京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21627号 / 医博第4433号 / 新制||医||1033(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 平井 豊博, 教授 松原 和夫, 教授 羽賀 博典 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

Carolina Rinse (CRS) has been shown to be effective in decreasing vascular permeability and neutrophil infiltration in reperfused equine small intestine. The objective of this study was to show that CRS applied topically and intraluminally could prevent immediate reperfusion injury after low flow ischemia or distention in the equine jejunum. Materials & Methods: Two groups of 5 horses were used to evaluate CRS treatment after low-flow ischemia (Group 1) and intraluminal distention (Group 2) of distal jejunum. Mesenteric blood flow, osmotic reflection coefficient (ORC), wet weight to dry weight ratios (WW/DW), and neutrophil accumulation in the serosa were measured. ORC is defined as the lymph protein concentration to plasma protein concentration ratio subtracted from one (1- Cl / Cp) at maximal lymph flow. The ORC from baseline values and at 60 minutes after initiating reperfusion was compared between Groups 1 and 2. Pair wise comparisons were made for mesenteric blood flow, tissue volume, neutrophil number, and WW/DW proximal control and CRS treated jejunal segments were made using a Mann Whitney U test (P< 0.05). Results: The mean ORC of bowel treated topically and intraluminally with CRS was similar to that recorded in normal bowel or ischemic intestine treated with CRS by arterial perfusion. The ORC after distention and decompression increased and was similar to that reported in untreated intestine. The WW/DW after both ischemia and distention increased compared to the proximal control segments. There was no difference in neutrophil number in either ischemic or distended intestine compared to the proximal control segments. Discussion: Carolina CRS was effective in preventing alterations in microvascular permeability during reperfusion afterischemia but not distention. Neutrophil migration curtailed in both groups suggesting that combined topical and intraluminal application of CRS to ischemic intestine may reduce the acute inflammatory responses during reperfusion thereby decreasing complications after ischemia or distention. / Master of Science

Many animal models have been established for the study of myocardial remodeling and heart failure due to its status as the number one cause of mortality worldwide. In humans, a pathologic occlusion forms in a coronary artery and reperfusion of that occluded artery is considered essential to maintain viability of the myocardium at risk. Although essential for myocardial recovery, reperfusion of the ischemic myocardium creates its own tissue injury. The physiologic response and healing of an ischemia/reperfusion injury is different from a chronic occlusion injury. Myocardial ischemia/reperfusion injury is gaining recognition as a clinically relevant model for myocardial infarction studies. For this reason, parallel animal models of ischemia/reperfusion are vital in advancing the knowledge base regarding myocardial injury. Typically, ischemia of the mouse heart after left anterior descending (LAD) coronary artery occlusion is confirmed by visible pallor of the myocardium below the occlusion (ligature). However, this offers only a subjective way of confirming correct or consistent ligature placement, as there are multiple major arteries that could cause pallor in different myocardial regions. A method of recording electrocardiographic changes to assess correct ligature placement and resultant ischemia as well as reperfusion, to supplement observed myocardial pallor, would help yield consistent infarct sizes in mouse models. In turn, this would help decrease the number of mice used. Additionally, electrocardiographic changes can continue to be recorded non-invasively in a time-dependent fashion after the surgery. This article will demonstrate a method of electrocardiographically confirming myocardial ischemia and reperfusion in real time.

Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Diabetes is considered a major threat to human health in both developed and developing nations. Cardiovascular disease which is common in diabetic patients has increased the overall disease affliction. Moreover, stress-induced hyperglycemia has led to increased mortality and morbidity in patients with an acute myocardial infarction (MI), whether the patient has diabetes or not. In addition, acute MI might stem from stress-induced hyperglycemia capability to increase inflammation and oxidative stress resulting in a worse functional cardiac outcome. Hyperglycemia-induced oxidative stress can similarly result in the formation of miss folded or damaged proteins that may be eliminated by the ubiquitin-proteasome system (UPS). Futhermore, hyperglycemia-induced oxidative stress can also result in dysregulation of the UPS that removes these misfolded proteins. Additionally, an increasing body of evidence implicates UPS dysfunction in cardiac diseases and hyperglycemia which has been associated with increased inflammation and blunted cardiac function in response to ischemia-reperfusion. Literature however is blurred whether a reduction or a rise in the UPS is damaging with hyperglycemia and in response to ischemia-reperfusion. In light of this, we hypothesized that UPS inhibitors such as Z-Leu-Leu-Leu-al (MG-132) and lactacystin, protects the rat heart against ischemia-reperfusion under hyperglycemic perfusion conditions. Isolated rat hearts were perfused ex vivo with Krebs-Henseleit buffer containing 33 mM glucose vs. controls (11 mM glucose) for 60 min, followed by 20 min global ischemia and 60 minutes reperfusion ± PI treatment (MG-132 and lactacystin), anti-inflammatory (Ibuprofen) and anti-oxidant (NAC). Infarct size was determined using Evans Blue dye and 1% 2,3,5-triphenyl tetrazolium chloride (TTC) staining with 20 minutes regional ischemia and 2 hours reperfusion ± PI’s treatments. Tissues were collected at the end of the global ischemia experiments and analyzed for UPS activity, oxidative stress, apoptosis and inflammation. Our data expressed a reduced cardiac contractile function in response to ischemia and reperfusion under hyperglycemic conditions as well as an increase in UPS activity. PI treatment resulted in cardio-protection for ex vivo rat heart model exposed to ischemia and reperfusion under hyperglycemic conditions as well as ibuprofen and NAC. In parallel lactacystin treatment significantly decreased myocardial oxidative stress, apoptosis, and inflammation which provided cardio-protection in response to ischemia and reperfusion under hyperglycemic conditions This study shows that acute hyperglycemia elicits myocardial oxidative stress, apoptosis and inflammation that in time results in an increase in contractile dysfunction following ischemia and reperfusion. However, we found that PI treatment with both MG-132 and lactacystin blunted high glucose-induced damaging effects which resulted in a robust cardio-protection in response to ischemia and reperfusion under hyperglycemic conditions, by reducing oxidative stress, decreasing apoptosis and limiting inflammation. A parallel outcome was observed at baseline although the underlying mechanisms driving this process still need to be clarified. Our findings indicate that the UPS may be a unique therapeutic target to treat ischemic heart disease in diabetic patients, and non-diabetic individuals that present with stress-induced hyperglycemia. In summary, this thesis established that PIs act as a novel cardio-protective intervention to treat acute hyperglycemia with associated cardiovascular complications. / AFRIKAANSE OPSOMMING: Diabeties word beskou as ‘n baie groot problem vir menslieke gesondhied vir biede die ontwikkel en onontwikkelende lande. Kardiovaskulêre siekte wat normaal met diabetiese pasiente geassoseerd word veroorsaak ‘n toeneemende druk, wat hierdie siekte laat toeneem. Verder meer vergroot stresgeïnduseerde hiperglukemie die mortaliteit van pasiente met of sonder diabeties wat akute miokardiale infarksie onder lede het. Akute miokardiale infarksie kan ook ontstaan van stresgeïnduseerde hiperglukemie se bekwaamheid om meer inflamasie en oksidante stress te veroorsaak wat in ‘n meer swakker funksionele kardiale toestand. Hiperglukemiegeïnduseerde oksidatiewe stres ook tot wanregulering van die ubikwitien-proteosoomsisteem (UPS) wat wangevoude protïene verwyder, aanleiding gee. Kontrasterende data bestaan van verhoogde/verlaagde UPS aktiwietiet, sowel as met hiperglukemie en/of in reaksie tot isgemie-reperfussie. As gewolg hiervan,, hipotetiseer ons dat Z-Leu-Leu-Leu-al (MG-132) and lactacystin as ‘n nuwe kardiobeskermingsmiddel kan optree deur miokardiale oksidatiewe stress, inflamasie en UPS aktiwiteit te verlaag in reaksie op isgemie-reperfussie tydens akute hiperglukemiese toestande kan verlaag. Geïsoleerde rotharte is ex vivo met Krebs-Henseleit buffer, wat, 33 mM glukose vs. kontrole (11 mM glukose) bevat, vir 60 min geperfuseer, daarna is dit deur 20 min globale isgemie gevolg en 60 min reperfussie ± PI behandeling (MG-132 and lactacystin), antiflammatoriese behandeling (Ibuprofen) en antioxidant behandeling (NAC). Infarkgrootte is bepaal deur Evans bou kleursel en 1% 2. 3-5 tripfeniel tetrazoloimcholierd (TTC) kleuring met 20 minute regionale ischemie, en 2 uur reprefussie ± PI’s behandeling. Weefsels is aan die einde van die globale isgemie eksperimente versamel, en vir oksidatewe stres, apoptose en inflammasie ontleed. Ons data toon aan dat kardiale kontraktiele funksie in reaksie op isgemie-reperfussie onder hiperglukemiese toestande verlaag het asook ‘n toename in UPS aktiwitiet veroorsaak. PI behandeling het gelei tot kardiale beskerming vir ex vivo rotharte wat aan isgemie-reperfussie onder hiperglukemiese toestande blootgestel was sowel as ibuprofen en NAC. Parallel hiermee het lactacystin oksidatiewe stres, apoptose, inflmasie, en UPS aktiwiteit na isgemie-reperfussie, verlaag in reaksie isgemie-reperfussie onder hiperglukemiese toestande. Hierdie studie het bevind dat akute hiperglukemie, miokardiale oksidatiewe stres lei tot oksidante stress, apoptose, en inflamasie na kontraktiele wanfunksionering na isgemie-reperfussie lei. Ons het bevind dat beide MG-132 en lactacystin behandeling, hoë glukose-geïnduseerde skadelike effekte onderdruk, en kardiale-beskerming in reaksie op isgemie-reperfussie onder hiperglukemiese toestande ondervind was deur oksidante stress, apoptose, en inflamasie te verlaag. ‘n Soorgelyke effek is tydens die basislyn waargeneem, alhoewel die onderliggende meganisme wat hierdie proses meer ondersoek instel. Ons bevinding dei dat die UPS ‘n nuwe behandeling teiken kan word in sgemie-geïnduseerde reperfussie onder aktute en chroniese hoë glukose toestande. In opsomming, het die tesis belowend bevindinge gevind wat ‘n nuwe terapeutiese intervensie vir die behandeling van akute hiperglukemie met geassosieërde kardiovaskulêre komplikasies gebruik kan word.

The modulation of the electron transport during ischemia-reperfusion has been shown to be protective. We hypothesized that metformin, a Complex I inhibitor, may exhibit characteristics of a pharmacological agent that could achieve long-term therapeutic intervention against ischemia-reperfusion injury. Mitochondria were harvested from adult male mice and incubated with or without metformin at 30oC for 15 minutes, while being shaken at 300 rpm. Metformin decreased Complex I oxidative phosphorylation and Complex I activity. However, metformin also increased injury and decreased the maximum membrane potential. Even though there was a decrease in maximum membrane potential, the proton motive force (PMF) was still intact as the ADP/O ratio was not affected. In conclusion, metformin does exhibit some characteristics of a drug that could achieve long-term therapeutic benefit against ischemia-reperfusion.

L'infarctus du myocarde est la première cause de mortalité cardiovasculaire dans les pays occidentaux. La reperfusion la plus précoce possible est actuellement le seul traitement validé pour réduire la taille d'infarctus, facteur pronostique fondamental de morbi-mortalité. Cependant, la reperfusion engendre des lésions d'ischémie-reperfusion (IR) irréversibles qui précipitent la mort par apoptose des cardiomyocytes. Une approche transcriptomique nous a permis d'identifier les gènes spécifiques du postconditionnement ischémique (PostC) dans le cœur de souris. Parmi ceux-ci, l'expression du gène codant pour le récepteur métabotrope de type 1 du glutamate (mGluR1) est augmentée par le PostC. L'objectif de mon travail de thèse a été d'étudier le rôle de mGluR1 au cours de l'IR myocardique. Notre stratégie, basée sur l'utilisation de souris knock-out, a permis de confirmer l'implication de mGluR1 dans la cardioprotection. L'injection de glutamate au moment de la reperfusion myocardique permet de diminuer significativement la taille de l'infarctus par inhibition de l'apoptose. Cet effet cardioprotecteur est diminué en présence de l'antagoniste spécifique YM 298198 ou en présence de wortmannin, inhibiteur de la PI3-kinase, activée dans la cascade de signalisation du récepteur. La réduction de la taille d'infarctus par le glutamate semble associée à une amélioration de la fonction contractile du ventricule gauche en échocardiographie (par speckle tracking, méthode de quantification de la déformation myocardique) dans un modèle murin d'IR myocardique. Ces résultats, bien que préliminaires, semblent prometteurs et nous permettent d'envisager une application clinique chez le patient coronarien. / Myocardial infarction is the major cause of cardiovascular mortality in western countries. Reperfusion as early as possible is the only treatment recognized to reduce infarct size, crucial prognostic factor of morbidity and mortality. However, reperfusion leads to ischemia-reperfusion (IR) injury leading to irreversible apoptotic death of cardiomyocytes. A transcriptomic approach has allowed us to identify genes specifically regulated upon ischemic postconditioning (PostC) in the mouse heart. Among them, the expression of the metabotropic glutamate receptor type 1 (mGluR1) gene is up-regulated by PostC. The aim of my thesis work was to study the role of mGluR1 during myocardial IR. Our strategy, based on the use of knockout mice, confirmed the involvement of mGluR1 in cardioprotection. Injection of glutamate at the time of reperfusion significantly reduced infarct size via apoptosis inhibition. This cardioprotective effect was reduced in presence of the specific antagonist YM 298198 or in presence of wortmannin, an inhibitor of PI3-kinase, which is activated downstream mGluR1. In our mouse model of myocardial IR injury, decrease in infarct size after glutamate treatment seems to be associated to an improved left ventricular contractile function assessed by echocardiography (speckle tracking method quantifying myocardial strain). These preliminary results are promising and allow us to consider a clinical trial for coronary patients.

Dans ce travail, nous avons montré qu'un nouveau ligand du TSPO, le TRO40303, possède des propriétés cardioprotectrices, confirmant que le TSPO joue un rôle important dans les effets délétères engendrés par l'ischémie-reperfusion myocardique. Des effets similaires ont déjà été observés avec d'autres ligands du TSPO, notamment le 4'-chlorodiazépam, mais le mécanisme d'action par lequel ces molécules exercent leur effet cardioprotecteur reste pour une large part encore mal connu. Nous avons montré chez le rat que la reperfusion d'un myocarde ischémié s'accompagne d'une augmentation du cholestérol mitochondrial, de la formation d'oxystérols et d'un stress oxydant majeur. Le 4'-chlorodiazépam inhibe ces effets et améliore les fonctions mitochondriales post-ischémiques, révélant que le TSPO est responsable du transport du cholestérol dans la mitochondrie cardiaque et que ce dernier, sous des formes oxydées ou non, pourrait participer aux effets délétères de la reperfusion. La limitation de l'entrée du cholestérol dans la mitochondrie lors de la reperfusion d'un myocarde ischémié est un mécanisme original qui pourrait donc contribuer à l'effet cardioprotecteur des ligands du TSPO. Enfin, nous avons montré que chez des rats génétiquement modifiés et associant hypercholestérolémie, obésité et diabète de type II, le cholestérol mitochondrial est d'une part très élevé, avec ou sans ischémie-reperfusion myocardique et d'autre part que le 4'-chlorodiazépam n'a pas d'effet sur le cholestérol mitochondrial après ischémie-reperfusion. Ces résultats suggèrent que les mécanismes d'action des ligands du TSPO sont probablement différents et que les traitements doivent être adaptés en présence de facteurs de co-morbidité. / In the present work we showed that a new TSPO ligand, TRO40303, has cardioprotective properties confirming that TSPO plays a key role in the deleterious effects of myocardial ischemia-reperfusion. Similar effects have been observed with other TSPO ligands, such as 4'-chlorodiazepam, but the mechanism of action of these molecules is not known. We showed that ischemia-reperfusion in rats increased mitochondrial concentration of cholesterol, oxysterol formation and oxidative stress. 4'-Chlorodiazepam inhibited these effects and improved post-ischemic mitochondrial functions, revealing that TSPO is responsible for cholesterol transport in cardiac mitochondria and that cholesterol, in oxidized or non-oxidized forms, could participate in the deleterious effects of reperfusion. The limitation of the increase in cholesterol in mitochondria during ischemia-reperfusion is an original mechanism which could contribute to the cardioprotective effect of TSPO ligands. We then showed, in genetically modified rats with hypercholesterolemia, obesity and type II diabetes, that the concentration of mitochondrial cholesterol is very high with or without ischemia-reperfusion. We further demonstrated that 4'-chlorodiazepam has no effect on mitochondrial cholesterol after ischemia-reperfusion. These results suggest that the mechanisms of action of TSPO ligands probably differ in these conditions and that treatment must be adapted in accordance with the presence of factors of co-morbidity.

Renal ischemia/reperfusion (I/R) is a main cause of acute kidney injury (AKI) and delayed graft function after renal transplantation. Previous studies in human and experimental models have identified that inflammation and oxidative stress are two key players in renal I/R injury. However, the underlying mechanisms remain speculative. The overall objective of the study was to investigate the biochemical and molecular mechanisms of I/R-induced renal injury and the effect of tyrosol supplementation on I/R-induced kidney oxidative stress damage. In the present study, renal I/R was induced in Sprague-Dawley rats and in a human kidney proximal tubular cell line. A significantly elevated expression of pro-inflammatory cytokine expression (MCP-1, IL-6) was observed. There was a significant decrease in mRNA and protein levels of two hydrogen sulphide (H2S)-producing enzymes, CBS and CSE, with a concomitant reduction of glutathione and H2S production. In the cell culture model, hypoxia–reoxygenation of proximal tubular cells led to a decrease in CBS and CSE expression and an increase in pro-inflammatory cytokine expression. Supplementation of glutathione or H2S donor (NaHS) effectively abolished cytokine expression in tubular cells. Experiments were conducted to detect oxidative stress markers. It was demonstrated that there was a significant increase in peroxynitrite formation and lipid peroxidation in the kidney after I/R insult, which might be caused by the elevation in nitric oxide (NO) metabolites and inducible nitric oxide synthase (iNOS). Administration of tyrosol, a natural phenolic compound, reduced peroxynitrite formation, lipid peroxidation and the level of NO metabolites via inhibiting NF-B activation and iNOS expression. Tyrosol treatment improved kidney function and had a protective effect against I/R-induced AKI. The present study has clearly demonstrated that (1) there is a reduction of H2S production via inhibition of CBS and CSE expression, which contributes to increased pro-inflammatory cytokine expression in the kidney and in tubular cells upon I/R insult; (2) restoration of endogenous H2S production would be of therapeutic value in regulating inflammatory response in I/R-induced kidney injury; (3) tyrosol treatment has a beneficial effect against renal I/R-induced oxidative stress, in part, through its inhibition on NF-B activation and iNOS-mediated NO production. / May 2016

Les objectifs de ce travail expérimental étaient : 1. Évaluer la place de la lactatémie capillaire comme dispositif de monitorage de l’ischémie et de la reperfusion tissulaire. 2. Comparer le suivi local du taux de lactate, au niveau du compartiment ischémié, par rapport au taux systémique du lactate lors des l’ischémie de membre, l’ischémie intestinale ainsi que durant le choc hémorragique. La mesure capillaire du taux de lactate pourrait permettre: 1. d’affirmer l’existence d’une ischémie tissulaire régionale. Le taux systémique du lactate ne permet ce diagnostic, 2. de confirmer l’efficacité d’une reperfusion au niveau d’un membre préalablement ischémique tandis que la mesure systémique des lactates ne le permet pas. L’affirmation de l’efficacité de la reperfusion et de sa constance, par une mesure aussi simple que la lactatémie capillaire compartimentale représente une avancée majeure en comparaison avec les autres méthodes existantes. La mesure capillaire du taux de lactate systémique dans une situation d’hypoperfusion par choc hémorragique n’est pas associée à augmentation du taux de lactate intra-musculaire. / Our objectives for this experimental work were: 1. Assessment of capillary lactate for tissue ischemia and reperfusion monitoring. 2. Compare the local and systemic capillary lactate time course during compartmental ischemia insults like limb ischemia, intestinal ischemia or during hemorrhagic shock. The capillary measurement of the lactate in IR could be interesting for: 1. Assessing a limb or intestinal tissue ischemia. On the opposite, the systemic measurement could not assess this diagnosis. 2. Assessing the efficiency of a limb reperfusion. On the opposite, the systemic measurement could not assess this diagnosis. The systemic capillary lactate measurement during haemorrhagic shock related hypo perfusion could not be associated with an increase in intra muscular lactate.