▼ Purpose: Ischemic preconditioning (IPC) has been used to protect myocardial cells against ischemia-reperfusion injury and is recently used for improving exercise performance. It is unknown whether a remote bout of IPC (RIPC) to tissue not involved in exercise can induce similar exercise improvements and what “dose” of IPC is necessary to induce exercise performance benefits. This study determined if unilateral and bilateral upper limb RIPC improves lower body anaerobic power output. Methods: Using two randomized, single blind, crossover study designs, we studied 43 young recreationally active adults. For study 1, unilateral RIPC was used and a sham control condition involved the inflation of blood pressure cuffs to 10 mm Hg. For study 2, the ischemic stimuli were increased to bilateral occlusion while the sham control condition used was 0 mm Hg of occlusion pressure. After the RIPC treatment, subjects completed four 30 s Wingate anaerobic tests on a Monark cycle ergometer with 2 min passive rest between trials. Results: In the unilateral occlusion trial, peak power, mean power, and fatigue index were not different between the two conditions at every Wingate test. In the bilateral occlusion trial, peak power was elevated in the RIPC condition than in the sham control for the fourth Wingate test (p<0.05). Additionally, compared with the sham control, mean power was greater in the RIPC condition during the first and fourth Wingate tests (both p<0.05). Conclusion: Remote ischemic preconditioning applied bilaterally increased lower body power output over a series of Wingate anaerobic tests. Unilateral RIPC, however, had no effect on any of the performance variables, suggesting that there is a threshold for the amount of target tissue needed to elicit anaerobic performance benefits. Advisors/Committee Members: Tanaka, Hirofumi, Ph. D. (advisor).

▼ Ischemic preconditioning is an endogenous cardioprotective mechanism in which short periods of ischemia and reperfusion provide protection when given before a subsequent ischemic event. Early mechanistic studies showed ATP-sensitive potassium (KATP) channels to play an important role in ischemic preconditioning. KATP channels link intracellular energy metabolism to membrane excitability and contractility. It is thought that KATP channels provide a cardioprotective role during ischemia by inducing action potential shortening, reducing an excessive Ca²⁺ influx, and by preventing arrhythmias. However, the mechanisms by which KATP channels protect during ischemic preconditioning are not known. In this study, we investigated a novel potential mechanism in which alterations in subcellular KATP channel trafficking during ischemia and ischemic preconditioning may result in altered levels of surface channel density, and therefore, a greater degree of cardioprotection. In the optimization of our experiments, we compared various antibodies for their specificity and sensitivity for channel subunit detection in immunoblotting. In addition, we examined the effects of varying salt concentrations during tissue homogenization in order to determine the optimal conditions for protein isolation. Furthermore, we examined the effect of heating the samples prior to SDS-PAGE for improved detection of channel proteins by immunoblotting. The subcellular trafficking of some membrane proteins is altered by ischemia. For example, the glucose transporter, Glut4, translocates from endosomal compartments to the sarcolemma (Sun, Nguyen, DeGrado, Schwaiger, & Brosius, 1994). Conflicting data exists regarding the effects of ischemia on KATP channel subcellular trafficking and the regulation of KATP channel surface density (Edwards et al., 2009 and Bao, Hadjiolova, Coetzee, & Rindler, 2011). We therefore, sought to test our hypothesis that KATP channels are internalized from the surface of cardiomyocytes to endosomal compartments during ischemia, and this internalization can be reduced and/or reversed by ischemic preconditioning. We subjected isolated Langendorff-perfused mouse hearts to ischemia, reperfusion, or ischemic preconditioning events and measured the density of KATP channels in the sarcolemmal and endosomal compartments. We also determined the degree of injury by staining heart slices with triphenyltetrazolium chloride and compared infarct sizes between hearts subjected to ischemia and ischemic preconditioning. Our data demonstrated that KATP channels are, in fact, internalized during ischemia and that reperfusion led to a slow recovery of surface KATP channel density. Interestingly, ischemic preconditioning reduced the size of infarcts induced by ischemia and also prevented the ischemia-induced decrease of KATP channel surface density, thereby, contributing to cardioprotection.

▼ Ischemic heart disease is the leading cause of death in Western countries. We demonstrated how alterations in energy metabolism and the activation of Reperfusion Injury Salvage Kinase (RISK) pathway induced by short-term calorie restriction (CR) contribute to protecting the diseased heart from ischemia/reperfusion (I/R) injury. Our findings using the Spontaneously Hypertensive Rat (SHR) validate that short-term CR exhibits cardioprotection in the rat model of hypertension and cardiac hypertrophy. Our data also suggest that improving glucose oxidation at the time of reperfusion and activating two members of the pro-survival anti-apoptotic RISK pathway, Akt and Erk1/2 MAPK, are possible mechanism by which short-term CR contributes to improving mechanical recovery of the heart during reperfusion following ischemia. Additionally, our data suggest that the effects of short-term CR in I/R injury may be mediated by an AMPK-independent mechanism as the hearts from SHRs exhibited improved metabolic status in presence of significantly reduced AMPK activity.

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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…

Advisors/Committee Members: Juvonen, T. (Tatu).

▼ Objective: Ischemic spinal cord injury is a serious complication of aortic surgery. The mechanism underlying ischemic preconditioning (IPC) protection against spinal cord ischemia/reperfusion (I/R) injury is unclear. We investigated the role of spinal cord autoregulation in tolerance to spinal cord I/R injury induced by IPC. Although the extracellular signal-regulated kinases 1 and 2 (ERK1/2) are generally regarded as related to cell survival and proliferation, increasing evidence suggests that the role of the ERK1/2 pathway in I/R injury is contributory to inflammation. We investigated the effect of blocking ERK1/2 pathway to inhibit inflammation reaction in tolerance to spinal cord I/R injury. Methods: In the part 1 study, Sprague-Dawley rats were randomly assigned to 4 groups. IPC (P) group animals received IPC by temporary thoracic aortic occlusion (AO) with a 2-F Fogarty arterial embolectomy catheter for 3 min. I/R injury (I/R) group animals were treated with blood withdrawal and temporary AO for 12 min, and shed blood reinfusion at the end of the procedures. (P+I/R) group animals received IPC, followed by 5 min reperfusion, and then I/R procedures for 12 min. Sham (S) group animals received anesthesia and underwent surgical preparation only. Neurological functions were evaluated, and lumbar segments were harvested for histopathological examination. To evaluate the role of autoregulation in IPC, spinal cord blood flow and tissue oxygenation were continuously monitored throughout the procedure duration. In the part 2 study, spinal cord ischemia rats was induced by occluding the thoracic descending aorta with a balloon catheter introduced through a femoral artery, accompanied by concomitant exsanguinations. Rats in the control group were given dimethyl sulfoxide (vehicle) before undergoing spinal cord ischemia/reperfusion injury. In the U0126-treated group, rats were pretreated with an inhibitor of ERK1/2, U0126, to inhibit ERK1/2 phosphorylation. The sham rats underwent aortic catheterization without occlusion. Parameters, including neurologic status, neuronal survival, inflammatory cell infiltration, and interleukin-1β production in the spinal cords, were compared between groups. Results: The Tarlov scores in the (I/R) group were significantly lower than those in the (S), (P), and (P+I/R) groups on days 1, 3, 5, and 7. The numbers of surviving motor neurons in the (S), (P), and (P+I/R) groups were significantly higher than those in the (I/R) group. The (P) group exhibited higher spinal cord blood flow and tissue oxygenation after reperfusion than the (S) group. The (P+I/R) group exhibited higher spinal cord blood flow and tissue oxygenation within the first 60 min after reperfusion than the (I/R) groups. In the part 2 study, early ERK1/2 phosphorylation was observed after injury in the control group, followed by abundant microglial accumulation in the infarct area and increased interleukin-1β expression. In the U0126 group, U0126 treatment completely blocked ERK1/2 phosphorylation. Microglial activation and… Advisors/Committee Members: Julie Y.H. Chan (chair), Han-Jung Chen (committee member), Aij-Lie Kwan (chair), Samuel H.H. Chan (committee member), Alice Y.W. Chang (chair).

▼ C-terminus of HSC70-interacting protein (CHIP, STUB1) is a ubiquitously expressed cytosolic E3-ubiquitin ligase. CHIP-deficient mice exhibit cardiovascular stress and motor dysfunction prior to premature death. This phenotype is more consistent with animal models in which master regulators of autophagy are affected rather than with the mild phenotype of classic E3-ubiquitin ligase mutants. The cellular and biochemical events that contribute to neurodegeneration and premature aging in CHIP KO models remain poorly understood. Electron and fluorescent microscopy demonstrates that CHIP deficiency is associated with greater numbers of mitochondria, but these organelles are swollen and misshapen. Acute bioenergetic stress triggers CHIP induction and re-localization to mitochondria where it plays a role in the removal of damaged organelles. This mitochondrial clearance is required for protection following low-level bioenergetic stress in neurons. CHIP expression overlaps with stabilization of the redox stress sensor PTEN-inducible kinase 1 (PINK1) and is associated with increased LC3-mediated mitophagy. Introducing human promoter-driven vectors with mutations in either the E3 ligase or TPR domains of CHIP in primary neurons derived from CHIP-null animals enhances CHIP accumulation at mitochondria. Exposure to autophagy inhibitors suggests the increase in mitochondrial CHIP is likely due to diminished clearance of these CHIP-tagged organelles. Proteomic analysis of WT and CHIP KO mouse brains from both sexes reveals proteins essential for maintaining energetic, redox and mitochondrial homeostasis undergo significant changes in expression dependent upon genotype. Together these data support the use of CHIP deficient animals as a predictive model of age-related degeneration with selective neuronal proteotoxicity and mitochondrial failure. Advisors/Committee Members: Joshua P. Fessel (committee member), Fiona E. Harrison (committee member), BethAnn McLaughlin (committee member), Kevin D. Niswender (Committee Chair).

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ENGLISH ABSTRACT: Tissue flaps form an important part of plastic and reconstructive surgery. They are transferred with their own blood supply and are typically applied to wounds where skin grafts are unsuitable. Monitoring of tissue flaps is an important practice as it can assist in determining flap condition and the detection of circulatory complications. Detecting complications timeously is beneficial if the flap is to be salvaged. In the case of pedicled groin flaps, a technique known as ischemic preconditioning (IP) has proven to be beneficial in promoting early flap separation. A literature survey revealed that IP has only been manually implemented and an automated procedure would benefit patient and staff, particularly in a South African public hospital setting. A device was designed and developed which makes use of pulse oximetry to assist in tissue flap monitoring and analyses of current IP protocols. The device is capable of monitoring and recording information including the oxygen saturation (SpO2) and photoplethysmogram (PPG) measured from three different sites. In addition to this, the device automated the IP process by controlling the inflation of a pneumatic tourniquet. Several device tests were performed prior to clinical trials, including functionality tests which indicated that the device is capable of measurements on those areas of the body that are relevant to tissue flaps. Tests indicated that the device should not be limited to the monitoring of a single tissue flap type. The completed device was delivered to a plastic and reconstructive surgeon who carried out the clinical trials at Chris Hani Baragwanath Hospital. Clinical testing was performed on four subjects who underwent pedicled groin flap surgery. Reasonable signal quality was obtained from the last three cases. Errors and shortcomings from the first case were addressed and corrected where possible. Analysis of the recorded data coincided with standard clinical observations, indicating that the device was able to assist determining flap condition. Two of the three patients undergoing IP benefited from early flap division. Additional clinical tests could further prove the function and efficacy of the device, as well as improving post-testing data analysis methods.

AFRIKAANSE OPSOMMING: Weefselflappe speel `n integrale rol in plastiese en rekonstruktiewe chirurgie. Weefselflappe beskik oor eie bloedtoevoer en geskik vir die behandeling van wonde waarvoor veloorplantings onmoontlik is. Die monitering van weefselflappe is ’n belangrike praktyk omdat dit ‘n bydrae kan lewer aan weefseltoestand metings en aan die vroegtydig waarneming van bloedsomloopkomplikasies. Om komplikasies vroegtydig waar te neem is voordelig wanneer die velweefsel gered moet word. ’n Tegniek genaamd isgemiese prekondisie (IP) is as voordelig bewys vir die aanmoediging van vroeë weefselonthegting in die geval van pedikel-liesflappe. Tot dusver is IP in literatuur slegs met die hand toegepas terwyl ’n geoutomatiseerde prosedure pasiënte en mediese personeel sal…

Advisors/Committee Members: Muller, Jacobus Hendrik, Van den Heever, David Jacobus, Stellenbosch University. Faculty of Engineering. Dept. of Mechanical and Mechatronic Engineering..

▼ This thesis investigated the effects of ischemic preconditioning (IPC), a blood flow restriction procedure demonstrated to exert cytoprotection and reduce metabolic demand during ischemia-reperfusion injury, on hemodynamic and neural (muscle sympathetic nerve activity; MSNA) responses to static handgrip and muscle metaboreflex activation (post-exercise circulatory occlusion; PECO). Thirty-seven men were randomized to a sham (n=16) or IPC (n=21) group. Participants completed a 2 min static handgrip followed by 3 min of PECO, and a static handgrip time-to-failure to elicit peak responses. IPC had no effect on hemodynamics during static handgrip or PECO, but significantly increased peak systolic blood pressure. IPC had no effect on MSNA during PECO, but significantly increased total MSNA during the first minute of static handgrip and peak MSNA burst frequency and incidence. These findings suggest that IPC does not attenuate the muscle metaboreflex, however, may facilitate sympathetic activation at exercise onset and volitional fatigue. Advisors/Committee Members: Millar, Philip J (advisor).

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Medicina - Kardiohirurgija / Medicine - Cardiac Surgery

Ranije studije su prikazale kontroverzne rezultate protektivnog dejstva udaljenog ishemijskog prekondicioniranja (RICP) na ishemični miokard u različitim populacijama bolesnika tokom kardiohirurških operacija. Cilj ovog rada je da prikaže efekte udaljenog ishemijskog prekondicioniranja na rane rezultate hirurške revaskularizacije miokarda u bolesnika sa akutnim koronarnim sindromom bez ST segment elevacije (NSTEMI AKS). Metode: Ova studija obuhvatila je 60 bolesnika koji su randomizovani u dve grupe: Grupa 1 koja je tertirana sa RICP i Grupa 2 bez RICP (kontrolna grupa). Poređeni su pre, intra i postoperativni klinički parametri ali je glavni cilj miokardna lezija koja se odražava kroz vrednosti koncentracije Troponina I merenih preoperativno i 1, 6, 12, 24, 48 i 72h postoperativno. Sekundarni ciljevi su bili hemodinamski parametri, krvarenje, vreme lečenja u jedinici intenzivne nege i mortalitet. Rezultati: Grupe 1 i 2 su bile slične po preoperativnim karakteristikama kao što su životna dob, NYHA klasa, EuroSCORE II, ejekciona frakcija leve komore i u zastupljenosti trosudovne koronarne bolesti. Vreme kardiopulmonalnog bajpasa (86.90±29.60 vs 66.47 ±20.90, p=0.003), vreme klemovanja aorte (57.50±18.32 vs 46.37±14.78, p=0.012) i broj graftova (3.5(3-4) vs 2,83(2-3), p<0.001) bili su različiti. Ostale intra i postoperativne varijable se nisu razlikovale među grupama. Nije bilo razlike u vrednostima C reaktivnog proteina (CRP) i postoperativnih hemodinamskih parametara. Vrednosti Troponina I su ispitivane u sedam vremenskih intervala i nisu pokazale značajnu razliku među grupama (preoperativno 0,61±1,45 vs 0,79±1,95; 1h 2,15±4,67 vs 1,14±1,33; 6h 4,59±6,36 vs 3,39±2,79; 12h 3,59±2,68 vs 3,87±3,65; 24h 2,94±3,02 vs 4,00±4,60; 48h 1,71±1,67 vs 2,13±2,32 i 72h postoperativno 1,18±1,40 vs 1,24±1,45). Takođe nije bilo značajne razlike u pojavi neželjenih događaja, dužini trajanja bolničkog lečenja i mortalitetu među grupama. Zaključak: Udaljeno ishemijsko prekondicioniranje tokom hirurške revaskularizacije miokarda u akutnom korornarnom sindromu bez ST segment elevacije ne obezbeđuje bolju protekciju miokarda i hemodinamske kararkteristike ali su neophodne veće randomizovane studije da bi se dokazao pravi efekat RICP.

Advisors/Committee Members: Milojević, Predrag, 1961- 6400359.

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Introduction: The past decade has seen strong progress in understanding the mechanisms of reperfusion injury and in developing strategies that render tissues more resistant to ischemia or ameliorate the reperfusion injury. Ischemic preconditioning is among the protective strategies currently used.Aim: The present experimental study was designed to investigate a possible protective effect of IPC against the liver inflammatory response to intestinal IR and also to elucidate the role of antithrombin in transient intestinal IPC by measuring TNF-a, IL-1b and IL-6 blood levels and examining MDA, MPO and the hepatocyte ultrastructural damage in liver specimens.Materials and Methods: Eighty rats were randomly allocated to 8 study groups (Control, Control AT, Sham, IR, IPC + IR, AT + IR, AT+ IPC+ IR, ATpreop + IPC + I/R) (n=10 per group). The ultrastructural damage of the hepatocytes was examined by Transmission Electron Microscope. Blood and liver specimens were obtained for studies. Results: Intestinal IR induced a significant increase of the proinflammatory cytokines IL-1b, IL-6 and TNF-a, in agreement with previous IR models. The activity of MPO and MDA were significantly elevated in liver samples in IR groups. TNF-a levels were lower in rats IPC pretreated after 30 minutes of ischemia and 60 minutes of reperfusion. The administration of AT reduced the inflammatory response in IR groups. However, it did not show a synergistic effect with IPC. IL-1b is improved in IPC groups compared to IR groups. However, IPC and AT did not show statistically significant synergistic effect. IL-6 also increased significantly during IR. IPC significantly decreased IL-6 levels and AT administration dampened the inflammatory response to all relevant groups. IPC and AT demonstrated synergistic effect. IPC has proved beneficial for the reduction of MDA levels and also, groups were treated with AT have decreased levels of MDA, as compared to the other groups. AT decreased MPO activity and in addition has proved synergistic with IPC.Conclusions: Our study shows that intestinal IPC has a beneficial remote effect on the liver injury. This finding was confirmed by biochemical and ultrastructural studies. AT treatment before intestinal ischemia attenuated or prevented histological damage from IR injury by inhibiting release of cytokines, lipid peroxidation and neutrophil infiltration. IPC and AT have independently proven beneficial in all groups. IL-6 and MPO levels have shown synergistic effect of IPC and AT, whereas this effect was not evident by TNF-a, IL-1b and MDA levels.

Εισαγωγή: Την τελευταία δεκαετία έχει επιτευχτεί σημαντική πρόοδος στην κατανόηση των μηχανισμών της βλάβης επαναιμάτωσης και στην ανάπτυξη στρατηγικών που είτε καθιστούν τους ιστούς πιο ανθεκτικούς σε ισχαιμία είτε ελαττώνουν τη βλάβη από επαναιμάτωση. Η ισχαιμική προετοιμασία είναι μεταξύ των προστατευτικών στρατηγικών που χρησιμοποιούνται σήμερα.Στόχος: Η παρούσα πειραματική μελέτη σχεδιάστηκε για να διερευνήσει μια πιθανή προστατευτική δράση της ισχαιμικής προετοιμασίας…

▼ Ischemic preconditioning is the innate ability of the heart to protect itself from an ischemic episode that can otherwise lead to a myocardial infarct. But only under the condition that the myocardium has been previously subjected to lesser severe episodes of ischemia. It has been discovered that those endogenous mechanisms involved in the come about of ischemic preconditioning can also be brought up by the use of specific drugs, one of which is not a stranger to the pharmacology of cardiovascular diseases; Nitrates. Advisors/Committee Members: Pórszász, Róbert (advisor), Debreceni Egyetem::Általános Orvostudományi Kar::Farmakológiai és Farmakoterápiai Intézet (advisor).

▼ The main aim of this thesis was to provide a systematic review of the complex pathophysiology of ischemia-reperfusion injury, and its effect on blood rheology and on the microcirculatory system. The phenomenon of ischemia-reperfusion injury is commonly witnessed in organ transplantation, major organ surgical resections and after shock, causing harmful local and systemic outcomes, and is associated with a high incidence of morbidity and mortality. Ischemic preconditioning, one of the most potent procedures that emerged to counteract ischemia-reperfusion injury, allows tissues to adapt and become tolerant to such conditions. Research has also more recently been conducted on a similar technique termed ischemic postconditioning, in which we have seen that timing to reperfusion is the key. Remote ischemic preconditioning, after application of brief ischemia-reperfusion cycles in a single organ, was found to provide systemic protection from a subsequent extensive period of ischemia. Similarly, remote ischemic postconditioning has also evolved as a protective measure. Ischemia-reperfusion affects the state of blood flow, at the microcirculatory level in many ways. However, the interactions between hemodynamic characteristics and alterations in hemorheology are complex and the effects of ischemia-reperfusion injury are difficult to determine because rheological properties of multiple blood elements are influenced simultaneously, each affecting different aspects of its rheological behavior, in turn. Advisors/Committee Members: Németh, Norbert (advisor), Debreceni Egyetem::Általános Orvostudományi Kar::Sebészeti Intézet::Sebészeti M?téttani Tanszék (advisor).

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Coronary heart disease (CHD) is the leading cause of morbidity and mortality inthe Western world and according to the World Health Organisation will be the majorcause of death in the world as a whole by the year 2020. A remarkable scientific efforthas focused on the molecular mechanism that associated with the pathophysiology ofthe disease. The greatest advance in our understanding of the cell survival machinerywas the discovery of endogenous mechanisms of protection, which they termed,Ischemic Preconditioning, (IPC) and Ischemic Postconditioning, (PostC). IPC andPostC describe the cardioprotection obtained from applying transient episodes ofmyocardial ischemia and reperfusion either before of after the index ischemic event,respectively. Both mechanisms appear to recruit a similar signaling pathway at time ofmyocardial reperfusion. The opening of mKATP channels, the release of nitric oxide(NO) and adenosine play pivotal role in cardioprotection.Novel therapeutic strategies, based on the knowledge of the molecular mechanismsof IPC and PostC and the elucidation of the intracellular signaling pathways, haveemerged in order to confer pharmaceutical protection to the ischemic heart. Atpresent, pharmacological PC and PostC represent ideal alternatives that may substitutethe short ischemic insults for pharmaceuticals means.Our group has already synthesized and assessed the cardioprotective effect ofheteroaromatic compounds with an attached nitrate ester group (NO donors). Theadministration of these compounds reduces the infarct size by triggeringpharmacological preconditioning in rabbits and protects the myocardium from theoxidative damage during ischemia/reperfusion.Here we report the design and synthesis of novel analogues of 6,9-disupstitutedpurine, which had the strongest cardioprotective activity, bearing an nitrate estergroup at positions 6, 8, 9 of purine moiety as well as adenosine derivatives, possessingstructural features of adenosine receptor agonists, with an attached nitrate ester group.The new compounds were evaluated for their ability to trigger the beneficial effectof IPC and PostC in vivo, in anesthetized rabbits, by means of myocardial infarct sizereduction. All the new analogues were administered either before or after thesustained ischemic insult.The novel compounds had a significant cardioprotective activity by means ofreducing infarct size, compared to control, IPC and PostC groups.

Η στεφανιαία νόσος αποτελεί την κύρια αιτία παθολογικών καταστάσεων καιθνησιμότητας στο δυτικό κόσμο και σύμφωνα με τον ιεθνή Οργανισμό Υγείας θαείναι η κύρια αιτία θανάτου μέχρι το 2020. Η μελέτη των κυτταρικών και μοριακώνμηχανισμών οι οποίοι συσχετίζονται με την παθοφυσιολογία της νόσου αποτελούναιχμή των ερευνητικών δραστηριοτήτων διεθνώς. Σημαντική πρόοδος στηνκατανόηση του μηχανισμού της κυτταρικής επιβίωσης σημειώθηκε με την αναφοράενδογενών μηχανισμών καρδιοπροστασίας, της ισχαιμικής προετοιμασίας (IschemicPreconditioning, IPC) και της μετισχαιμικής προστασίας (Ischemic Postconditioning,PostC), σύμφωνα με τους…

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Abstract The repair of thoracoabdominal aortic aneurysms carries a substantial risk of ischaemic perioperative spinal cord injury. Although several protective methods have been developed, the risk of paraplegia has not been eliminated. Moreover, aortic aneurysms, including arch aneurysms, are complex clinical challenges requiring cerebral protection with hypothermic circulatory arrest (HCA). Hypothermia lowers the rate of cerebral metabolism and allows a temporary halt of the systemic circulation. However, there is still a risk for cerebral damage and a need for additional neuroprotective methods. During the last 15 years, our research group has used a porcine model to investigate a variety of neuroprotective tools. In this thesis, an animal model was utilized to study the efficacy of remote ischaemic preconditioning (RIPC) to ameliorate ischaemic damage to the central nervous system, and to shed light on the potential mechanism. Moreover, diazoxide, the pharmacological mimetic of RIPC, was tested in the HCA animal model. In the first Study (I), RIPC showed beneficial effect on the spinal cord against ischaemic insult as recorded with motor-evoked potentials. Strikingly, the beneficial effect of RIPC was observed even before the ischaemia. In the second Study (II), some beneficial effect of RIPC was seen in the immunohistochemical analysis of the spinal cord ischemia but the result remains inconclusive. Similarly, the diazoxide-treated animals had better hemodynamic status postoperatively and mildy better antioxidant activity of the brain in the third Study (III). The fourth study (IV) was a review of the current knowledge of RIPC from the cardiovascular point of view. Our studies indicate that RIPC might be a potential adjunct for preventing neuronal ischaemic injury in the setting of thoracoabdominal aortic surgery. Our result indicates that further preclinical studies with diazoxide are required before studies can be conducted in humans.

Tiivistelmä Torakoabdominaalisen aortan aneurysman kirurginen korjaaminen sisältää riskin iskeemiselle selkäytimen vauriolle. Vaikka useita suojaavia tekniikoita on kehitetty, paraplegian riskiä ei ole saatu poistettua kokonaan. Kirurgisen korjaamisen haasteellisuus moninkertaistuu, jos aneurysma on laajentunut myös aortan kaareen. Tällöin vaaditaan hypotermista verenkierron pysäytystä (HCA). Hypotermia alentaa aivojen metabolista aktiivisuutta merkittävästi ja siten verenkierron väliaikainen pysäytys on mahdollista. Tästä huolimatta hypotermiseen verenkierron pysäytykseen liittyy riski aivokudoksen vauriolle. Meidän tutkimusryhmämme on tutkinut useita keskushermostoa suojaavia tekniikoita ja lääkeaineita viimeisen 15 vuoden aikana. Käytämme sikaa koe-eläin mallina, jota on tämänkin väitöskirjan osajulkaisuissa käytetty. Tämän väitöskirjatyön tarkoituksena on ollut tutkia sekä esialtistavan raajaiskemian (RIPC) että farmakologisen mimeetin, diazoxiden, keskushermostoa suojaavia vaikutuksia sekä niiden mahdollista vaikutusmekanismia. Ensimmäisessä osajulkaisussa esialtistava…

Advisors/Committee Members: Juvonen, T. (Tatu), Anttila, V. (Vesa).

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A ototoxicidade pode ser descrita como a perda da funÃÃo auditiva e ou vestibular decorrente de lesÃes celulares das estruturas da orelha interna por substÃncias quÃmicas. Apesar do potencial ototÃxico de algumas drogas antineoplÃsicas, como a cisplatina, nÃo se deve desprezÃ-las como uma alternativa terapÃutica para o paciente portador de neoplasia maligna, devido a sua eficÃcia e escassez de opÃÃes para protocolos terapÃuticos. O prÃ-condicionamento isquÃmico à distÃncia (dPCI) foi proposto a partir do estudo de que o prÃ-condicionamento isquÃmico (PCI) de uma Ãrea vascular cardÃaca poderia proteger outra totalmente distinta. Dentre os estudos com dPCI, os estudos com induÃÃo de isquemia em animais atravÃs da isquemia e reperfusÃo de patas anteriores ou posteriores à bastante utilizado, devido à sua fÃcil aplicabilidade e ao baixo custo. Baseando-se nesses conhecimentos, propÃs-se um estudo com induÃÃo de ototoxicidade em ratos por cisplatina na dose de 32mg/kg, dividida em 04 aplicaÃÃes de 08mg/kg/dia, que mostrou ser uma dose tÃxica e com baixa mortalidade a partir da experiÃncia de nosso grupo de pesquisa, e otoproteÃÃo com prÃ-condicionamento isquÃmico em pata traseira direita. Os ratos Wistar foram submetidos à anestesia. Aqueles com otoscopia normal realizaram avaliaÃÃo auditiva por meio do potencial evocado auditivo de tronco encefÃlico (PAETE). ApÃs essa avaliaÃÃo, administrou-se cisplatina via intraperitoneal, grupo 1(n=08 animais), e salina via intraperitoneal, grupo 2 (n=08 animais). Nos grupos 3 (n=09 animais) e 4 (n=07 animais), realizou-se uma isquemia de pata traseira direita por 10 minutos seguida de reperfusÃo por 30 minutos, quando apÃs administrou-se via intraperitoneal cisplatina (grupo 3) e salina (grupo 4). Ao final (D4), todos foram avaliados por PAETE. Foi removido o osso temporal direito, apÃs eutanÃsia. A cÃclea foi dissecada para realizaÃÃo das tÃcnicas de microscopia Ãptica e imuno-histoquÃmica. Baseado nos resultados deste estudo, encontrou-se que o dPCI, mecanismo jà amplamente estabelecido, protegeu de forma significante a lesÃo funcional na cÃclea por cisplatina, atravÃs da avaliaÃÃo funcional por PAETE (p=0,0477). NÃo se observou diferenÃa estatÃstica na anÃlise por microscopia Ãptica (p>0,05). Foi observado reversÃo de imunomarcaÃÃo, no grupo 3, de fator de necrose tumoral α e Ãxido nÃtrico sintase induzida da lesÃo em estria vascular por cisplatina. Obteve-se proteÃÃo na ototoxicidade sistÃmica da cisplatina. Conclui-se que o dPCI protegeu a ototoxicidade por cisplatina na avaliaÃÃo funcional por PAETE e protegeu a toxicidade sistÃmica pela avaliaÃÃo do peso.

Ototoxicity can be decribed as a lost of auditory or vestibular function as a consequence of injury in cells of the inner ear. Despite of this effect, same antineoplastic drugs, as cisplatin, can not be excluded as an option for the pacient with a malignant tumor, because of its efficiency and the lack of options for terapeutic protocols. Remote ischemic preconditioning (RIPC) was proposed from the study that the…

Advisors/Committee Members: Ronaldo de Albuquerque Ribeiro, Marcos Rabelo de Freitas, MIGUEL ANGELO HYPPOLITO.

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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…

Advisors/Committee Members: Santos, Jorge Luiz dos.

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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…

Advisors/Committee Members: Hueb, Whady Armindo.

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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…

Advisors/Committee Members: Juvonen, T. (Tatu), Anttila, V. (Vesa).

▼ ABSTRACT Background: Ischemic heart disease is one of the leading causes of death globally. This thesis explores endogenous mechanisms protecting against myocardial ischemia in context of epigenetics (changes in gene activity not caused by changes in DNA sequences). Epigenetic regulation of vascular thromboprotective mechanism was assessed, as well as the capacity of extracellular vesicle (EV) involvement in mediating epigenetic changes related to cardioprotection in ischemic preconditioning (IPC). Aims: The aim of Papers I and II was to evaluate if histone deacetylase inhibition, by valproic acid (VPA) treatment, increases stimulated tissue plasminogen activator (t-PA) release capacity and affects plasminogen activator inhibitor-1 (PAI-1) levels in vivo, in healthy large animals and in an atherosclerotic cohort. The aim of Papers III and IV was to assess if coronary venous EV genetic content is affected by myocardial IPC in vivo. Methods: In a porcine myocardial ischemia model transcoronary t-PA release was measured and compared between VPA treated (n=12) and untreated animals (n=10). In the clinical cross-over study (n=16), the perfused forearm model was used to measure single and repeated t-PA release capacity by isoprenaline provocation with and without VPA. PAI-1 was also measured. In a porcine model, EV were collected from coronary venous blood before and after myocardial IPC. The EV were isolated by differential ultracentrifugation and the preparation was evaluated by western blot, electron microscopy and nanoparticle tracking analysis. Changes in EV genetic content after IPC were identified by microarray and DNA sequencing. Results: Animals treated with VPA demonstrated a significantly higher cumulative transcoronary t-PA release compared to controls. In the clinical study, VPA treatment resulted in increased cumulative t-PA release capacity during repeated isoprenaline stimulation, though there was no difference when comparing single stimulation sequences. Levels of PAI-1 were reduced after VPA treatment. Among 11678 mRNA sequences detected in EV, about 10% were up or down regulated after IPC. Among these, over half were increased, including several with association to cardioprotection and IPC. DNA fragments, representing all porcine chromosomes, were identified in EV. The DNA content in EV changed after myocardial IPC. Conclusions: Intervention of HDACi, by VPA treatment, may improve actions of the fibrinolytic system by enhancing t-PA release capacity and reducing PAI-1 levels in vivo. In a future perspective, this may have clinical relevance as novel means of preventive strategies for ischemic heart disease. Myocardial IPC influences the composition of EV genetic content, including increases in gene transcripts associated to cardioprotecion. This may reflect a biological relevance of EV in delivering cardioprotective signals in IPC, although further studies are necessary to confirm such connection.

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Aim: The present experimental study was designed to investigate a possible protective effect of IPC against the renal inflammatory response after intestinal IR and also to elucidate the role of antithrombin in transient intestinal IPC by measuring TNF-a, IL-1b and IL-6 blood levels and examining MDA, MPO. Materials and Methods: Eighty rats were randomly allocated to 8 study groups (Control, Control AT, Sham, IR, IPC + IR, AT + IR, AT+ IPC+ IR, ATpreop + IPC + I/R) (n=10 per group). The ultrastructural damage of the renal cells was examined by Transmission Electron Microscope. Results: Intestinal IR induced a significant increase of the cytokines IL-1b, IL- 6 and TNF-a, and also MPO and MDA. TNF-a and IL-1b levels did not show a synergistic effect of AT and IPC. IL-6 also increased significantly during IR. IPC significantly decreased IL-6 levels and AT administration dampened the inflammatory response. IPC and AT demonstrated synergistic effect. IPC has proved beneficial for the reduction of MDA levels and also, groups were treated with AT have decreased levels of MDA. AT decreased MPO activity and in addition has proved synergistic with IPC. Conclusions: Our study shows that intestinal IPC has a beneficial remote effect on the kidney injury. AT treatment before intestinal ischemia attenuated or prevented histological damage from IR injury. IPC and AT have independently proven beneficial in all groups.

Στόχος: Η παρούσα πειραματική μελέτη σχεδιάστηκε για να διερευνήσει μια πιθανή προστατευτική δράση της ισχαιμικής προετοιμασίας (ischemic preconditioning - IPC) έναντι της νεφρικής φλεγμονώδους απόκρισης μετά από ισχαιμία επαναιμάτωση (IR) του λεπτού εντέρου και επίσης για να διαλευκάνει το ρόλο της αντιθρομβίνης μετά από παροδική εντερική IPC, εξετάζοντας τις MDA και ΜΡΟ σε δείγματα νεφρού και μετρώντας τα επίπεδα των IL-1β, IL-6 και TNF-α στο αίμα.Υλικά και Μέθοδος: Ογδόντα επίμυες χωρίστηκαν τυχαία σε 8 ομάδες: Φυσιολογικοί, Ελέγχου, Εικονικής επέμβασης, Ισχαιμίας/επαναιμάτωσης (IR), Ισχαιμικής προετοιμασίας + Ισχαιμίας/επαναιμάτωσης (IPC+IR), Αντιθρομβίνης + Ισχαιμίας/επαναιμάτωσης (AT+IR), Αντιθρομβίνης + Ισχαιμικής προετοιμασίας + Ισχαιμίας/επαναιμάτωσης, Αντιθρομβίνη προεγχειρητικά + Ισχαιμικής προετοιμασίας + Ισχαιμίας/επαναιμάτωσης. Οι δομικές βλάβες των νεφρικών κυττάρων μελετήθηκαν με ηλεκτρονικό μικροσκόπιο. Αποτελέσματα: Η εντερική IR προκάλεσε σημαντική αύξηση των κυτοκινών IL-1β, IL-6 και TNF-α και των ΜΡΟ και MDA. Τα επίπεδα TNF-α και IL-1β έδειξαν η IPC και η AT δεν εμφάνισαν στατιστικώς σημαντική συνεργική δράση. Η IPC μείωσε σημαντικά τα επίπεδα της IL-6 και η AT άμβλυνε τη φλεγμονώδη απόκριση, αποδεικνύωντας συνεργική δράση. Η IPC αποδείχτηκε ευεργετική για τη μείωση των επιπέδων MDA, όπως επίσης, ομάδες που υπέστησαν αγωγή με AT έχουν μειωμένα επίπεδα MDA. Η AT μείωσε την δραστικότητα MPO και επίσης απεδείχθη συνεργική με την IPC. Συμπέρασμα: Η μελέτη μας δείχνει ότι η εντερική IPC έχει ευεργετική επίδραση στην νεφρική βλάβη που προκαλείται από ισχαιμία-επαναιμάτωση του εντέρου. Η θεραπεία με ΑΤ…

▼ Perinatal hypoxic-ischemic (HI) brain injury remains a major cause of newborn mortality and morbidity. Preconditioning with mild hypoxia can protect the brain against HI insults, while it has recently been shown that mild hypoxia administered after a brain injury (postconditioning) can protect the adult mouse brain. Furthermore, pre-injury treatment with compounds that mimic the effects of hypoxia (hypoxia-mimetics) can protect the brain against HI. The neuroprotective effects of hypoxic preconditioning, postconditioning and post-injury treatment with the hypoxia mimetics desferoxamine (DFX, 200mg/kg), cobalt chloride (CoCl2, 60mg/kg) and ethyl-3,4-dihydroxybenzoate (EDHB, 200mg/kg) were examined in a neonatal rat model of HI brain injury, at 7 days post-injury. Hypoxic treatments consisted of either 3 hr of 8% oxygen performed 24 hr prior to injury (preconditioning); or 1 hr of 8% oxygen 24 hr post-injury, performed once a day for 5 days (postconditioning). HI reduced ipsilateral brain tissue, while hypoxic preconditioning, postconditioning, DFX, CoCl2 and EDHB treatment, were all able to reduce the extent of brain damage to the ipsilateral hemisphere. NeuN immunohistochemistry in regional brain areas was performed to examine neuronal loss after HI injury. Hypoxic preconditioning reduced cortical, hippocampal and striatal neuronal loss, while hypoxic postconditioning reduced cortical and striatal loss. Treatment with DFX reduced neuronal loss in the cortex and striatum, whereas EDHB reduced cortical and hippocampal neuronal loss. The long-term neuroprotective actions of hypoxic postconditioning and EDHB treatment were also examined (37d post-injury), with sensorimotor and memory assessments following HI. Hypoxic postconditioning and EDHB treatment prevented long-term brain damage, after HI. Furthermore, behavioural testing (35d post-injury) revealed that both hypoxic postconditioning and EDHB treatment improved memory function in the novel object recognition test, whilst hypoxic postconditioning was also able to increase forearm muscle strength and improve fore/hindlimb placement in a grid-walking test after HI injury. The ability for hypoxic postconditioning and EDHB treatment to enhance tissue repair was also examined through post-injury neovascularisation and cell proliferation; however, evidence that hypoxic postconditioning or EDHB treatment could stimulate a reparative response through these mechanisms, after HI brain injury was not observed. Advisors/Committee Members: Jones, Nicole, Medical Sciences, Faculty of Medicine, UNSW.

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A interrupção do fluxo sanguíneo, ou isquemia, representa um dos problemas mais importantes de doenças cardiovasculares e cerebrovasculares enfrentados pelos médicos na sua rotina. Em relação ao miocárdio muitos estudos têm sido realizados nessa área e sabe-se que os anestésicos inalatórios e os opiódes podem protege as células cardíacas contra a lesão de isquemia e reperfusão. O propofol por sua parece não ter efeito de précondicionamento, porém apresenta características similares as ações antioxidantes da vitamina E, neutralizando os efeitos nocivos da produção de radicais livres. A associação de sevoflurano, sufentanil e propofol não está descrita na literatura. O objetivo deste estudo foi examinar a potencialização de cardioproteção entre sevoflurano, propofol e sufentanil por meio de análise do tamanho da área de infarto e inibição de apoptose em células miocárdicas. Ratos foram submetidos a 5 protocolos de pré-condicionamento diferentes e divididos em grupos agudos e crônicos. Os resultados indicaram que a associação destes anestésicos não conferiu proteção maior do que quando administrados isoladamente. Além disso, o sevoflurano conferiu proteção ao miocárdio no pós-infarto agudo e crônico. Já o propofol conferiu cardioproteção no pós-infarto crônico

The interruption of blood flow, or ischemia, represents one of the major problems of cardiovascular and cerebrovascular diseases seen by physicians in their routine. With respect to the myocardium, many studies have been conducted in this area and it is a known fact that inhaled anesthetics and opiates may protect cardiac cells against the ischemia and reperfusion injury. Propofol, in turn, seems to have no preconditioning effect, but it has similar characteristics to the antioxidant actions of vitamin E by neutralizing the harmful effects of free radical production. The combination of sevoflurane, sufentanil and propofol has not been described in literature. The aim of this study was to survey cardioprotection potentiation among sevoflurane, propofol and sufentanil by analyzing the size of infarct area and the inhibition of apoptosis in cardiac cells. Rats were subjected to five different preconditioning protocols and divided into acute and chronic groups. Results indicated that the combination of these anesthetics did not confer greater protection than when they were administered alone. Furthermore, sevoflurane conferred myocardial protection in the postacute and chronic infarction stage. Propofol, in turn, conferred cardioprotection in the chronic post-infarction stage

Advisors/Committee Members: Auler Junior, Jose Otavio Costa.

▼  Remote preconditioning is a promising and robust treatment for myocardial ischemia/reperfusion injury that evokes cardioprotection through endogenous neural and/or humoral signaling. A recent study has reported that protective signaling is mediated by exosomes through the circulation; however this concept is supported by limited and inconsistent evidence. Despite overwhelming success in preclinical studies, the efficacy of remote preconditioning in human studies is inconclusive. Importantly, the majority of remote preconditioning studies use healthy animal models despite growing evidence that comorbidities, such as type-2 diabetes, may negatively influence outcomes. Nonetheless, the efficacy of remote preconditioning in the setting of type-2 diabetes has not been investigated. Using an established model of myocardial ischemia/reperfusion in the Zucker model of type-2 diabetes and a model of hypoxia/reoxygenation in cultured HL-1 cardiomyocytes we tested four hypotheses: i. remote preconditioning is ineffective in early-stage type-2 diabetes in vivo; ii. the traditional ultracentrifugation technique for exosomes isolating is inadequate to isolate protective factor(s) from remote preconditioning; iii. enhanced ultracentrifugation technique for exosome isolation sequesters a protective fraction of serum; iv. the humoral component of remote preconditioning is defective in type-2 diabetes. In support of Hypothesis I, we demonstrate that remote preconditioning failed to reduce infarct size caused by ischemia/reperfusion in the Zucker model of early-stage type-2 diabetes. Our results illustrate that the loss in efficacy is not the result of hyperglycemia per se nor sensitization of the myocardium to ischemia/reperfusion. Subsequently, we sought to isolate a subfraction of serum from remote preconditioned rats which contained exosomes that could communicate protection and render HL-1 cardiomyocytes resistant to hypoxia/reoxygenation-induced cell death. In agreement with Hypothesis II, we report that the traditional ultracentrifugation isolation technique (100,000 xg for 2 hr) did not isolate the protective component with the exosome-rich pellet from serum, suggesting that the protective component remained in the supernatant. In accordance with these observations, we enhanced the ultracentrifugation technique to improve exosome sedimentation and obtain a protective sub-fraction of serum. In agreement with Hypothesis III, the enhanced ultracentrifugation technique (300,000 xg for 12 hr) isolated a protective… Advisors/Committee Members: Karin Przyklenk.

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O tempo para 1,0mm de depressão do segmento ST (T-1,0mm) adotado para caracterizar o fenômeno do aquecimento, uma expressão do precondicionamento isquêmico (PCI), em testes ergométricos sequenciais é consistente e reprodutível, porém, possui várias limitações. O objetivo deste estudo foi aplicar um escore eletrocardiográfico de isquemia miocárdica em testes ergométricos sequenciais comparando com o clássico índice T-1,0mm. Avaliamos 61 pacientes, com idade média de 62,2+7,5 anos, 86,9% homens, portadores de diabetes mellitus tipo 2 e coronariopatia multiarterial. Foram analisados 151 exames, destes 116 de pacientes completaram as duas fases de avaliação. A primeira fase compreendia dois testes ergométricos sequenciais para documentação do PCI e a segunda fase, após 1 semana, mais dois testes sob efeito de repaglinida oral. Dois observadores aplicaram o escore de forma cega. Observou-se concordância perfeita inter e intraobservador (Kendall Tau-b = 0,96, p<0,0001, Kendall Tau-b=0,98, p<0,0001, respectivamente). Os valores de sensibilidade, especificidade, valor preditivo negativo, valor preditivo positivo e acurácia, foram respectivamente de 72,41%, 89,29%, 75,8%, 87,5% e 81%. Concluímos que o escore de isquemia é um método consistente e reprodutível para documentação do fenômeno do aquecimento, representando uma alternativa factível ao índice T-1,0mm.

The time to 1.0mm ST-segment depression (T-1.0mm), adopted to document the warm-up phenomenon, an expression of the ischemic preconditioning (IPC), during sequential exercise tests is considered reliable and reproductible, although with several limitations. The main goal of this study was to apply an electrocardiographic ischemic myocardium score to sequential exercise tests, comparing with the standard T-1.0mm. We evaluated 61 patients, mean age 62,2+7,5 years-old, 86.9% male, with type 2 diabetes mellitus and multivessel coronary disease. We analyzed 151 exercise tests, being 116 tests from patients who fulfilled the two phases of the study. The first phase enrolled the patients for two sequential exercise tests to document the IPC and the second phase, after 1 week, two additional sequential exercise tests were performed under repaglinide treatment. We observed a perfect concordance inter and intraobserver (Kendall Tau-b=0.96, p<0.0001; Kendall Tau-b=0,98, p<0,0001, respectively). The sensibility, specificity, positive predictive value and negative predictive value were also determined: 72.41%, 89.29%, 75.8%, 87.5% and 81%, respectively. In conclusion, the electrocardiographic ischemic score is a consistent and reproductible tool to document the warm-up phenomenon, representing a reliable alternative to the T-1.0mm.

Advisors/Committee Members: Moffa, Paulo Jorge.

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O fenômeno do precondicionamento isquêmico é definido como o aumento da tolerância à isquemia e à lesão de reperfusão, induzida por curtos e sucessivos episódios de isquemia prévios a período de isquemia prolongada. A angina do aquecimento e a de pré-infarto são duas condições clínicas relacionadas ao precondicionamento. Este fenômeno apresenta duas fases distintas, clássica ou precoce e tardia. A atenuação do infradesnível do segmento ST provocada pelo precondicionamento precoce está bem documentada, porém sua expressão cintilográfica permanece controversa. O objetivo desta pesquisa foi avaliar se as atenuações eletrocardiográficas do precondicionamento durante testes sequenciais estão associadas a modificações simultâneas das imagens de cintilografia de perfusão miocárdica em indivíduos com doença coronariana. Vinte e três pacientes foram selecionados entre março de 2009 e julho de 2011. A média de idade foi 64,5 anos (dp=7,0), 19 (82,6%) do sexo masculino e todos tinham lesão coronária em pelo menos um vaso superior a 60%. A medicação antiisquêmica foi suspensa por três a cinco dias. Os pacientes foram submetidos a três testes ergométricos a partir do exame de seleção, sendo dois deles sequenciais e o terceiro realizado após sete dias. A injeção do radiofármaco sestamibi-Tc-99m no teste de precondicionamento e contraprova foi administrado no tempo de aparecimento do infradesnível de ST de -2,0 mm na derivação MC5 e/ou dor precordial anotados no teste inicial ou de seleção. A imagem cintilográfica foi adquirida entre 60 a 90 minutos após o esforço. Os resultados do segundo teste (precondicionamento) mostraram aumento significativo do tempo para o aparecimento da depressão do segmento ST de 1,0 mm (338±130) e 2,0 mm (431±126), em relação ao teste inicial (245±96; 366±103) p<0,001. A diferença na redução do valor máximo de infradesnível de ST entre os três testes foi significativa (3,8±0,8; 2,3±0,6; 3,1±1,0) p<0,001. Houve redução significativa nos escores de perfusão de estresse (p=0,045) entre o primeiro e o segundo testes, bem como para o escore da diferença entre o estresse e repouso (p= 0,03), sem diferença na extensão da área de isquemia entre as três etapas detectadas pela cintilografia (p=0,691). Em conclusão, houve redução significativa das alterações eletrocardiográficas induzidas pelo precondicionamento isquêmico precoce em maior proporção do que as observadas nas respectivas imagens de cintilografia de perfusão miocárdica; não se observou associação entre a redução da depressão do ST e a redução do escore de perfusão na fase de precondicionamento, nem correlação entre a magnitude do infradesnível máximo de ST e a redução do escore de perfusão (r=0,07 e p=0,75).

The phenomenon of ischemic preconditioning is defined as the increase of tolerance to ischemia and injury of reperfusion induced by short and consecutive episodes of isquemia prior to prolonged arterial occlusion. Warm-up and pre-infarction angina are two clinical conditions regarding this phenomenon. The ischemic preconditioning has two…

Advisors/Committee Members: Batlouni, Michel.

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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…

Advisors/Committee Members: Chagas, Antonio Carlos Palandri.

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Abstract In pathologies of the ascending aorta or in congenital heart defects, circulation may be temporarily halted during surgical intervention. This is achieved by operating under deep hypothermic circulatory arrest or under hypothermia combined with isolated perfusion techniques. For deep hypothermic circulatory arrest (DHCA), the patient is cooled below 18°C using an extracorporeal heart-lung machine, and circulation and breathing are stopped. The advantage of hypothermia is that it decreases oxygen and glucose consumption and provides the surgeons the time required to repair complex heart defects. However, there is still a relatively high risk of neurological complications that can affect the quality of life of patients and their families. One of the methods to mitigate ischaemia-reperfusion injury is remote ischaemic preconditioning. In this work, the neuroprotective mechanisms of remote ischaemic preconditioning (RIPC) were studied in acute and surviving chronic animal models. In study I, we used an acute model, and studied the effects of RIPC in cerebral microcirculation using an intravital microscope and samples analysed by transmission electron microscope. In study II, a chronic model was used to evaluate whether the effects of remote ischaemic preconditioning can be seen in the markers of oxidative stress or in redox-regulating enzymes. Study III was conducted to supplement the findings of study II, considering the markers of oxidative stress. Findings in all studies were consistent with one another. Study I showed the effect of remote ischaemic preconditioning on leukocyte activation and adhesion to cerebrocortical vessels in piglets after prolonged DHCA. Additionally, cellular preservation of endoplasmic reticulum was present in transmission electron microscope analysis of the central nervous system. In studies II and III, the remote ischaemic preconditioning lowered markers of ischaemia-reperfusion-related oxidative stress. In study III the remote ischemic preconditioning lowered oxidative stress already during cardiopulmonary bypass.

Tiivistelmä Hoidettaessa nousevan aortan sairauksia ja synnynnäisiä sydänvikoja verenkierto voidaan tilapäisesti pysäyttää kirurgisten toimenpiteiden ajaksi. Tämä saavutetaan jäähdyttämällä elimistö verenkierron pysäytyksen ajaksi tai jäähdytettynä isoloitujen perfuusiotekniikoiden avulla. Potilas jäähdytetään alle 18 °C lämpötilaan käyttäen kehonulkoista sydän-keuhkokonetta, minkä jälkeen verenkierto ja hengitys voidaan väliaikaisesti pysäyttää. Elimistön viilentäminen vähentää hapen ja glukoosin kulutusta ja antaa kirurgeille aikaa korjata monimutkaisia sydänsairauksia. Verenkierron pysäytyksestä ja palauttamisesta voi ilmaantua keskushermostoon iskemia-reperfuusiovaurioita, mitkä vaikuttavat potilaiden ja heidän läheistensä elämänlaatuun. Esialtistava raajaiskemia on yksi tutkituista menetelmistä lieventää iskemia-reperfuusiovauriota. Tässä työssä esialtistavan raajaiskemian hermostoa suojaavia mekanismeja tutkittiin akuutilla ja kroonisilla koe-eläinmalleilla.…

Advisors/Committee Members: Juvonen, T. (Tatu), Anttila, V. (Vesa).

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Background-Aims: Hepatic steatosis is an independent aggravating factor for liver surgery and transplantation, causing severe liver injury and has deleterious effects when associated with ischemia-reperfusion mechanism. Ischemic preconditioning significantly improves survival in lean and fatty livers after prolonged hepatic ischemia and reperfusion, by improving the micro-circulation and the reduction of lipid peroxidation. Our objective was to investigate the effect of ischemic preconditioning to prolonged liver ischemia and extensive hepatectomy in severe hepatic steatosis. Methods: Severe hepatic steatosis was performed by 12-14 weeks choline-free diet in Wistar rats. The effect of ischemic preconditioning was examined in steatotic and lean livers who subjected to hepatic ischemia-reperfusion injury and extented hepatectomy. We induced 30-minute ischemia-reperfusion manipulation and extensive hepatectomy with or without prior ischemic preconditioning in steatotic and lean livers, and after 6 and 24 hours of reperfusion IL6, TNFα, endogenous nitric oxide, lactic acid and glutathion in liver tissue and blood were measured. Also, measurements of AST, ALT, and ALP, γGT, triglycerides, cholesterol and total lipids were made in the blood at 6 and 24 hours after the end of the experimental manipulations.Results: Steatotic rats subjected only to prolonged hepatic ischemia and reperfusion after extensive hepatectomy, showed extensive liver damage at significantly increased values of TNFα and significantly reduced values of IL6 and NO, while no one rat survived for more than 29 hours. On the contrary, steatotic and lean rats, which 24 hours before ischemia-reperfusion were subjected to intermittent ischemic preconditioning, presented a significant improvement regarding survival (30-day survival for steatotic rats was 67%, and for lean rats was 83.3%) and significantly lower values of TNFα, and Lactic acid, as well as significantly increased values of IL6 and NO. Also, the lean rats that weren’t subjected to prophylactic manipulation of ischemic preconditioning, before the induction of ischemia reperfusion injury and extensive hepatectomy, showed significantly greater survival (30-day survival was 67%) compared to steatotic rats, with significantly lower values of TNFα, and lactic acid, as well as significantly increased values of IL6 and NO. The increase of IL6 and NO and the reduction of TNFα and Lactic acid following intermittent ischemic preconditioning are related to the protective effect of the manipulation in ischemic reperfusion injury in steatotic rats.Conclusions: The ischemia-reperfusion injury (IRI) after major hepatic surgery is the main pathogenic factor determining largely morbidity and mortality. Our results demonstrate the possible positive role played by ischemic preconditioning to improve the outcomes and the survival after prolonged hepatic ischemia, reperfusion and extensive hepatectomy.

Σκοπός: η ηπατική στεάτωση αποτελεί ανεξάρτητο επιβαρυντικό παράγοντα για την ηπατική χειρουργική, προκαλώντας…

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The aim of this study was to assess whether ischemic preconditioning protects adequately an already ischemic myocardium. Isolated rat hearts were used according to the Langendorff technique. They were subjected to a final ischemic period of 20 minutes, preceded by an ischemic period of 20, 15 and 10 minutes, depending on which group they belonged. They were divided into subgroups depending on whether preconditioning was applied before or after the first ischemic period or not at all. Post-ischemia function of the left ventricle was assessed by measuring left ventricular end-diastolic pressure, left ventricular developed pressure and its negative and positive difference over time. Our results were that ischemic preconditioning did not produce significant cardioprotection in rat hearts which were subjected to 20 or 15 minutes of ischemia prior to the application of preconditioning, while a 10 minute first ischemic period acted as preconditioning by itself. These findings may have practical and clinical applications.

Στόχος της διατριβής ήταν η διερεύνηση του κατά πόσον η ισχαιμική προπόνηση προστατεύει επαρκώς το ισχαιμικό μυοκάρδιο. Χρησιμοποιήθηκαν απομονωθείσες καρδιές επιμύων σε πειραματική διάταξη Langendorff. Υπεβλήθησαν σε ισχαιμία 20 λεπτών, της οποίας προηγήθηκαν περίοδοι ισχαιμίας 20, 15 και 10 λεπτών στις τρείς ομάδες επιμύων, με διαχωρισμό σε υποομάδες αναλόγως της μη εφαρμογής ισχαιμικής προπονήσεως ή της εφαρμογής της προ ή μετά την αρχική ισχαιμία. Ως δείκτης μετισχαιμικής ανάκαμψης χρησιμοποιήθηκαν η τελοδιαστολική πίεση της αριστεράς κοιλίας, η ανάκαμψη της αναπτυσσόμενης πιέσεως της αριστεράς κοιλίας και της θετικής και αρνητικής πρώτης παραγώγου της. Ως δείκτης νέκρωσης χρησιμοποιήθηκε η συγκέντρωση της CK στο εκρέον διάλυμα διαπότισης. Το συμπέρασμα ήταν ότι η ισχαιμική προπόνηση δεν παρείχε αξιόλογη προστασία στα ζώα που υπεβλήθησαν αρχικά σε ισχαιμία 20 ή 15 λεπτών. Στα ζώα που υπεβλήθησαν σε ισχαιμία 10 λεπτών αυτή είχε η ίδια θέση ισχαιμικής προπονήσεως. Τα αποτελέσματα μπορούν να έχουν πρακτικές κλινικές προεκτάσεις.