Thesis (MScMed)--Stellenbosch University, 2008. / ENGLISH ABSTRACT: Protein kinases and phosphatases play important roles in the phosphorylation state of intracellular proteins under both physiologic and pathophysiologic conditions. Compared to the large number of studies investigating the significance of kinases, in particular the mitogen-activated protein kinases (MAPKs) in myocardial ischaemia/reperfusion and ischaemic preconditioning, relatively few studies have been done on the protein phosphatases in this scenario. Although several role players in the signal transduction cascade of ischaemia/reperfusion and ischaemic preconditioning have been identified thus far, the exact mechanism of cardioprotection still remains unclear. Previous studies from our laboratory have shown that the stress kinase, p38 MAPK, has a dual role in preconditioning: it acts as trigger of the process, while attenuation of its activation during sustained ischaemia and reperfusion is required for cardioprotection. Since the activation of p38 MAPK is dependent on both the upstream kinases for phosphorylation and phosphatases for dephosphorylation, we hypothesized that the balance between the activation state of the MAPKs and the induction of phosphatases may play a major role in determining the fate of cardiomyocytes exposed to ischaemic stress. The objectives of this study were: (i) to assess the activity of the myocardial protein phosphatases (PSPs and PP1) during sustained ischaemia and during reperfusion of non-preconditioned and ischaemic preconditioned hearts; (ii) to evaluate the significance of these phosphatases in ischaemia/reperfusion as well as in ischaemic preconditioning using available appropriate inhibitors; (iii) to give particular attention to the role of the phosphatase, mitogen-activated protein kinase phosphatase-1 (MKP-1), in ischaemia/reperfusion. MKP-1 is upregulated by stress conditions and selectively inactivates p38 MAPK by dephosphorylation of the regulatory Thr and Tyr residues. The glucocorticoid, dexamethasone which increases MKP-1 expression, was used as agonist to upregulate MKP-1 experimentally. The isolated perfused working rat heart was used as experimental model. After stabilization, hearts were subjected to either a one-cycle or multi-cycle ischaemic preconditioning protocol, followed by sustained global or regional ischaemia and reperfusion. Non-preconditioned hearts were subjected to ischaemia/reperfusion only. For Western blot analysis of MAPKs, PKB/Akt and MKP-1, hearts were freeze-clamped at different times during the perfusion protocol. Endpoints were infarct size, functional recovery and phosphorylation of the MAPKs (ERK and p38 MAPK) and PKB/Akt during reperfusion. Expression of MKP-1 was monitored. The results obtained showed that activation of PSPs and PP1 does not occur during sustained global ischaemia or reperfusion of non-preconditioned and preconditioned hearts. The role of the phosphatases was subsequently further investigated using two inhibitors namely cantharidin (5 μM, a concentration which inhibits both PP1 and PP2A) and okadaic acid (7.5 nM, a concentration which inhibits PP2A selectively). Administration of cantharidin or okadaic acid during the preconditioning phase, completely abolished preconditioning induced cardioprotection as indicated by mechanical failure during reperfusion and increased infarct size, associated with increased phosphorylation of p38 MAPK and PKB/Akt and dephosphorylation of ERK42/44. These results suggest a role for PP2A in the trigger phase of preconditioning. Administration of cantharidin or okadaic acid during early reperfusion of preconditioned hearts improved functional recovery. This was associated with increased phosphorylation of ERK42/44 and PKB, but not p38 MAPK. Dexamethasone, administered intraperitoneally to rats for 10 days (3mg/kg/day) or directly added to the perfusate (1 μM) resulted in significant cardioprotection of hearts subjected to 20 min sustained global ischaemia, followed by 30 min reperfusion. This is associated with a marked upregulation of MKP-1 and dephosphorylation of p38 MAPK during reperfusion. These studies suggest that the phosphatases are definitely involved in the phenomenon of ischaemia/reperfusion and ischaemic preconditioning. However, it also become clear that extensive further research is required to fully elucidate which phosphatases are involved and the mechanisms thereof. Due to the large size of the protein phosphatase family, this may prove to be a formidable task and far beyond the scope of this thesis. The results also suggested that pharmacological targetting of phosphatases involved in phosphorylation of the reperfusion injury salvage kinase (RISK) pathway (e.g. ERK42/44 and PKB/Akt) or dephosphorylation of pro-apoptotic kinases, such as p38 MAPK, may have significant clinical potential. / AFRIKAANSE OPSOMMING: Proteïenkinases en fosfatases speel 'n belangrike rol in die fosforileringstatus van intrasellulêre proteïene in beide fisiologiese en patofisiologiese toestande. In teenstelling met die groot aantal studies gedoen ten einde die rol van die kinases, veral die mitogeen-geaktiveerde proteïenkinases (MAPKs), in iskemie/herperfusie en iskemiese prekondisionering te ondersoek, is relatief min bekend aangaande die rol van die fosfatases in hierdie scenario. Hoewel verskeie rolspelers in die seintransduksieprosesse van iskemie/herperfusie en iskemiese prekondisionering reeds geïdentifiseer is, is die presiese meganisme van miokardiale beskerming steeds onbekend. Vroeëre studies vanuit ons laboratorium het getoon dat die streskinase, p38 MAPK, 'n tweeledige rol in prekondisionering speel: dit is 'n sneller ("trigger") van die proses, terwyl verlaagde aktivering tydens volgehoue iskemie en herperfusie, noodsaaklik vir beskerming is. Ons hipotese is dus dat die balans tussen die aktiveringstatus van die MAPKs en induksie van fosfatases die oorlewing van kardiomiosiete blootgestel aan iskemiese stres, bepaal. Die doelwitte van hierdie studie was: (1) bepaling van die aktiwiteit van miokardiale proteïen fosfatases (PSPs en PP1) tydens volgehoue iskemie en herperfusie van nie-geprekondisioneerde en iskemies-geprekondisioneerde harte; (ii) evaluering van die belang van fosfatases in iskemie/herperfusie beskadiging sowel as in iskemiese prekondisionering deur van geskikte inhibitore gebruik te maak; (iii) ondersoek na die rol van die fosfatase, mitogeen-geaktiveerde proteïen kinase fosfatase-1 (MPK-1) in iskemie/herperfusie beskadiging. Dit is bekend dat MKP-1 deur strestoestande opgereguleer word en p38 MAPK selektief deur defosforilasie van die regulatoriese Thr en Tyr residue inaktiveer word. Die glukokortikoïed, deksametasoon, wat MKP-1 uitdrukking stimuleer, is as agonis gebruik ten einde MKP-1 eksperimenteel op te reguleer. Die geïsoleerde, geperfuseerde werkende rothart is as eksperimentele model gebruik. Na stabilisasie, is die harte aan 'n enkel- of veelvuldige siklus iskemiese prekondisioneringsprotokol onderwerp, gevolg deur volgehoue globale of streeksiskemie. Nie-geprekondisioneerde harte is slegs aan iskemie/herperfusie onderwerp. Harte is op verskillende tye tydens die perfusieprotokol gevriesklamp vir Western blot analise van die MAPKs, PKB/Akt en MKP-1. Infarktgrootte en funksionele herstel tydens herperfusie is as indikators van iskemiese beskadiging gebruik. Fosforilasie van MAPKs en PKB/Akt sowel as uitdrukking van MKP-1 tydens vroeë herperfusie is gemonitor. Die resultate toon dat aktivering van PSP en PP1 tydens volgehoue iskemie en herperfusie nie plaasvind nie. Die rol van die fosfatases is verder ondersoek deur van twee inhibitore gebruik te maak, naamlik cantharidin (5 μM inhibeer beide PP1 en PP2A) en okadaic suur (7.5 nM inhibeer PP2A selektief). Toediening van of cantharidin of okadaic suur tydens die prekondisioneringsprotokol, hef prekondisionering-geïnduseerde beskerming totaal op, soos aangetoon deur hartversaking tydens herperfusie en 'n toename in infarktgrootte, tesame met 'n toename in die fosforilering van p38 MAPK en PKB/Akt en defosforilering van ERK42/44. Hierdie waarnemings dui op 'n rol vir PP2A as sneller in prekondisionering. Toediening van hierdie inhibitore tydens vroeë herperfusie het ook die miokardium beskerm, soos aangetoon deur 'n verbeterde meganiese herstel van geprekondisioneerde harte, tesame met ‘n verhoogde fosforilering van ERK42/44 en PKB (maar nie p38 MAPK nie). Deksametasoon, intraperitoneaal toegedien, vir 10 dae (3mg/kg/dag) of direk by die perfusaat gevoeg (1μM), het tot 'n hoogs beduidende beskerming teen iskemiese beskadiging gelei van harte blootgestel aan 20 min globale iskemie en 30 min herperfusie. Hierdie toename in funksionele herstel en afname in infarktgrootte het met 'n toename in MKP-1 uitdrukking en defosforilasie van p38 MAPK gepaard gegaan. Bogenoemde resultate dui op 'n definitiewe betrokkenheid van fosfatases in iskemie/herperfusie en iskemiese prekondisionering. Dit is egter ook duidelik dat intensiewe verdere navorsing benodig word om die presiese rol van die fosfatases te bepaal. Vanweë die grootte van die fosfatase familie, val dit egter buite die beskek van hierdie studie. Ten slotte, die resultate toon dat farmakologiese manipulasie van fosfatases betrokke by die fosforileringstatus van anti-apoptotiese kinases soos ERK42/44 en PKB/Akt en defosforilasie van pro-apoptotiese kinases, soos p38 MAPK, besondere kliniese toepassings mag hê.

Orientador: Norma Sueli Pinheiro Módolo / Banca: Leandro Gobbo Braz / Banca: Laís Helena Navarro e Lima / Banca: Rogério Luiz da Rocha Videira / Banca: Alexandra Assad / Resumo: O pós-condicionamento em modelo de isquemia e reperfusão já mostrou evidências de efeito renoprotetor, mas ainda há alguma controvérsia sobre os protocolos e seus resultados. A administração de cetamina S(+) em dose subanestésica em infusão contínua, como estratégia anti-inflamatória, ainda não foi testada na lesão renal aguda, bem como sua interação com o pós-condicionamento isquêmico não é conhecida. Testamos a hipótese de que a cetamina S(+) atenua o dano tubular e melhora a função renal em ratos sob pós-condicionamento. Quarenta e um ratos machos Wistar (≥300g) foram divididos aleatoriamente em quatro grupos: GS-Sham; GC-Cetamina S(+) em dose subanestésica em infusão contínua; GP-Pós-condicionamento isquêmico; GCP-Cetamina S(+) em dose subanestésica em infusão contínua e pós-condicionamento. Todos os animais foram submetidos à nefrectomia direita. Nos ratos submetidos ao pós-condicionamento (GP e GCP) foi realizada oclusão da artéria renal esquerda por 30 minutos. A reperfusão plena foi precedida por três ciclos de 2 min de reperfusão, seguido por 2 min de reoclusão. A pressão arterial, a frequência cardíaca e a temperatura foram controladas durante o experimento. A hidratação foi realizada com solução de Ringer lactato em infusão contínua intravenosa (3,0 mL.Kg-1.h-1), além de bolus após cada coleta. A função renal foi avaliada pela dosagem plasmática de NGAL, creatinina e ureia em três momentos: C1 (após estabilização), C2 (após 30 min de reperfusão completa) e C3 (após 24h). Dano tubular foi avaliado pela histologia renal. Foram utilizados os critérios de RIFLE e AKIN para avaliação evolutiva da creatinemia entre momentos. A creatinina e a ureia apresentaram aumento estatisticamente significativo nos grupos com pós-condicionamento isquêmico (GS e GC), mas não a NGAL (p = 0,08). Dano tubular significativo foi encontrado apenas nos ... / Abstract: Postconditioning against ischaemia-reperfusion injury has shown renoprotective effects, but there is still some controversy about protocols and its outcomes. The potencial application of subanesthetic S(+) ketamine continuous infusion as an antiinflammatory strategy, is not yet available in acute kidney injury, as well as the interaction with ischaemic postconditioning (IP). We tested the hypothesis that it attenuates tubular damage and improves renal function in IP in rats. Forty-one male Wistar rats (≥300g) were randomized into four groups: GS-sham; GK-subanesthetic S(+) ketamine; GP-posconditioning and GKP-subanesthetic S(+) ketamine and postconditioning. All animals were subjected to right nephrectomy but only in postconditioned rats 30-min left kidney arterial occlusion was performed, in which complete reperfusion were preceded by three cycles of 2 min of reperfusion followed by 2 min of reocclusion. Animals were studied for 24 h. Renal function was assessed by measurement of serum NGAL, creatinine and blood urea nitrogen (BUN) at three moments: C1 (after stabilization), C2 (after 30-min complete reperfusion) and C3 (after 24h). Tubular damage was evaluated by renal histology. RIFLE and AKIN criteria were used to evaluate creatinine among moments. Creatinine and BUN significantly increased in IP groups as compared to rats in GS and GK, but not NGAL (P=0,08). Despite significant tubular damage found only in IP groups, there was no significant difference between IP and S(+) ketamine/IP. RIFLE and AKIN criteria showed identical functional lesions. S(+) ketamine infusion does not attenuate tubular damage or improve renal function. However, IP groups show identical results and postconditioning is unable to show a renoprotective effect in this model / Doutor

Orientador: Norma Suelei Pinheiro Módolo / Banca: Geraldo Rolim Rodrigues Junior / Banca: Luiz Antonio Vane / Banca: Antonio Argolo Sampaio Filho / Banca: Rodrigo Leal Alves / Resumo: Justificativa e Objetivo: a lesão renal aguda(LRA) determina um aumento na morbidade e na mortalidade de pacientes hospitalizados. O diagnóstico precoce e a realização de medidas de proteção renal são essenciais. Foi observado que o pantoprazol possui efeito similar ao précondicionamento isquêmico em corações de ratos, promovendo efeito protetor. O objetivo deste experimento foi analisar o emprego do pantoprazol na proteção de rins submetidos à lesão de isquemia e reperfusão em ratos sob anestesia geral inalatória com isoflurano. Método: quarenta ratos, Wistar, machos, foram distribuídos aleatoriamente em quatro grupos: PIR (pantoprazol + isquemia artéria renal esquerda), P (pantoprazol), IR (isquemia renal esquerda) e Sham. O tempo de isquemia renal foi de 20 minutos e o de reperfusão, 30 minutos. Anestesia inalatória com isoflurano foi a técnica anestésica realizada. A dose administrada de pantoprazol foi de 192 mcg/kg. Todos os animais foram submetidos à nefrectomia direita e ao final do experimento procedeu-se nefrectomia esquerda, para posterior avaliação histológica e classificação por meio de escala de necrose tubular. Os atributos estudados foram: pressão arterial média (PAM), temperatura retal, os critérios de RIFLE e AKIN, dosagem urinária dos biomarcadores NGAL, KIM-1 e IL-8 e avaliação histológica. A creatinina sérica foi coletada em três momentos: M1, após monitorização; M2, após a reperfusão; e M3, 24 horas após o inicio do experimento. As coletas de urina para dosagem dos biomarcadores ocorreram antes (Urina 1) e após o experimento (Urina 2). Os atributos foram submetidos à análise estatística e as diferenças foram consideradas estatisticamente significativas quando p<0,05. Resultados: diferenças estatisticamente significativas foram observadas entre os grupos com aplicação dos critérios de RIFLE (p=0,007) e AKIN (p-0,003). Os grupos PIR e IR evoluíram com maior incidência de... / Abstract: Background and Objective: acute kidney injury (AKI) determines an increase in morbidity and mortality of hospitalized patients. Early diagnosis and the realization of renal protection measures are essential. It was observed that pantoprazole has effect similar to ischemic preconditioning in rat hearts, promoting protective effect. The objective was to analyze the use of pantoprazole in protecting kidneys subjected to ischemia and reperfusion in rats under inhalation anesthesia with isoflurane. Method: forty rats, Wistar male rats, were randomly divided into four groups: RIP (pantoprazole + left renal ischemia), P (pantoprazole), IR (left renal ischemia) and Sham. Renal ischemia time was 20 minutes and reperfusion 30 minutes. The anesthetic technique performed: inhalation anesthesia with isoflurane. The pantoprazole dose was 192 mcg/kg. All animals underwent right nephrectomy and at the end of the experiment proceeded to left nephrectomy for subsequent histological evaluation and classification through tubular necrosis scale. The parameters were: mean arterial pressure (MAP), rectal temperature, the criteria of RIFLE and AKIN, urinary dosage of biomarkers NGAL, KIM-1 and IL-8 and histological evaluation. Serum creatinine was collected in three moments: M1, after monitoring, M2, and M3 after reperfusion, 24 hours after the beginning of the experiment. The urine samples to test for biomarkers occurred before (Urine 1) and after the experiment (Urine 2). The attributes were subjected to statistical analysis and differences were considered statistically significant when p <0,05. Results: significant differences were observed between the groups with application of RIFLE criteria (p=0.007) and AKIN (p=0.003). The PIR and IR groups evolved with higher incidence of AKI. The NGAL and KIM-1 biomarkers increased in all groups throughout the experiment, but with higher values in the PIR and IR groups. IL-18 revealed no group effect (p=0.38) nor the ... / Doutor

Made available in DSpace on 2014-06-11T19:30:29Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-12-13Bitstream added on 2014-06-13T19:19:20Z : No. of bitstreams: 1 000742453.pdf: 1981037 bytes, checksum: 4a9056658a30d5e113687815220ac620 (MD5) / O pós-condicionamento em modelo de isquemia e reperfusão já mostrou evidências de efeito renoprotetor, mas ainda há alguma controvérsia sobre os protocolos e seus resultados. A administração de cetamina S(+) em dose subanestésica em infusão contínua, como estratégia anti-inflamatória, ainda não foi testada na lesão renal aguda, bem como sua interação com o pós-condicionamento isquêmico não é conhecida. Testamos a hipótese de que a cetamina S(+) atenua o dano tubular e melhora a função renal em ratos sob pós-condicionamento. Quarenta e um ratos machos Wistar (≥300g) foram divididos aleatoriamente em quatro grupos: GS-Sham; GC-Cetamina S(+) em dose subanestésica em infusão contínua; GP-Pós-condicionamento isquêmico; GCP-Cetamina S(+) em dose subanestésica em infusão contínua e pós-condicionamento. Todos os animais foram submetidos à nefrectomia direita. Nos ratos submetidos ao pós-condicionamento (GP e GCP) foi realizada oclusão da artéria renal esquerda por 30 minutos. A reperfusão plena foi precedida por três ciclos de 2 min de reperfusão, seguido por 2 min de reoclusão. A pressão arterial, a frequência cardíaca e a temperatura foram controladas durante o experimento. A hidratação foi realizada com solução de Ringer lactato em infusão contínua intravenosa (3,0 mL.Kg-1.h-1), além de bolus após cada coleta. A função renal foi avaliada pela dosagem plasmática de NGAL, creatinina e ureia em três momentos: C1 (após estabilização), C2 (após 30 min de reperfusão completa) e C3 (após 24h). Dano tubular foi avaliado pela histologia renal. Foram utilizados os critérios de RIFLE e AKIN para avaliação evolutiva da creatinemia entre momentos. A creatinina e a ureia apresentaram aumento estatisticamente significativo nos grupos com pós-condicionamento isquêmico (GS e GC), mas não a NGAL (p = 0,08). Dano tubular significativo foi encontrado apenas nos... / Postconditioning against ischaemia-reperfusion injury has shown renoprotective effects, but there is still some controversy about protocols and its outcomes. The potencial application of subanesthetic S(+) ketamine continuous infusion as an antiinflammatory strategy, is not yet available in acute kidney injury, as well as the interaction with ischaemic postconditioning (IP). We tested the hypothesis that it attenuates tubular damage and improves renal function in IP in rats. Forty-one male Wistar rats (≥300g) were randomized into four groups: GS-sham; GK-subanesthetic S(+) ketamine; GP-posconditioning and GKP-subanesthetic S(+) ketamine and postconditioning. All animals were subjected to right nephrectomy but only in postconditioned rats 30-min left kidney arterial occlusion was performed, in which complete reperfusion were preceded by three cycles of 2 min of reperfusion followed by 2 min of reocclusion. Animals were studied for 24 h. Renal function was assessed by measurement of serum NGAL, creatinine and blood urea nitrogen (BUN) at three moments: C1 (after stabilization), C2 (after 30-min complete reperfusion) and C3 (after 24h). Tubular damage was evaluated by renal histology. RIFLE and AKIN criteria were used to evaluate creatinine among moments. Creatinine and BUN significantly increased in IP groups as compared to rats in GS and GK, but not NGAL (P=0,08). Despite significant tubular damage found only in IP groups, there was no significant difference between IP and S(+) ketamine/IP. RIFLE and AKIN criteria showed identical functional lesions. S(+) ketamine infusion does not attenuate tubular damage or improve renal function. However, IP groups show identical results and postconditioning is unable to show a renoprotective effect in this model

Made available in DSpace on 2016-08-12T18:48:42Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-02-27. Added 1 bitstream(s) on 2016-08-12T18:50:55Z : No. of bitstreams: 1 000865111.pdf: 1312003 bytes, checksum: 38fab956c6701f8def4faf35f56489ff (MD5) / Justificativa e Objetivo: a lesão renal aguda(LRA) determina um aumento na morbidade e na mortalidade de pacientes hospitalizados. O diagnóstico precoce e a realização de medidas de proteção renal são essenciais. Foi observado que o pantoprazol possui efeito similar ao précondicionamento isquêmico em corações de ratos, promovendo efeito protetor. O objetivo deste experimento foi analisar o emprego do pantoprazol na proteção de rins submetidos à lesão de isquemia e reperfusão em ratos sob anestesia geral inalatória com isoflurano. Método: quarenta ratos, Wistar, machos, foram distribuídos aleatoriamente em quatro grupos: PIR (pantoprazol + isquemia artéria renal esquerda), P (pantoprazol), IR (isquemia renal esquerda) e Sham. O tempo de isquemia renal foi de 20 minutos e o de reperfusão, 30 minutos. Anestesia inalatória com isoflurano foi a técnica anestésica realizada. A dose administrada de pantoprazol foi de 192 mcg/kg. Todos os animais foram submetidos à nefrectomia direita e ao final do experimento procedeu-se nefrectomia esquerda, para posterior avaliação histológica e classificação por meio de escala de necrose tubular. Os atributos estudados foram: pressão arterial média (PAM), temperatura retal, os critérios de RIFLE e AKIN, dosagem urinária dos biomarcadores NGAL, KIM-1 e IL-8 e avaliação histológica. A creatinina sérica foi coletada em três momentos: M1, após monitorização; M2, após a reperfusão; e M3, 24 horas após o inicio do experimento. As coletas de urina para dosagem dos biomarcadores ocorreram antes (Urina 1) e após o experimento (Urina 2). Os atributos foram submetidos à análise estatística e as diferenças foram consideradas estatisticamente significativas quando p<0,05. Resultados: diferenças estatisticamente significativas foram observadas entre os grupos com aplicação dos critérios de RIFLE (p=0,007) e AKIN (p-0,003). Os grupos PIR e IR evoluíram com maior incidência de... / Background and Objective: acute kidney injury (AKI) determines an increase in morbidity and mortality of hospitalized patients. Early diagnosis and the realization of renal protection measures are essential. It was observed that pantoprazole has effect similar to ischemic preconditioning in rat hearts, promoting protective effect. The objective was to analyze the use of pantoprazole in protecting kidneys subjected to ischemia and reperfusion in rats under inhalation anesthesia with isoflurane. Method: forty rats, Wistar male rats, were randomly divided into four groups: RIP (pantoprazole + left renal ischemia), P (pantoprazole), IR (left renal ischemia) and Sham. Renal ischemia time was 20 minutes and reperfusion 30 minutes. The anesthetic technique performed: inhalation anesthesia with isoflurane. The pantoprazole dose was 192 mcg/kg. All animals underwent right nephrectomy and at the end of the experiment proceeded to left nephrectomy for subsequent histological evaluation and classification through tubular necrosis scale. The parameters were: mean arterial pressure (MAP), rectal temperature, the criteria of RIFLE and AKIN, urinary dosage of biomarkers NGAL, KIM-1 and IL-8 and histological evaluation. Serum creatinine was collected in three moments: M1, after monitoring, M2, and M3 after reperfusion, 24 hours after the beginning of the experiment. The urine samples to test for biomarkers occurred before (Urine 1) and after the experiment (Urine 2). The attributes were subjected to statistical analysis and differences were considered statistically significant when p <0,05. Results: significant differences were observed between the groups with application of RIFLE criteria (p=0.007) and AKIN (p=0.003). The PIR and IR groups evolved with higher incidence of AKI. The NGAL and KIM-1 biomarkers increased in all groups throughout the experiment, but with higher values in the PIR and IR groups. IL-18 revealed no group effect (p=0.38) nor the ...

Orientador: Ivan Felizardo Contrera Toro / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-07T09:33:33Z (GMT). No. of bitstreams: 1 Mussi_RicardoKalaf_D.pdf: 7974637 bytes, checksum: 40bf555137ac51738964d4e90337bddf (MD5) Previous issue date: 2006 / Resumo: Objetivos: A proposta deste estudo foi avaliar os efeitos do treinamento físico na resposta inflamatória (permeabilidade vascular e influxo neutrofílico) após isquemia-reperfusão pulmonar (IRP) em ratos. Considerando que o treinamento físico tem propriedades protetoras em determinadas doenças cardiovasculares, hipotetizamos que animais submetidos a treinamento com exercício físico podem ficar protegidos contra a lesão causada pela isquemia e reperfusão pulmonar. Métodos: ratos Wistar machos (pesando entre 350g e 450g) foram divididos em três grupos: sham operados (SHAM); animais sedentários submetidos à isquemia-reperfusão (IR/SD) e animais treinados submetidos à isquemia-reperfusão (IR/TR). Animais treinados foram submetidos a um programa de oito semanas de treinamento em corrida, com sessões de 60min por dia, cinco dias por semana. Os ratos foram anestesiados e ventilados artificialmente. Após a toracotomia, a artéria pulmonar esquerda, o brônquio principal esquerdo e a veia pulmonar esquerda foram clampeados, mantendo o pulmão inflado parcialmente por 90 minutos. O clampe foi então removido e os pulmões voltaram a ventilar, e foram reperfundidos por duas horas. A circulação pulmonar foi perfundida através do tronco arterial pulmonar com solução salina. O extravasamento de proteína plasmática foi medido através do acúmulo de albumina sérica marcada com I. em ambos os pulmões, enquanto que a infiltração neutrofílica foi medida através da atividade de mieloperoxidase (MPO). 125Resultados: Após oito semanas de treinamento em corrida, o peso corpóreo foi significativamente menor em 15% no grupo treinado comparado com o do grupo sedentário (p<0,05). A contagem de células mononucleares e polimorfonucleares em ratos treinados não se altera significativamente quando comparada à dos ratos sedentários. A IRP causou acentuado aumento (p< 0,05) na permeabilidade vascular e na atividade da mieloperoxidase do pulmão direito dos animais sedentários. O precondicionamento físico dos animais atenuou significativamente (p< 0,05) a elevação da permeabilidade vascular, sem afetar o influxo neutrofílico. O protocolo de isquemia e reperfusão também resultou em níveis elevados de IL-1 ß e TNF-a no grupo sedentário, e foram revertidos pelo programa de treinamento em corrida. Os níveis séricos de IL-10 não foram alterados nos três grupos. Conclusões: Os dados deste estudo mostram que o precondicionamento físico atenua a elevação da permeabilidade vascular, mas não o influxo neutrofílico induzido pela isquemia-reperfusão em ratos. Estes dados sugerem que o treinamento físico age no componente vascular mas não no celular da resposta inflamatória, deste modelo, protegendo contra a lesão pulmonar causada pela isquemia-reperfusão / Abstract: Objective: The purpose of this study was to evaluate the effects of exercise training on the inflammatory response (vascular permeability and neutrophil influx) after lung ischemia-reperfusion in rats. Considering that exercise training have protective properties in certain cardiovascular disease, we hypothesized that animals undergoing exercise physical training would be protective against direct lung ischemia-reperfusion injury. Methods: Male Wistar rats (weighing 350g and 450g) were divided into tree groups: Sham operated (SHAM); sedentary animals submitted to ischemia-reperfusion (IR/SD) and trained animals submitted to ischemia-reperfusion (IR/TR). Trained animals underwent 8 weeks of run training program in sessions of 60 min/day, 5 days/week. Rats were anesthetized and artificially ventilated. After thoracotomy, the left pulmonary artery, bronchus and pulmonary vein were occluded, maintaining the lung in a partially inflated state for 90min. The clamp was then removed, and lungs were allowed to ventilate and reperfuse for 2h. The pulmonary circulation was flushed through the main pulmonary artery with salin solution. Protein plasma extravasation was measured as accumulation of human serum albumin labeled with I in the botht lung tissues, whereas neutrophil infiltration was measured as the myeloperoxidase (MPO) activity.125 Results: After 8 weeks of run training, the body weight was significantly 15% lower in trained groups compared with the sedentary group (p<0.05). Counts of mononuclear and polymorphonuclear cells in trained rats did not significantly changed compared with sedentary rats. The lung ischemia-reperfusion caused a marked increase (p<0.05) in vascular permeability and MPO activity in the right lung of sedentary animals. The physical preconditioning of the animals significantly attenuated (p<0.05) the increased vascular permeability, without affecting the neutrophil influx. The ischemia-reperfusion protocols also resulted in increased levels of serum IL-1ß and TNF-a in sedentary group, and that was reversed by run training program. The serum levels IL-10 were not altered in all groups. Conclusions: Our data show that physical preconditioning attenuate the increased vascular permeability but not the neutrophil influx induced by rat lung ischemia-reperfusion. This data suggests that physical training act in the vascular (but not in the cellular) component of the inflammatory response to protect against the injury caused by lung ischemia-reperfusion / Doutorado / Cirurgia / Doutor em Cirurgia

This study investigated if exogenous adenosine (ADO) improves recovery of cardiac function during repetfusion (RPF) after global ischemia (ISC), and if lidocaine is required for. ADO-mediated cardioprotection during reperfusion. Isolated rabbit hearts, retrogradely perfused with erythrocyte-enriched Krebs-Henseleit buffer at constant left ventricular (LV) volume and physiologic flow rates, were subjected to 20 min. of global no-flow ischemia, and reperfused at the same rate as before ischemia. Hearts received one of the following treatments: 1) control (CON; no drug treatment), 2) adenosine (ADO; 200J.1M before and after ISC), or 3) adenosine+lidocaine (NL; 200 JlM ADO before and after ISC, 1 J.Lg/ml/min LIDO during first 20 min. of RPF). Myocardial function (e.g., using developed LV pressure, DP) declined as expected during no-flow ischemia and gradually returned during reperfusion. Functional recovery in ADO and NL groups were significantly improved from CON during early RPF (p<0.05 at 2 min RPF), but not at later RPF times(> 10 min). Differences did not exist between ADO and NL groups at any RPF time except at 10 min. RPF. Additionally, myocardial ATP content was measured before ischemia, after ischemia, and after 10 and 30 min of reperfusion. ATP content decreased significantly during ischemia; ADO hearts showed a increased repletion (85% of pre-ischemia level) of ATP at 30 min. of reperfusionas compared to CON (60%). These data suggest that ADO alone improves cardiac functional recovery during early repetfusion; LIDO does not appear to be required for ADO-mediated cardioprotection. ADO and LIDO do not improve cardiac function, however, ADO appears to improve myocardial ATP repletion at later RPF times in this blood-perfused rabbit model of global myocardial ISC/RPF.

Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Introduction: Coronary heart disease (CHD) is the leading cause of death worldwide with 3.8 million men and 3.4 million women dying globally each year. Although existing myocardial reperfusion strategies such as thrombolysis and percutaneous coronary intervention (PCI), if applied in a timely manner, limit myocardial infarct size, the mortality and morbidity remains significantly high. Ischaemic preconditioning (IPC) may offer the potential to attenuate myocardial ischaemia/reperfusion injury through cardioprotective signaling pathways which is recruited at the time of myocardial reperfusion, thereby improving clinical outcomes in patients with coronary artery disease. Ischaemic preconditioning is a phenomenon whereby short intermittent episodes of coronary occlusion followed by reperfusion protect the myocardium against a subsequent period of sustained ischaemia. This protection is reflected in the limitation of infarct size and improved functional recovery of the ischaemic heart during reperfusion. Despite intensive research efforts, the promise of an effective cardioprotective strategy using the endogenous protective mechanisms of the heart which underlies IPC, has not yet been materialized. Although progress has been made in terms of signaling mechanisms in the preconditioned heart, the identification of the myocardial reperfusion phase as the critical “window” for cardioprotection, requires the elucidation of the signal transduction pathways during the reperfusion phase after IPC. In view of the above, the aims of the present study were to investigate: i. the involvement of the RISK pathway and p38 MAP kinase pathway in IPC during early and late reperfusion ii. the involvement of heat shock protein-27 (HSP-27), heat shock protein-70 (HSP-70), GSK-3β, CAMKII, AMPK and the transcription factor CREB in the context of IPC during early reperfusion iii. the involvement of autophagy and apoptosis during early and late reperfusion after IPC iv. the correlation of the protein kinases with the hemodynamic parameters of the heart v. the mechanism of IPC by means of two-dimensional (2D) proteomics Methods: The isolated perfused working rat heart model was used with functional recovery as end-point. Hearts were preconditioned (IPC) for 3x5 min global ischaemia, alternated with 5 min reperfusion. Hearts were subjected to 25 min sustained global ischaemia, followed by 5, 10, 15 or 30 min reperfusion when hearts were snap-frozen for western blotting analysis. Alternatively, hearts were reperfused for 30 min to record hemodynamic parameters and measure functional recovery. Non-preconditioned (Non-IPC) hearts were stabilized for 30 min and subjected to 25 min sustained global ischaemia followed by 5, 10, 15 or 30 min reperfusion when hearts were snap-frozen. Alternatively Non-IPC hearts were reperfused for 30 min to serve as control for the 30 min reperfused IPC group. Activation of the protein kinases was determined by western blotting analysis. For the proteomic study mitochondrial and cytosolic proteins were isolated from heart tissue and separated in the first dimension by isoelectric focusing, followed by separation in the second dimension by two dimensional gel electrophoresis. The PD Quest software programme was used to identify significantly expressed protein spots. Protein spots of interest were excised and subjected to in-gel digestion and the resulting peptides were analysed by mass spectrometry. Proteins were identified by Mascot and the Swiss Prot database. Results: Western blotting analysis demonstrated that the RISK pathway and p38 MAPK are activated very early in reperfusion, but the activation is not sustained during the reperfusion period. Autophagy is also upregulated during this early reperfusion phase; it is attenuated in the middle reperfusion phase and increase for a second peak of upregulation in the late reperfusion phase. In addition, we identified CAMKII as a novel marker of functional recovery in IPC after reperfusion. The proteomic analysis identified twenty differentially expressed mitochondrial and thirty six differentially expressed cytosolic proteins between Non-IPC and IPC hearts. Functions ascribed to the majority of these individual proteins were directly related to cardiac metabolism. Conclusion: Activation of the majority of the protein kinases investigated in the present study is associated with the hemodynamic parameters of the heart instead of functional recovery. Results indicated that the variable signaling patterns could be attributed to differences in heart rate and the effect thereof (ejection fraction, minimum and maximum rate of contraction), as a result of sympathetic stimulation due to psychological stress in the animals before slaughtering. Proteomics results demonstrated that IPC hearts which failed after ischaemia /reperfusion are metabolically compromised and “worse off” compared to non-IPC hearts. / AFRIKAANSE OPSOMMING: Inleiding: Koronêre hartsiekte is die vernaamste oorsaak van sterftes wêreldwyd met 3.8 miljoen mans en 3.4 miljoen vrouens wat jaarliks sterf. Alhoewel bestaande miokardiale herperfusie strategieë soos trombolise en perkutane koronêre intervensie (PKI), wanneer betyds toegepas, miokardiale infarktgrootte beperk, bly mortaliteit en morbiditeit steeds hoog. Isgemiese prekondisionering (IPK) beskik oor die potensiaal om miokariale isgemie/herperfusie skade te verminder deur beskermende seinoordragpaaie tydens miokardiale herperfusie te aktiveer en sodoende die pasiënte wat aan koronêre arterie siekte ly, se prognose te verbeter. Isgemiese prekondisionering verwys na die verskynsel waartydens kort episodes van isgemie opgevolg deur herperfusie, die miokardium teen ‘n daaropvolgende langdurige isgemiese insident beskerm. Hierdie beskerming word gereflekteer in die beperking van infarktgrootte en verbeterde funksionele herstel van die isgemiese hart tydens herperfusie. Ten spyte van intensiewe navorsingspogings is die presiese meganisme van endogene beskerming tydens IPK nog nie ten volle ontrafel nie. Die identifisering van die miokardiale herperfusie fase se kritiese “vensterperiode” van beskerming, noodsaak ‘n volledige analise van die seinoordragpaaie wat geaktiveer word tydens die herperfusie fase na IPK. In die lig van bogenoemde, was die doel van die huidige studie om die volgende te ondersoek: i. die betrokkenheid van die RISK seinoordragpad en p38 MAP kinase tydens vroeë en laat herperfusie na IPK ii. die betrokkenheid van “heat shock protein-27” (HSP-27), “heat shock protein- 70” (HSP-70), GSK -3β, CAMKII, AMPK en die transkripsie faktor, CREB, in die konteks van IPK tydens vroeë herperfusie iii. die betrokkenheid van outofagie en apoptose tydens vroeë en laat herperfusie na IPK iv. die korrelasie van die proteïenkinases met die hemodinamiese parameters van die hart v. die meganisme van IPK deur middel van twee dimensionele proteomika Metodes: Die geïsoleerde werkende rothart model, met funksionele herstel as eindpunt, is gebruik. Harte is geprekondisioneer (IPK) met 3x5 min globale isgemie, afgewissel met 5 min herperfusie. Daarna is harte blootgestel aan 25 min volgehoue globale isgemie, gevolg deur 5, 10, 15 of 30 min herperfusie, waartydens harte gevriesklamp is. Alternatiewelik, is harte blootgestel aan 30 min herperfusie ten einde funksionele herstel te meet en hemodinamiese parameters te registreer. Nie-geprekondisioneerde (Non-IPK) harte is gestabiliseer vir 30 min, waarna dit onderwerp is aan 25 min volgehoue globale isgemie, gevolg deur 5, 10, 15 of 30 min herperfusie, waartydens harte gevriesklamp is vir westelike klad analise. Alternatiewelik, is Non-IPK harte onderwerp aan 30 min herperfusie om te dien as kontrole vir die 30 min IPK groep. Aktivering van die proteïenkinases is bepaal deur westelike klad analise. Vir die proteomiese studie, is onderskeidelik mitokondriale en sitosoliese proteïene geïsoleer en geskei in die eerste dimensie met behulp van isoelektriese fokusering, gevolg deur skeiding in die tweede dimensie met behulp van twee dimensionele gel elektroforese. Die PDQuest sagteware program is gebruik om proteïenkolle te identifiseer wat statisties beduidende verskille toon. Proteïenkolle van belang is uitgesny en onderwerp aan in-gel tripsinering en die peptiede wat sodoende verkry is, is deur middel van massa spektrometrie geanaliseer. Proteïene is geïdentifiseer deur Mascot en die Swiss Prot databasis. Resultate: Westelike klad analise het aangetoon dat die RISK pad en p38 MAPK geaktiveer is tydens vroeë herperfusie, maar die aktivering word nie volgehou tydens die hele herperfusie periode nie. Outofagie word gestimuleer tydens die vroeë herperfusie fase; dit word onderdruk in die middel herperfusie fase en bereik ‘n tweede piek van stimulering in die laat herperfusie fase. Die proteomiese analise het onderskeidelik twintig differensieel gereguleerde mitokondriale proteïene en ses en dertig differensieel gereguleerde sitosoliese proteïene geïdentifiseer tussen Non-IPK en IPK. Die grootste persentasie van hierdie proteïene is direk betrokke by miokardiale energie metabolisme. CAMKII is geidentifiseer as ‘n unieke merker van funksionele herstel in IPK tydens reperfusie. Gevolgtrekking: Aktivering van die meeste van die proteïenkinases wat ondersoek is in die huidige studie, is geassosieer met die hemodinamiese parameters van die hart, in plaas van funksionele herstel. Die resultate het aangetoon dat die varierende patrone van kinase aktivering toegeskryf kan word word aan verskille in harttempo en die effek daarvan (ejeksie fraksie, minimum en maksimum tempo van kontraksie), as gevolg van simpatiese stimulasie toegeskryf aan sielkundige stres in die diere voor slagting. Proteomiese analise het getoon dat IPK harte wat faal na isgemie/reperfusie metabolies gekompromiseer is en “slegter daaraan toe” is, in vergelyking met Non-IPK harte.

Made available in DSpace on 2015-06-17T19:33:40Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-08-26. Added 1 bitstream(s) on 2015-06-18T12:48:14Z : No. of bitstreams: 1 000825977.pdf: 523697 bytes, checksum: d2f8ec7342d5b6db7422e962ecea6b53 (MD5) / Justificativas e objetivos: os anti-inflamatórios não esteroidais (AINEs) têm mostrado sua eficácia na abordagem multimodal da dor, reduzindo o consumo de opioides na dor aguda pós-operatória. Embora os AINEs possam afetar a função renal, existem poucos dados da utilização de AINEs coxibes disponíveis nesse contexto. Os biomarcadores precoces de lesão renal aguda, como o NGAL (neutrophil gelatinase-associated lipocalin), podem ser importantes aliados na determinação de lesão real associada ao uso de AINEs coxibes no período perioperatório. Materiais e métodos: após aprovação pelo Comitê de Ética em Pesquisa Experimental, 40 ratos Wistar foram distribuídos aleatoriamente em quatro grupos. Sob anestesia geral, e dependendo do grupo a qual os animais foram incluídos, eles foram submetidos a isquemia e reperfusão renal. Dois grupos receberam parecoxibe para avaliar a influência deste medicamento na função renal. Dosagem de NGAL e histologia renal bilateral foram realizadas para a avaliação da existência e grau da lesão renal. Resultados: o grupo isquemia (que não recebeu injeção de parecoxibe) apresentou os maiores níveis de NGAL e maior frequência de lesão renal. O grupo que sofreu isquemia e recebeu parecoxibe apresentou níveis de NGAL e frequência de lesão renal similares aos outros grupos que não sofreram tal injúria. Conclusão: neste modelo experimental, parecoxibe exibiu propriedades em promover proteção renal / Background and aims: nonsteroidal anti-inflammatory drugs (NSAIDs) have been proved to be effective, in a multidimensional approach, at reducing the opioid consumption during the postoperative acute pain. Although the NSAIDs may affect the renal function, there are few published data related to the use of coxibs and the kidney function. The early biomarkers of acute renal injury (EBARI) may be important tool to determining the actual risk associated with the use of coxibs NSAIDs in the perioperative period. Methods: after approval by Experimental Ethics Committee, 40 male Wistar rats were randomly assigned into four groups. Under general anesthesia, and depending on the assigned group, rats underwent renal ischemia and reperfusion. Parecoxib was injected in two of the groups to evaluate the coxib intluence on the renal function. Parametric data, serum neutrophil gelatinase-associated lipocalin (NGAL), an EBARI, and right and left renal histology were used to evaluate there renal function. Results: the animals of ischemia group which did not receive parecoxib showed the highest NGAL serum level, as well as the most severe tubular injury. The animals on ischemia group which received parecoxib showed NGAL plasmatic levels and tubular injury similar to the groups not subjected to renal ischemia. Conclusion: on this experimental model, parecoxib showed protective renal properties

Orientador: Guilherme Antonio Moreira de Barros / Banca: Eliana Marisa Ganen / Banca: Eunice Sizue Hirata / Resumo: Justificativas e objetivos: os anti-inflamatórios não esteroidais (AINEs) têm mostrado sua eficácia na abordagem multimodal da dor, reduzindo o consumo de opioides na dor aguda pós-operatória. Embora os AINEs possam afetar a função renal, existem poucos dados da utilização de AINEs coxibes disponíveis nesse contexto. Os biomarcadores precoces de lesão renal aguda, como o NGAL (neutrophil gelatinase-associated lipocalin), podem ser importantes aliados na determinação de lesão real associada ao uso de AINEs coxibes no período perioperatório. Materiais e métodos: após aprovação pelo Comitê de Ética em Pesquisa Experimental, 40 ratos Wistar foram distribuídos aleatoriamente em quatro grupos. Sob anestesia geral, e dependendo do grupo a qual os animais foram incluídos, eles foram submetidos a isquemia e reperfusão renal. Dois grupos receberam parecoxibe para avaliar a influência deste medicamento na função renal. Dosagem de NGAL e histologia renal bilateral foram realizadas para a avaliação da existência e grau da lesão renal. Resultados: o grupo isquemia (que não recebeu injeção de parecoxibe) apresentou os maiores níveis de NGAL e maior frequência de lesão renal. O grupo que sofreu isquemia e recebeu parecoxibe apresentou níveis de NGAL e frequência de lesão renal similares aos outros grupos que não sofreram tal injúria. Conclusão: neste modelo experimental, parecoxibe exibiu propriedades em promover proteção renal / Abstract: Background and aims: nonsteroidal anti-inflammatory drugs (NSAIDs) have been proved to be effective, in a multidimensional approach, at reducing the opioid consumption during the postoperative acute pain. Although the NSAIDs may affect the renal function, there are few published data related to the use of coxibs and the kidney function. The early biomarkers of acute renal injury (EBARI) may be important tool to determining the actual risk associated with the use of coxibs NSAIDs in the perioperative period. Methods: after approval by Experimental Ethics Committee, 40 male Wistar rats were randomly assigned into four groups. Under general anesthesia, and depending on the assigned group, rats underwent renal ischemia and reperfusion. Parecoxib was injected in two of the groups to evaluate the coxib intluence on the renal function. Parametric data, serum neutrophil gelatinase-associated lipocalin (NGAL), an EBARI, and right and left renal histology were used to evaluate there renal function. Results: the animals of ischemia group which did not receive parecoxib showed the highest NGAL serum level, as well as the most severe tubular injury. The animals on ischemia group which received parecoxib showed NGAL plasmatic levels and tubular injury similar to the groups not subjected to renal ischemia. Conclusion: on this experimental model, parecoxib showed protective renal properties / Mestre