Made available in DSpace on 2016-12-06T17:07:04Z (GMT). No. of bitstreams: 1 Kayo Pereira.pdf: 116613 bytes, checksum: 0b670635cacde86cdf673047846f82d8 (MD5) Previous issue date: 2015-07-30 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The present study aim understand the influence of variables in iscquemic preconditioning (PCI), and the enhancement performance after PCI. First, a meta-analise was forged to situate the PCI estate of art. After, an experimental study was conducted to advance knowledge in performance after PCI, and PCI application guidelines. Nine men was submitted to 3 PCI application methods in different days. The PCI methods consisted in different application: superior limbs (PCISUP), inferior limbs (PCIINF) and all limbs (PCIAMB). Besides, more one condition was performed without PCI applications for de control condition (CON). Thirty minutes late PCI the protocol of maximal voluntary contraction and electroestimulation was performed. The central activation rate (TAC) was significant different between CON and: PCIINF and PCAMB, but PCISUP show only tendency. The force evoked in rest (FER) was significant different only between CON and PCIAMB. The PCI have a little benefit of aerobic and anaerobic performance. However, more studies are needed for improve PCI guidelines. In present study PCI modify both peripheral and central variables in exercise. The effect of PCI seem depend of spinal level. Our results suggest that PCI effect can be dependent of occluded mass and local of PCI application. Therefore, the PCI effect was more evident when more mass or the PCI was applied in the exercise engaged limb. / Este estudo visou entender de maneira ampla as variáveis que influenciam na aplicação do Precondicionamento isquêmico (PCI) e como o PCI influência no desempenho. Para isso foi realizado uma meta-analise para situar o atual estado da arte sobre as pesquisas sobre esse tema. Após, foi realizado um procedimento experimental na tentativa de avançar no conhecimento sobre o tema e propor novas diretrizes sobre a aplicação do PCI. Para isto 9 sujeitos foram submetido em dias diferentes a 3 métodos de PCI com diferentes massas envolvidas, aplicado em diferentes membros: membros superiores (PCISUP), inferiores (PCIINF) e ambos (PCIAMB) e uma condição controle (CON). Então foram submetidos a um protocolo de contração máxima voluntária e eltroestimulação sobreposta. A Taxa de ativação central (TAC) foi significativamente diferente entre CON e: PCIINF e PCIAMB e PCISUP apresentou apenas uma tendência em relação a CON. A Força evocada em repouso (FER) foi significativamente diferente somente entre CON e PCIAMB. O PCI possui um pequeno efeito porém com potencial benéfico tanto em desempenhos aeróbios e anaeróbios. Porém mais pesquisas são necessárias para aprimoramento da técnica. No presente estudo o PCI foi capaz de alterar variáveis centrais e periféricas durante o exercício, parecendo o efeito residir a nível espinal. Nossos resultados sugerem que estas alterações podem ser dependentes da massa envolvida e do local de aplicação, sendo o efeito mais evidente em maiores massas e quando o PCI é aplicado no membro envolvido no exercício.

Introdução: A hipotermia tópica (HT) e o pré-condicionamento isquêmico (PCI) são métodos utilizados para diminuir a lesão de isquemia/reperfusão (I/R). A eficácia do uso concomitante da HT e PCI (HT+PCI) no fígado em relação à inflamação e à citoproteção antioxidante não está elucidada. Objetivo: Avaliar o processo inflamatório e os mecanismos de segunda linha de defesa antioxidante na lesão de I/R hepática em ratos em relação à utilização das técnicas de HT e PCI de forma isolada ou associada. Métodos: Ratos Wistar (n=32) foram submetidos à isquemia hepática parcial (70%) durante 90 minutos seguida por 120 minutos de reperfusão. Os animais foram alocados nos grupos sham (n=4), isquemia normotérmica (IN, n=7), PCI (n=7), HT (n=7) e HT+PCI (n=7). O PCI consistiu na aplicação consecutiva de 10 minutos de isquemia e reperfusão antes do insulto isquêmico. A HT foi induzida pela superfusão de solução salina a 26°C sobre os lobos isquêmicos. A eutanásia foi realizada ao término do experimento e as amostras foram coletadas para a realização das análises moleculares utilizando as técnicas de ELISA e Western Blot, com o objetivo de comparar os perfis pró-inflamatório, anti-inflamatório e antioxidante. Resultados: O grupo HT comparado ao grupo IN apresentou diminuição da concentração do fator de necrose tumoral (TNF)-α, interleucina (IL)-1β, IL-6 e IL-12 e um aumento dos níveis de IL-10. O grupo HT apresentou menor expressão da óxido nítrico sintase induzível (iNOS) e um aumento da expressão da óxido nítrico sintase endotelial (eNOS). A expressão da NAD(P)H quinone oxidoreductase-1 (NQO1) foi menor no grupo HT. O PCI não demonstrou diferença significativa em relação a esses marcadores quando comparado ao grupo IN. O grupo HT+PCI apresentou menor concentração de IL-12 e menor expressão da iNOS e NQO1, mas em relação a estas moléculas a utilização de HT isolada demonstrou um comportamento semelhante. O grupo HT+PCI apresentou maior expressão da Kelch-like ECH-associated protein (Keap)-1 e menor expressão do nuclear erythroid 2-related factor 2 (Nrf2) nuclear e citoplasmático em relação ao grupo IN. Conclusão: O método de HT foi eficaz na proteção contra a lesão inicial de I/R. O uso de PCI isolado desencadeou a ativação da segunda linha de defesa antioxidante. A aplicação combinada de HT+PCI não confere benefício adicional em relação ao processo inflamatório quando comparado ao grupo HT, mas apresenta a vantagem de evitar a ativação da segunda linha de defesa antioxidante. / Background: Topical hypothermia (TH) and ischemic preconditioning (IPC) are used to decrease ischemia/reperfusion (I/R) injury. The effectiveness of using concomitantly TH and IPC (TH+IPC) in liver, regarding inflammation and antioxidant cytoprotection, is lacking. Aim: To evaluate the process inflammatory and second-line antioxidant defense mechanisms in hepatic I/R injury in rats in relation to the use of techniques TH and IPC isolate or associated. Methods: Wistar rats (n=32) subjected to partial (70%) hepatic ischemia during 90 minutes followed by 120 minutes of reperfusion. Livers from the animals allocated in sham (n=4), normothermic ischemia (NI, n=7), IPC (n=7), TH (n=7) and TH+IPC (n=7) groups. IPC consisted of consecutive 10-minute periods of ischemia and reperfusion before the ischemic insult. TH was induced by the superfusion of cooled saline at 26oC onto the ischemic lobes. Euthanasia was undertaken exactly at the end of the experiment and samples were collected for molecular analyses by ELISA and Western Blot assays, aiming to compare pro-inflammatory, anti-inflammatory and antioxidant profiles. Results: Compared with NI, TH presented decreased tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6 and IL-12 concentrations and increased IL-10 levels. TH displayed lower inducible nitric oxide synthase (iNOS), higher endothelial nitric oxide synthase (eNOS) expressions. NAD(P)H-quinone oxidoreductase-1(NQO1) expression was also lower in TH. Isolate IPC showed no differences regarding all these markers compared to NI. TH+IPC showed decreased IL-12 concentration and reduced iNOS and NQO1 expressions, but regarding these molecules isolate TH behaved similarly. TH+IPC showed higher Kelch-like ECH-associated protein (Keap)-1 and diminished nuclear and cytosolic nuclear erythroid 2-related factor 2 (Nrf2) expressions than NI. Conclusion: TH was the effective method of protection against early I/R injury. Isolated IPC entailed triggering of second-line antioxidant defense enzymes. Combined TH+IPC seemed to confer no additional advantage over isolated TH in relation to the inflammatory process, but had the advantage of avoid activation second-line antioxidant defense enzymes.

Introdução: A hipotermia tópica (HT) e o pré-condicionamento isquêmico (PCI) são métodos utilizados para diminuir a lesão de isquemia/reperfusão (I/R). A eficácia do uso concomitante da HT e PCI (HT+PCI) no fígado em relação à inflamação e à citoproteção antioxidante não está elucidada. Objetivo: Avaliar o processo inflamatório e os mecanismos de segunda linha de defesa antioxidante na lesão de I/R hepática em ratos em relação à utilização das técnicas de HT e PCI de forma isolada ou associada. Métodos: Ratos Wistar (n=32) foram submetidos à isquemia hepática parcial (70%) durante 90 minutos seguida por 120 minutos de reperfusão. Os animais foram alocados nos grupos sham (n=4), isquemia normotérmica (IN, n=7), PCI (n=7), HT (n=7) e HT+PCI (n=7). O PCI consistiu na aplicação consecutiva de 10 minutos de isquemia e reperfusão antes do insulto isquêmico. A HT foi induzida pela superfusão de solução salina a 26°C sobre os lobos isquêmicos. A eutanásia foi realizada ao término do experimento e as amostras foram coletadas para a realização das análises moleculares utilizando as técnicas de ELISA e Western Blot, com o objetivo de comparar os perfis pró-inflamatório, anti-inflamatório e antioxidante. Resultados: O grupo HT comparado ao grupo IN apresentou diminuição da concentração do fator de necrose tumoral (TNF)-α, interleucina (IL)-1β, IL-6 e IL-12 e um aumento dos níveis de IL-10. O grupo HT apresentou menor expressão da óxido nítrico sintase induzível (iNOS) e um aumento da expressão da óxido nítrico sintase endotelial (eNOS). A expressão da NAD(P)H quinone oxidoreductase-1 (NQO1) foi menor no grupo HT. O PCI não demonstrou diferença significativa em relação a esses marcadores quando comparado ao grupo IN. O grupo HT+PCI apresentou menor concentração de IL-12 e menor expressão da iNOS e NQO1, mas em relação a estas moléculas a utilização de HT isolada demonstrou um comportamento semelhante. O grupo HT+PCI apresentou maior expressão da Kelch-like ECH-associated protein (Keap)-1 e menor expressão do nuclear erythroid 2-related factor 2 (Nrf2) nuclear e citoplasmático em relação ao grupo IN. Conclusão: O método de HT foi eficaz na proteção contra a lesão inicial de I/R. O uso de PCI isolado desencadeou a ativação da segunda linha de defesa antioxidante. A aplicação combinada de HT+PCI não confere benefício adicional em relação ao processo inflamatório quando comparado ao grupo HT, mas apresenta a vantagem de evitar a ativação da segunda linha de defesa antioxidante. / Background: Topical hypothermia (TH) and ischemic preconditioning (IPC) are used to decrease ischemia/reperfusion (I/R) injury. The effectiveness of using concomitantly TH and IPC (TH+IPC) in liver, regarding inflammation and antioxidant cytoprotection, is lacking. Aim: To evaluate the process inflammatory and second-line antioxidant defense mechanisms in hepatic I/R injury in rats in relation to the use of techniques TH and IPC isolate or associated. Methods: Wistar rats (n=32) subjected to partial (70%) hepatic ischemia during 90 minutes followed by 120 minutes of reperfusion. Livers from the animals allocated in sham (n=4), normothermic ischemia (NI, n=7), IPC (n=7), TH (n=7) and TH+IPC (n=7) groups. IPC consisted of consecutive 10-minute periods of ischemia and reperfusion before the ischemic insult. TH was induced by the superfusion of cooled saline at 26oC onto the ischemic lobes. Euthanasia was undertaken exactly at the end of the experiment and samples were collected for molecular analyses by ELISA and Western Blot assays, aiming to compare pro-inflammatory, anti-inflammatory and antioxidant profiles. Results: Compared with NI, TH presented decreased tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6 and IL-12 concentrations and increased IL-10 levels. TH displayed lower inducible nitric oxide synthase (iNOS), higher endothelial nitric oxide synthase (eNOS) expressions. NAD(P)H-quinone oxidoreductase-1(NQO1) expression was also lower in TH. Isolate IPC showed no differences regarding all these markers compared to NI. TH+IPC showed decreased IL-12 concentration and reduced iNOS and NQO1 expressions, but regarding these molecules isolate TH behaved similarly. TH+IPC showed higher Kelch-like ECH-associated protein (Keap)-1 and diminished nuclear and cytosolic nuclear erythroid 2-related factor 2 (Nrf2) expressions than NI. Conclusion: TH was the effective method of protection against early I/R injury. Isolated IPC entailed triggering of second-line antioxidant defense enzymes. Combined TH+IPC seemed to confer no additional advantage over isolated TH in relation to the inflammatory process, but had the advantage of avoid activation second-line antioxidant defense enzymes.

Introdução: O aumento da tolerância do miocárdio isquêmico observado durante o segundo de dois testes de esforços seqüenciais, o fenômeno do pré-aquecimento, foi proposto como um modelo clínico do pré-condicionamento isquêmico. Bloqueadores dos canais de K-ATP dependentes, tais como as sulfoniluréias, podem induzir a perda do pré-condicionamento isquêmico, o qual poderia estar envolvido no aumento dos eventos cardiovasculares. A repaglinida é um agente hipoglicemiante oral, pertencente à família da meglitinida e supostamente dotada de menor efeito no pré-condicionamento isquêmico, ainda que o fármaco tenha seu principal mecanismo de ação nos canais de K-ATP dependentes. Objetivos e Métodos: O objetivo foi investigar os efeitos da repaglinida no fenômeno do pré-condicionamento isquêmico em pacientes diabéticos com doença coronariana estável. Foram estudados 42 pacientes diabéticos tipo 2, com angina estável e doença arterial documentada. Todos os pacientes tinham testes ergométricos positivos para isquemia. Na primeira fase do teste, a sulfoniluréia e os betabloqueadores foram suspensos por trinta dias e sete dias, respectivamente. Os pacientes foram submetidos a dois testes ergométricos seqüenciais, com intervalo de trinta minutos (testes 1 e 2). Na segunda fase, os pacientes receberam repaglinida por sete dias e mais dois testes ergométricos foram repetidos (testes 3 e 4). Resultados: Todos os pacientes alcançaram ST >1 mm na primeira fase (Teste 1 e 2). O tempo alcançado no teste 2 foi maior que aquele alcançado no teste 1 (4:44s. x 5:37s. p=0,001), como também foi maior a duração do exercício (6:15s x 6:29s. p=0,008), denotando pré-condicionamento isquêmico. Após o uso da repaglinida, nos testes 3 e 4, observou-se menor tempo alcançado para atingir isquemia no teste 4 (5:37s. x 4:58s. p=0,001). Observou-se, ainda, menor tempo de tolerância ao exercício na fase 2 (6:57s x 6:34s. p=0,007). Em relação ao surgimento de angina, não se constataram diferenças estatísticas entre as duas fases. Conclusão: Nos pacientes diabéticos com doença coronariana estável, a repaglinida bloqueou o pré-condicionamento isquêmico. / Background: The increase of tolerance to myocardial ischemia observed during the second of two sequential exercise tests, the warm-up phenomenon, has been proposed as a clinical model of ischemic preconditioning. Blockers of K-ATP channels, such as the Sulfonylurea drugs, can induce loss of ischemic preconditioning, what could be involved in an increase of cardiac events. Repaglinide is a hypoglycemic agent with supposedly lower influence on ischemic preconditioning, despite acting in K-ATP channels. Objectives and Methods: This study investigated the effects of repaglinide on the ischemic preconditioning in diabetic patients with CAD. There were 42 patients and inclusion criteria were positive treadmill test for myocardial ischemia. Sulphonylureas and beta-blocking agents were withdrawn 30 and 7 days respectively before phase 1 of the study. In this phase, the patients underwent two consecutive treadmill exercise tests at 30 minute intervals (test 1 and test 2). In phase 2 of the study, all patients received repaglinide 2 mg three times daily during 7 days before treadmill exercise test (test 3 and test 4). Results: All patients achieved 1.0 mm ST-segment depression during phase 1. The time achieved to ST depression during test 2 was greater than that during test 1 (4:44s vs. 5:37s. p=0.001) as well as the duration of the exercise (6:15s vs.6: 29s. p=0.008), suggesting a higher ischemic threshold. In phase 2 after repaglinide, all patients achieved 1 mm ST-segment depression. However, the time achieved to ST depression, as well as the duration of the exercise, was lower in test 4 comparing with test 3. There were no statistical differences regarding angina episodes in phase 1 or phase 2. Conclusions: In diabetic patients with stable coronary disease, the oral hypoglycemic agent repaglinide abolished the myocardial ischemic preconditioning.

No presente trabalho, estudaram-se os efeitos da l-alanil glutamina (Ala-Gln), do precondicionamento isquÃmico (PCI) e das duas tÃcnicas concomitantemente sobre a lesÃo pulmonar provocada por isquemia e reperfusÃo (I/R) causada por pinÃamento da aorta infra-renal em ratos. Foram utilizados 60 ratos machos Wistar, randomizados em cinco grupos (n = 12) divididos em dois tempos (n = 6): Grupo Simulado, Grupo I/R, Grupo PCI + I/R, Grupo Ala-Gln + I/R, Grupo Ala-Gln + PCI + I/R. Tempos: T1 (4h de isquemia) e T2 (4 horas de isquemia e 1h de reperfusÃo). Todos os grupos receberam soluÃÃo salina previamente, menos os grupos prÃ-tratados com Ala-Gln que receberam o dipeptÃdeo e soluÃÃo salina em igual volume. Foi utilizado o modelo de pinÃamento da aorta infra-renal com 4 horas de isquemia e 1 hora de reperfusÃo. Determinaram-se as concentraÃÃes de mieloperoxidase (MPO) pulmonar, substÃncias reativas ao Ãcido tiobarbitÃrico (TBARS) e glutationa reduzida (GSH) no sangue e pulmÃo para avaliar os grupos em estudo. O teste de Kolmogorov-Smirnoff mostrou distribuiÃÃo normal dos dados. Dados expressos como mÃdia acompanhada pelo seu desvio padrÃo (MÃdia  DPM) sendo realizado teste t de Student. Para anÃlise comparativa simultÃnea de trÃs grupos utilizou-se o teste Anova com pÃs-teste de Tukey. Em todos os casos foi adotado o nÃvel de significÃncia de p<0,05. Houve elevaÃÃo das concentraÃÃes de MPO pulmonar tanto no grupo submetido à isquemia quanto no grupo que realizou a I/R. Ocorreu reduÃÃo significante das concentraÃÃes de MPO pulmonar nos grupos submetidos à isquemia prÃ-tratados com Ala-Gln e com PCI. Na avaliaÃÃo dos grupos que sofreram I/R nÃo foi observada alteraÃÃo nas concentraÃÃes de MPO nos grupos prÃ-tratados Ala-Gln ou PCI. O grupo prÃ-tratado com as duas tÃcnicas apresentou aumento significante da MPO nos tempos estudados. A Ala-Gln como prÃ-tratamento isolado reduziu TBARS plasmÃtico na isquemia e o aumentou no pulmÃo na I/R. Jà no pulmÃo durante isquemia e no plasma na I/R houve reduÃÃo da GSH. O PCI como prÃ-tratamento isolado elevou o TBARS pulmonar na I/R e reduziu a GSH pulmonar na I/R. A associaÃÃo da Ala-Gln e PCI acresceu o TBARS plasmÃtico na isquemia, tambÃm o elevando no pulmÃo e mÃsculo na I/R. Jà a GSH, com os dois prÃ-tratamentos, sofre reduÃÃo plasmÃtica na isquemia e pulmonar na I/R, com elevaÃÃo plasmÃtica na I/R. O presente estudo demonstra que tanto o prÃ-tratamento com Ala-Gln como o PCI protegem contra a lesÃo isquÃmica à distÃncia, em modelo murino de pinÃamento da aorta infra-renal quando avaliado MPO pulmonar. O mesmo nÃo ocorre na lesÃo por I/R. NÃo hà benefÃcio, e sim agravamento de lesÃo à distÃncia pulmonar, na associaÃÃo dos dois prÃ-tratamentos ao mensurar a MPO pulmonar. / The present work determined the effects of pre-treatment with L-alanyl glutamine (Ala-Gln) and ischemic preconditioning (IPC), alone and in combination, against lesions caused by I/R by clamping the infrarenal aorta in rats. Sixty Wistar rats were distributed into five groups (n = 12) divided into two times (n = 6): Control, Group I/R, Group IPC + I/R, Group Ala-Gln + I/R, Group Ala-Gln + IPC + I/R. Times: T1 (infrarenal-aorta clamping ischemia-4h); T2 (ischemia-4h plus reperfusion-1h). Pulmonary myeloperoxidase (MPO) and plasma TBARS concentrations were measured. Data expressed as mean  standard-deviation, analyzed by Studentâs t-test and ANOVA/Tukeyâs post-test. P-values < 0,05 were considered significant. Increased MPO concentrations in ischemic group and in I/R group occurred as compared to control. Reduction in MPO concentrations happened in ischemic groups pre-treated with either Ala-Gln or IPC. I/R induced no change in MPO concentrations in groups pre-treated with either Ala-Gln or IPC. Pre-treating with the two procedures showed increased MPO at both times studied. Reduction in TBARS concentrations occurred in Ala-Gln pre-treated group, whereas significant elevation was observed when Ala-Gln and IPC were associated in ischemic animals. Ischemia/reperfusion induced elevation of plasma TBARS. Pre-treatment with either Ala-Gln or IPC protects against distant pulmonary lesion due to ischemia. The same did not occur in I/R lesion. Combining the two procedures aggravated inflammation indicated by increased MPO concentrations. Elevated TBARS concentrations in ischemic animals pre-treated with the two procedures indicate increased lipid peroxidation, whereas pre-treatment with Ala-Gln induced decreased TBARS concentrations.

Abstract Open aortic arch surgery almost always requires a bloodless operating field which necessitates the use of hypothermic circulatory arrest. Hypothermic circulatory arrest is a technique where the core temperature of a patient is lowered so that the systemic blood circulation can be stopped momentarily. This can cause unwanted damage to the brain. The risk for neurological impairment is at its highest when corrective surgery has to be performed in emergency situations. This highlights the need for additional neuroprotective methods. Our research group has used a porcine model described in this thesis for about 12 years in various setups to study many neuroprotective hypotheses. We have tested and researched surgical and CPB strategies that could be useful in a HCA and aortic arch reconstruction setting. In this thesis we have combined both chronic surviving animal data with acute experiments and aim to shed light on the mechanisms and efficacy of RIPC as neuroprotective method. In our experimental model, RIPC provided a mitigation of inflammatory response and cerebral injury after prolonged HCA. In general, the collected data showed homogeneity as similar biochemical results were seen in study I and II. Also interestingly, study III and IV possibly shed some light as to the mechanisms of the neuroprotective effect seen in Study II. These results seem to corroborate each other in a logical way. In study I which was acute experiment we saw faster EEG recovery rates in the intervention group. Additionally we recorded beneficial biochemical changes from samples that were collected from the brain. In our chronic study, were the animals were followed for a 7 day period after hypothermic circulatory arrest, we saw a statistically significant neuroprotective effect of remote ischemic preconditioning. In studies III and IV we attempted to shed light on the mechanisms. Study III revealed that an altered oxygen usage profile during hypothermic circulatory arrest and recovery phase might have a role in the neuroprotection. In study IV we saw a reduced microcirculatory leukocyte accumulation in cerebrocortical vessels was noted using an intravital microscope. The intravital microscope also provided results that indicated a difference in the redox state of the mitochondria via NAD+/NADH autofluorescence measurements. / Tiivistelmä Sydän- ja aorttakirurgiassa tarvitaan jossain tilanteissa täysin veretöntä leikkausaluetta. Verettömän leikkausalueen saavuttamiseksi joudutaan joskus turvautumaan potilaan elimistön jäähdytyksen jälkeiseen verenkierron pysäytykseen. Tämän menetelmän haittana on kuitenki aivokudokselle aiheutuva hapenpuute ja tästä mahdollisesti seuraava vaurioituminen. Vaurioitumisen riski on korkeimillaan erityisesti päivystyksellisissä tilanteissa. Tämän tutkimuksen tavoitteena on ollut selvittää, onko esialtistavalla raajaiskemialla kykyä suojata aivokudosta hapenpuutostilanteissa. Tutkimusryhmämme on viimeisen 12 vuoden aikana tutkinut sianporsailla eri keinoja, joilla voitaisiin parantaa aivojen suojausta sydän- ja aorttakirurgian aikana. Esialtistava raajaiskemia toteutetaan kiristämällä mansetti eläimen oikean takajalan ympärille. Tämän jälkeen mansetti täytetään viiden minuutin välein neljästi. Täyttökertojen välissä pidettään viiden minuutin tauko, jolloin mansetti on avatuna ja jalan verenkierto palautuu normaaliksi. Ensimmäisessä tutkimuksessamme totesimme, että esialtistava raajiskemia vaikuttaa aivojen sähkökäyrän toipumista nopeuttavasti. Toisessa tutkimuksessamme seurasimme koe-eläimiä seitsemän päivän ajan kokeen jälkeen. Tämän tutkimuksen yhteydessä toteutessa aivokudoksen mikroskooppiananalyysissä havaitsimme, että raajaiskemia vaikutti suojaavan aivokudosta hapenpuutteen aiheuttamilta aivovaurioilta. Kolmanessa tutkimuksessa selvitimme, että raajaiskemia vaikuttaa aivojen happipitoisuuteen sekä verenkierron pysäytyksen aikana että toipumisvaiheessa. Viimeisessä tutkimuksessa kuvasimme aivojen pintaverisuonia mikroskoopilla. Seurasimme kokeessa valkosolujen käyttäytymistä aivokudoksessa käyttäen fluoresoivia lääkeaineita. Havaitsimme, että raajaiskemiaryhmässä valkosoluja oli aivokudoksen pintaverisuonissa merkittävästi vähemmän. Lisäksi samalla menetelmällä tutkimme sitruunahappokiertoon osallistuvan NAD+/NADH parin suhteita autofluoresenssi ilmiöllä. Autofluorensenssi tutkimuksen tulokset viittaavat siihen, että mitokondrioiden hapetus/pelkistys kyky oli parempi raajaiskemia ryhmässä. Kokeissamme esialtistava raajaiskemia vähensi tulehdussolujen määrää aivokudoksessa sekä vähensi aivovauriota hapenpuutteen jälkeen.

Abstract During cardiac and aortic surgery, disturbance of the blood supply in the central nervous system occurs when the repair of aortic pathologies is performed or a bloodless operation field is needed in complex cardiac surgery. To enable the suitable operation environment, the technique named hypothermic circulatory arrest (HCA) has been utilized via heart-lung machine. In this method, the core temperature is lowered to the target temperature, after which blood circulation is halted for a certain period of time. A challenge is that the successful usage of HCA, however still involves the risks of postoperative neurological complications and mortality. In cardiac and aortic arch surgery, the brain is at the highest risk for deficits, whereas in the repair of thoracoabdominal aortic aneurysms (TAAAs), spinal cord injury remains the most severe adverse outcome. Adjunctive protective strategies are required to reduce ischemic injury in these settings. In this thesis, Studies I and II focused on the spinal cord and the Study III on the brain. The studies were performed using acute (II, III) or subacute (I) experimental porcine models, primarily aiming to assess the effectiveness of remote ischemic preconditioning (RIPC) in spinal cord protection along with the aim of studying the underlying mechanisms of RIPC in neuroprotection. Studies I and II demonstrated enhanced motor evoked potential (MEP) responses in both hind limbs, indicating spinal cord protection by RIPC. The faster recovery of brain damage marker S100B along with higher cardiac index and lower systemic lactate levels confirmed the cardio- and neuroprotective properties of RIPC in Study III. The protective mechanism of RIPC was associated with increased antioxidant response (II, III). / Tiivistelmä Sydän- ja aorttakirurgiassa, keskushermoston verenkiertoa joudutaan häiritsemään toteutettaessa aortan korjausleikkauksia tai vaikeissa sydänkirurgisissa toimenpiteissä verettömän leikkausalueen saavuttamiseksi. Sydän-keuhkokoneen avulla toteutettava täydellinen verenkierron pysäytys mahdollistaa vaaditut olosuhteet. Tässä menetelmässä ydinlämpötilaa lasketaan ja verenkierron pysäytys toteutaan tavoitellussa kohdelämpötilassa tietyssä aikaikkunassa. Kyseisen menetelmän onnistunut käyttö sisältää kuitenkin riskejä operaatioiden jälkeisiin neurologisiin komplikaatioihin ja kuolleisuuteen. Sydämen ja aortankaaren kirurgiassa aivot ovat suurimmassa vaarassa vaurioille. Rinta- ja vatsa-aortan aneurysmien eli pullistumien korjausleikkauksiin liittyvä selkäydinvaurio on puolestaan yksi vakavimmista ja vaikeimmista seurauksista. Lisäsuojausmenetelmiä tarvitaan vähentämään iskeemistä vauriota näissä asetelmissa. Väitöskirjan osatyöt I ja II keskittyivät selkäytimeen. Osatyö III käsitteli puolestaan aivojen suojausta. Osatyöt toteutettiin akuutteina (II, III) ja subakuutteina (I) kokeellisina porsasmalleina. Tutkimusten tavoitteina oli arvioida esialtistavan perifeerisen raajaiskemian vaikuttavuutta selkäytimen suojauksessa sekä tutkia raajaiskemian taustalla olevia mekanismeja hermokudoksen suojauksessa. Osatöissä I, II havaittiin motoristen herätepotentiaalien parantuneita vasteita molemmissa takajaloissa osoittaen esialtistavan raajaiskemian suojaavan selkäydintä simuloidussa rinta-aortan korjaustoimenpiteessä. Osatyö III keskittyi alhaisessa lämpötilassa toteutettavaan täydelliseen verenkierron pysäytykseen. Tässä tutkimuksessa todetut aivovauriomarkkeri S100B tason nopeampi lasku, korkeampi sydänindeksi ja alhaisemmat laktaattitasot varmistivat raajaiskemian sydän- ja hermokudossuojausvaikutusta. Esialtistavan perifeerisen raajaiskemian suojaava mekanismi voidaan liittää parantuneeseen solujen antioksidanttivasteeseen (II, III).

Abstract Interest in off-pump coronary surgery and ischaemic preconditioning has been increasing. The aim of this study was to evaluate surrogate indicators of haemodynamic, myocardial, and cerebral outcome during off-pump surgery and preconditioning. Haemodynamics and myocardial preservation were monitored in a pilot study of twelve patients undergoing off-pump coronary surgery. Indicators of myocardial metabolism and tissue injury as well as cerebral damage were evaluated in a randomized study of thirty-three patients undergoing on-pump (11) or off-pump surgery with (11) or without (11) preceding myocardial ischaemic preconditioning for five minutes followed by reperfusion for five minutes. The pilot study showed minimal haemodynamic changes and myocardial derangements during off-pump surgery as evaluated intraoperatively based on transcardiac differences of ATP degradation products and lactate and postoperatively based on MB mass of creatine kinase and troponin T. In the following studies, myocardial ischaemic metabolism was evaluated intraoperatively by measuring transcardiac differences of ATP degradation products, lactate, and pH, which increased significantly from the baseline values in all study groups. However, the maximum values of lactate and pH were significantly higher in the cardiopulmonary bypass group (p = 0.02 and p = 0.007, respectively). There were no statistical differences between the preconditioning and non-preconditioning groups. Myocardial tissue injury was evaluated by postoperative leakage of MB mass of creatine kinase and troponin I. Their peak values were significantly higher (p &lt; 0.001 and p = 0.008) after cardiopulmonary bypass (15.1 μg/l and 13.8 μg/l) than after off-pump surgery without preconditioning (6.3 μg/l and 5.2 μg/l). The respective values were 14.8 μg/l and 7.4 μg/l after preconditioning, and there were no statistically significant differences between the off-pump groups with and without preconditioning. Cerebral damage was evaluated based on the intra- and postoperative serum concentrations of neuron-specific enolase, which were corrected with respect to haemolysis. The corrected values were significantly higher after on-pump than off-pump surgery (p = 0.003 and p = 0.005). In conclusion, multi-vessel off-pump coronary artery surgery is a haemodynamically feasible procedure offering better myocardial preservation compared to on-pump surgery. Ischaemic preconditioning of the myocardium does not seem to improve myocardial preservation in off-pump surgery. The slightly lower levels of neuron-specific enolase also suggest less cerebral damage.

INTRODUÇÃO: Baseados em estudo que evidenciou menor área de infarto do miocárdio (IM) em ratos submetidos a treinamento físico (TF),na ausência de reperfusão; e na liberação de endorfinas que ocorre durante o TF, nossos objetivos são: demonstrar se não só TF, mas também opióides e isquemia/reperfusão (IR) intermitente são capazes de reduzir área de IM, na ausência de reperfusão; se TF e opióides exibem efeito sinérgico e se o mecanismo de redução da área de IM pelo TF envolve receptores opióides. MATERIAIS E MÉTODOS: Ratos Wistar machos (n=76) foram divididos em 7 grupos:1- controle;2- TF (esteira elétrica,1 hora/dia,5 vezes/semana,por 12 semanas), antes do IM; 3- morfina antes do IM; 4- morfina+TF; 5- grupo com 3 ciclos de IR antes do IM; 6- naloxone antes da morfina; 7- naloxone antes de cada dia de TF. Todos os ratos foram submetidos à mensuração da pressão diastólica final (PDF) e a IM através da oclusão da artéria descendente anterior. A eficácia do TF foi avaliada através do consumo de oxigênio (VO2) e da distância máxima percorrida. Os ratos foram sacrificados no 8o pós-IM e a área de IM mensurada por planimetria. RESULTADOS: Não houve diferença no peso inicial (p=0,94), mortalidade (p=0,99), e relação peso cardíaco/peso corporal (p=0,29) entre os grupos. Entretanto, houve aumento do deltaVO2 (VO2 pico - VO2repouso) (p=0,0001)e da distância máxima percorrida (p=0,0001), nos grupos treinados. A PDF aumentou no pós-IM, em todos os grupos (p=0,0001). Os grupos tratados tiveram menor área de IM (p=0,0001), com exceção dos grupos morfina + naloxone e TF+ naloxone sendo que não houve maior redução no grupo TF+morfina. Os grupos TF e TF+morfina apresentaram maior espessura do septo inter-ventricular, em relação ao grupo controle (p=0,0008). Já o grupo TF + naloxone não apresentou maior espessura do septo IV, em relação aos outros grupos. Também não houve diferença na densidade capilar (p=0,88). CONCLUSÃO: Não só TF, mas também morfina e IR reduzem a área de IM, na ausência de reperfusão, sendo que não há efeito sinérgico entre TF e morfina. Esta redução não ocorre através do aumento da densidade capilar. Além disto, a ação do TF sobre a área de IM provavelmente ocorre através do estímulo de receptores opióides, visto que seu bloqueio anulou o efeito cardioprotetor do TF / BACKGROUND AND OBJECTIVES: Studies have shown a decrease in infarcted area in rats submitted to exercise training (ET), in the absence of reperfusion. Based on that, we tested four hypotheses: 1- not only ET but also another stimulus that causes myocardial protection, like opioid infusion and brief periods of ischemia-reperfusion (IR) before irreversible left anterior descending (LAD) coronary occlusion could reduce infarct area, 2- ET plus opioid infusion could have additive effects in reducing infarct size, 3- blocking the opioid system we could lose the myocardial protection caused by ET, 4-myocardial protection given by different strategies could occur due to the increase in capillary density. METHODS: Male Wistar rats (n=76) were randomly assigned to 7 groups: control (n=11); ET (n=12); morphine infusion before myocardial infarction (MI) (n=14); ET plus morphine (n=11); naloxone (a non selective opioid receptor blocker) plus morphin (n=9); intermittent IR (n=12) before MI; naloxone before each ET session (n=7). All groups were submitted to MI by LAD ligation technique and to measurement of left ventricular end-diastolic pressure (LVEDP) before and 5 min after MI. ET was performed on a treadmill for 60 min, 5 times/week for 12 weeks at 60% peak oxygen (peak VO2). To evaluate the efficacy of ET, we tested the exercise capacity and the peak VO2 before and after experimental period. Seven days after MI induction, rats were killed and hearts were harvested. Infarct size was expressed by evaluation of necrotic area, expressed as a % of the risk region (total left ventricle area). RESULTS: There were no differences in initial weight, cardiac/animal weight or mortality among groups. Exercise training increased exercise capacity (p=0.0001) and delta VO2 (VO2 peak-VO2 rest) (p=0.0001). Inter-ventricular septum thickness was higher in the ET and ET plus morphine groups, compared to the control group (p=0.0008). The LVEDP increased in the post-MI period, for all groups (p=0.0001). All treatment groups but not morphine plus naloxone and ET plus naloxone showed a decrease in infarcted area (p=0.0001). There was no additional decrease in infarct size in the ET+ morphine group, comparing with each group alone . There was no difference in capillary density for all groups. CONCLUSION: Not only ET, but also morphine and IR decrease infarcted area, in the absence of reperfusion. There is no additional effect between ET and morphine. Moreover, this reduction is not due to an increase in capillary density. The effect of ET in decreasing infarct size might occur by opioid receptor stimulus

Cell swelling may contribute to acute cell injury subsequent to ischemia/reperfusion. The potential role of mitochondrial uncoupling and the resultant mitochondrial swelling, due to opening of the mitochondrial permeability transition pore (MPTP), were examined in an in vitro ischemically pelleted isolated rabbit cardiomyocyte model using the protonophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP) to uncouple mitochondria. Cyclosporin A (CsA) was employed to inhibit MPTP opening. Cell volume was determined by a cell-flotation, density-gradient assay, using bromododecane. Cell viability, subsequent to an osmotic stress, was determined by trypan blue permeability. Ischemic preconditioning (IPC) facilitated volume regulation following an osmotic stress. Ischemic-cell swelling was reduced by IPC. IPC protected ischemically pelleted cells, but CsA had no significant effects on injury or IPC protection. CCCP ischemia accelerated rates of ischemic contracture and injury, and abolished IPC protection. IPC protection was restored by CsA. In CCCP-ischemic-uncoupled cells, subjected to a reduced (170 mOsm) osmotic stress, CsA and IPC afforded independent and additive protection. Chelerythrine and 5-hydroxydecanoate (5-HD) blocked IPC, but did not reduce CsA protection. Electron microscopy confirmed that CCCP ischemia induced mitochondrial matrix swelling that was reduced by CsA. Cardioprotection by IPC and CsA was accompanied by proportional reductions in cell swelling. Morphometric analysis of the electron photomicrographs demonstrated that the mitochondrial volume fractions were significantly reduced in the CsA/CCCP (29.8 ± 2.3%, P < 0.004) and IPC/CsA/CCCP (31.5 ± 1.7%, P < 0.0008) groups as compared to the CCCP-ischemic group (40.5 ± 1.7%) The IPC/CCCP group (39.5 ± 4.2%) was not significantly different from the CCCP-ischemic group. NIM 811, a CsA analogue MPTP blocker with no calcineurin inhibitory activity, afforded protection similar to CsA. The results suggest that CsA protection may, in part, be mediated by reduction of mitochondrial swelling.