e CO na modulaÃÃo da proteÃna de adesÃo celular (&#946;2-integrina), utilizou-se inibidores da iNOS (1400W, 3 mg/kg ou Amg, 50 mg/kg, sc), HO-1 (ZnPPIX, 10 mg/kg, sc) eGuanilato Ciclase (ODQ (5 Âmol/kg, ip)) em prÃ-tratamento de 30 min antes do PCI. ApÃs peritonite, o sangue foi colhido e a expressÃo de CD11bem neutrÃfilos foi determinada por citometria de fluxo. Para anÃlise estatÃstica, utilizou-se ANOVA/Bonferroni. P<0,05 foi aceito. Resultados:Os inibidores de CO e NO preveniram o efeito inibitÃrio do PCIsobre a mn (p <0,05). AlÃm disso, os neutrÃfilos de animais prÃ-condicionados apresentaram reduÃÃo de quimiotaxia (p <0,05),achado que se correlacionou com a diminuiÃÃo da expressÃode CXCR2 na membrana dos neutrÃfilos (p <0,05) e comaumento da expressÃo de GRK2. NÃo houve alteraÃÃo da quimiotaxia, nem da expressÃo de GRK2 quando os neutrÃfilos foram obtidos a partir de animais prÃ-condicionados e prÃ-tratados com inibidores de de CO, NO e GCs. Os animais prÃ-condicionados apresentaram reduÃÃo nos neutrÃfilos circulantes e da aderÃnciana miv, as quais foram prevenidas pelo prÃ-tratamento com os inibidores (p <0,05). O efeito inibitÃrio do PCI persistiu em animais nocautes para &#946;2-integrina, o que nÃo se observou nos animais nocautes para ICAM-1. AlÃm disso, os neutrÃfilos de animais prÃ-condicionados apresentaram reduÃÃo significativa na expressÃo de CD11b, que foi prevenida nos animais prÃ-tratados com os inibidores de iNOS, HO-1 e GCs.ConclusÃes: Os resultados sugerem que o NO e CO atuam no efeito inibitÃrio do PCI via iNOS e HO-1 em sitio distante, modulando ICAM-1, &#946;2-integrina e CXCR2 via GRK2. / Introduction: Ischemic preconditioning (IPC) has been considered as a potent endogenous mechanism capable of inhibiting the inflammatory response. The migration of neutrophils (mn) is a central event in the development of inflammation. Our group has demonstrated that PCI inhibits mn in experimental models, however the mechanisms and mediators involved are not yet known. Aim: To study the involvement of mediators nitric oxide and carbon monoxide, adhesion proteins (ICAM-1 and &#946;2-integrin) and expression of CXCR2 in the inhibitory effect of PCI on the distance min. Methods: The model of distance PCI was performed with a tourniquet in the right hind limb of mice for 10 minutes followed by 30 reperfusion. Involvement of NO and CO was investigated using inhibitors of iNOS (1400 W, 3 mg/kg or aminoguanidine (Amg), 50 mg/kg) and HO-1 (ZnPPIX, 10 mg/kg) as pretreatment 30 min. Later, peritonitis was induced by carrageenan (Cg) (500 mg/cav). Four hours later the peritoneal cavity (pc) was washed and leukocytes were counted. After that same procedure, the animals were subjected to intravital microscopy (IVM) to evaluate the effects of NO and CO in the mesenteric venules of 3rd order. Neutrophils from animals preconditioned and pre-treated or untreated, were used for testing chemotaxis in vitro, using the stimulus to chemokine KC (30ng/ml). The expression of GRK2 and CXCR2 in neutrophils was determined by flow cytometry and immunohistochemistry, respectively. The stakes and ICAM-1/CD54 &#946;2-integrina/CD11b been investigated in knockout mice for genes of these molecules. The mn in these animals was evaluated according to protocol previously described by washed peritonela. In the investigation of the role of NO and CO in the modulation of cell adhesion protein (&#946;2-integrin), we used iNOS inhibitor (1400W, 3 mg/kg or Amg, 50 mg/kg, sc), HO-1 (ZnPPIX, 10 mg/kg, sc) and guanylate cyclase (ODQ (5 Âmol/kg, ip)) in pre-treatment 30 min before PCI. After peritonitis, blood was collected and the expression of CD11b on neutrophils was determined by flow cytometry. For statistical analysis, we used ANOVA/Bonferroni. P<0,05 was accepted. Results: The CO and NO inhibitors prevented the inhibitory effect of PCI on mn (P<0,05). Moreover, neutrophils from animals preconditioned showed reduced chemotaxis (p<0,05), a finding that correlated with decreased expression of CXCR2 in the membrane of neutrophils (p<0,05) and increased expression of GRK2. There was no change in chemotaxis or the expression of GRK2 when neutrophils were obtained from animals pre-conditioned and pretreated with inhibitors of CO, NO and sGC. Preconditioned animals showed a reduction in circulating neutrophils and the grip on IVM, which were prevented by pretreatment with inhibitors (P<0,05). The inhibitory effect of PCI was shown in knockout animals for &#946;2-integrin, which was not observed in the knockout animals to ICAM-1. Moreover, neutrophils from animals preconditioned showed a significant reduction in the expression of CD11b, which was prevented in animals pretreated with inhibitors of iNOS, HO-1 and GCs. Conclusions: The results suggest that NO and CO act in the inhibitory effect of PCI via iNOS and HO-1 in place apart by modulating ICAM-1, &#946;2-integrin and CXCR2 via GRK2.