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The Langendorff perfused murine heart has become an increasingly important research model in cardiovascular physiology and pharmacology. However, the model remains relatively poorly characterised when compared with the widely employed rat preparation. The purpose of the research within this thesis was initially two-fold: 1) to characterise the functional and substrate-dependent properties of the murine model; and 2) to characterise the relationships between glycolysis, ischaemic tolerance and adenosine-mediated cardioprotection in the mouse. Initial studies, confirmed by simultaneous/subsequent work in other laboratories, revealed the frequent occurrence of regular cyclic oscillations in contractile function and coronary flow in glucose-perfused isovolumically contracting hearts. This phenomenon (labelled 'cycling') was unaltered by inhibition of ?-adrenergic receptors, prostaglandins, and nitric oxide synthase. However, A1/A2 adenosine receptor agonism did abolish the oscillations in flow and reduced contractile oscillations by 50%. Importantly, cycling was eliminated by addition of 50 IU/l insulin to perfusion fluid, or provision of 5 mM pyruvate as a co-substrate with glucose. These data suggest that functional 'cycling' in glucose-perfused murine hearts likely occurs as a result of a mismatch between substrate metabolism (energy supply) and myocardial energy demand. It may be that glycolysis with exogenous glucose is insufficient to ensure appropriate matching of myocardial energy supply and demand. For this reason, it is advisable to employ a co-substrate such as pyruvate in studies of murine hearts. Further studies performed within this thesis generally employ this co-substrate addition. Addition of pyruvate as co-substrate removes 'cycling' but is also known to inhibit/modify glycolysis, which may affect ischaemic tolerance and/or cardioprotection mediated by adenosine. Experiments throughout this thesis demonstrated that pyruvate-perfusion improved tolerance to both ischaemia (delayed time to onset of ischaemic contracture; TOC) and reperfusion (reduced diastolic dysfunction and cell death). The delay in TOC as a result of pyruvate-perfusion also suggests that contracture is not solely influenced by anaerobic glycolysis (as outlined in current paradigms). To test the relevance of glycolysis to ischaemic injury hearts were subjected to various forms of glycolytic inhibition. Glycolysis was inhibited by use of 10 mM pyruvate, (iodoacetic acid) IAA treatment, and glycogen depletion by pre-ischaemic substrate-free perfusion (all groups employing pyruvate as sole-substrate). Each form of glycolytic modification resulted in significant delays in TOC, in complete contrast to findings from other models and species. Glycogen depletion also reduced the peak level of contracture. These findings indicate that the mouse is either unique in terms of substrate metabolism and mechanisms of contracture (an unlikely possibility), or raise serious questions regarding current models of contracture development during ischaemia (theorised to be delayed by prolonging anaerobic glycolysis). Modification of glycolysis also altered post-ischaemic outcome, with pyruvate perfusion and glycogen depletion both enhancing functional recoveries. However, IAA treated hearts, despite near-identical ischaemic tolerance (ie contracture development) to pyruvate-perfused hearts, displayed very poor functional recovery, which was below that for all other groups. These data clearly reveal that blocking glycolysis improves tolerance to ischaemia (as evidenced by reduced contracture), provide evidence of dissociation of ischaemic injury or contracture from post-ischaemic recovery, and confirm the key importance of glycolysis in enhancing recovery from ischaemia. Since tolerance to ischaemia/reperfusion was shown to be glycolysis dependent, and since it has been theorised that adenosine protects hearts through modulating glycolysis, the relationships between glycolytic inhibition and adenosine-mediated cardioprotection was tested. In a number of studies, exogenously applied adenosine was shown to protect both glucose- and pyruvate-perfused hearts (supporting no dependence of adenosinergic protection on glycolysis). However, to more equivocally test the role of glycolysis effects of IAA were studied and were shown to markedly limit protection with adenosine. The effects of adenosine during ischaemia were abolished by IAA treatment, and effects on post-ischaemic recovery were reduced (but not eliminated). Similar results were acquired for protection with endogenous adenosine (using iodotubercidin to block adenosine phosphorylation). Collectively, these data reveal that adenosinergic protection during ischaemia depends entirely upon glycolysis while protection during reperfusion likely involves glycolysis dependent and independent processes. However, glycolysis is required for full recovery of function during reperfusion. Further studies assessed the involvement of glycolysis in cardioprotection afforded by transgenic A1 adenosine receptor (A1AR) overexpression. It was found that pyruvate-perfusion provided the same protection as A1AR overexpression, and the two responses (to pyruvate and A1AR overexpression) were not additive. Thus, it is probable that common mechanisms are targeted in both responses (likely glycolysis). Finally, the effects of adenosine and pyruvate on oxidant injury were studied, testing whether interactions between adenosine and pyruvate observed in prior work within this thesis could be explained by alterations in anti-oxidant responses. It was found that adenosine has quite profound anti-oxidant responses in glucose-perfused hearts, with very selective effects on markers of damage. Pyruvate also had some anti-oxidant effects but interestingly it reduced the anti-oxidant effects of adenosine. In conclusion, the work entailed within this thesis demonstrates that the isolated mouse heart model may possess unique properties and should be further characterised by potential users in order to improve its utility, and the reliability of experimental findings (chiefly when studying ischaemia-reperfusion). Other work within thesis demonstrates that modification of glycolysis is important in dictating recovery from ischaemia-reperfusion, and also impacts on adenosine-mediated protection (principally but not exclusively during ischaemia itself). The manner in which glycolysis is modified and contributes to protection remains unclear.

This study describes the development of a novel method of three dimensional perfused cell culture for in vitro toxicity testing. Multiple parallel perfused microbioreactors (TissueFlex^TM) were adopted to provide a well-controlled cell culture environment. Alginate and collagen type I, commonly used as hydrogel scaffolds to support cell culture, were tested as the scaffolding materials for this application. Alginate supports cell proliferation, but does not support cell attachment. Collagen gel (type I), good for cell attachment but with poor mechanical strength, could be used at the high concentration of 5mg/ml to prevent the degradation of the gel. Improvement of collagen biomechanical property by a purpose-designed compressor to physically induce cross-linking showed promising results and merits further study. The suitability of alamarBlue® assay, a common non-toxic non-destructive viability assay method, was confirmed for this study and the protocol was optimised. To demonstrate the effectiveness of three dimensional perfused cell culture, human mesenchymal stem cells (MSC) seeded in collagen type I were employed to test the cell inhibition of two antibiotics, trimethoprim and pyrimethamine. The results displayed the perfusion system has greater advantage and sensitivity than the static system, as does these of 3D scaffolds, compared with 2D. Such differences are related to the continuous supply of fresh culture medium to keep cells at a stable pH, temperature, oxygen, and a more physiological like environment. The cytotoxicity of two stereoisomer compounds, obtained confidentially from Pfizer. Ltd., was assessed using the developed method and compared to conventional 2D static and perfused culture by using rat adipose mesenchymal stem cells. The results successfully distinguished toxic and non-toxic compounds and also demonstrated that the 3D perfused system improved the prediction of drug toxicity over 2D culture. 3D perfused bioreactors were applied to hepatotoxicity study using freshly isolated rat hepatocytes. Only algimatrix^TM supported hepatocyte spheroid formation among those tested including collagen type I, alginate beads, poly lactic acid fibres, and Algimatrix^TM. A new variation of TissueFlex^TM bioreactor with micro-patterned surface, designed specifically for hepatocyte self-assembly culture without use of any scaffold, was tested. The results demonstrated that, compared with the standard sandwich culture, the self-assembly culture in the micro-patterned bioreactors showed high cell viability, biomarkers expression, as well as more physiological immunocytochemistry. Moreover, the differential gene expression indicated that self-assembly culture could provide more relevant information regarding metabolising processes than the 2D sandwich culture, which would potentially improve hepatotoxicity prediction. In conclusion, 3D perfused cell culture for in vitro toxicity testing improved the predictivity, reliability and physiological relevance of drug toxicity compared to traditional 2D culture.

CT is a powerful method for noninvasive assessment of the lung. Advancements to CT technology have guided the high-resolution structural and functional assessment of lung diseases. This has helped make the transition from characterizing the severity of lung disease to novel phenotyping of disease subtypes. Chronic obstructive pulmonary disease (COPD) is a spectrum of inflammatory lung disease affecting lung parenchyma, airways, and the pulmonary and systemic vasculature. Quantitative CT-based measures have largely focused on quantifying the extent of airway and parenchymal damage with disease. Recently perfusion CT method has been used to assess the pulmonary vascular bed. This technique was used to demonstrate a vascular etiology of smoking-associated centriacinar emphysema (CAE), a subtype of the COPD spectrum. However, technical challenges have limited the transition of this CT method to clinical studies to assess pulmonary vascular physiology. In this thesis, we introduce dual energy CT-perfused blood volume (DECT-PBV) as a novel image-based biomarker to assess peripheral pulmonary vascular dysfunction. Using this technique, we show that smoking-associated pulmonary perfusion heterogeneity, a marker of abnormal blood flow is a reversible process, in the midst of smoking-associated lung inflammation, and not a product of advanced lung disease. We demonstrate, via regional PBV measures and structural measures of the central pulmonary vessels, that the reversibility of pulmonary perfusion heterogeneity is a direct result of increased peripheral (downstream) parenchymal perfusion. We validate our quantitative imaging approach in a unique cohort of early CAE-susceptible smokers using a pharmaceutical intervention to dilate the pulmonary parenchymal vascular bed. The validated DECT approach and our novel DECT imaging findings extend our characterization of the vascular phenotype in inflammatory lung disease and provide a framework for future quantitative imaging studies of the lung to assess early intervention targeted to pulmonary vessels.

Prostaglandins, released from Kupffer cells, have been shown to mediate the increase in hepatic glycogenolysis by various stimuli such as zymosan, endotoxin, immune complexes, and anaphylotoxin C3a involving prostaglandin (PG) receptors coupled to phospholipase C via a G(0) protein. PGs also decreased glucagon-stimulated glycogenolysis in hepatocytes by a different signal chain involving PGE(2) receptors coupled to adenylate cyclase via a G(i) protein (EP(3) receptors). The source of the prostaglandins for this latter glucagon-antagonistic action is so far unknown. This study provides evidence that Kupffer cells may be one source: in Kupffer cells, maintained in primary culture for 72 hours, glucagon (0.1 to 10 nmol/ L) increased PGE(2), PGF(2 alpha), and PGD(2) synthesis rapidly and transiently. Maximal prostaglandin concentrations were reached after 5 minutes. Glucagon (1 nmol/L) elevated the cyclic adenosine monophosphate (cAMP) and inositol triphosphate (InsP(3)) levels in Kupffer cells about fivefold and twofold, respectively. The increase in glyco gen phosphorylase activity elicited by 1 nmol/L glucagon was about twice as large in monocultures of hepatocytes than in cocultures of hepatocytes and Kupffer cells with the same hepatocyte density. Treatment of cocultures with 500 mu mol/L acetylsalicylic acid (ASA) to irreversibly inhibit cyclooxygenase (PGH-synthase) 30 minutes before addition of glucagon abolished this difference. These data support the hypothesis that PGs produced by Kupffer cells in response to glucagon might participate in a feedback loop inhibiting glucagon-stimulated glycogenolysis in hepatocytes.

Increased vascular reactivity is a common observation in human and experimental hypertension. In this study, reactivity of the perfused mesenteric arterial/arteriolar vascular bed was examined during the development of renal and renal/salt hypertension in the rat. Increased reactivity to noradrenaline was observed in tissues from rats 1-12 weeks after the induction of renal/salt hypertension. In the early (1-2 week) stages, preparations were supersensitive to noradrenaline but not to KCl. In the later (4-6 week) stages, the increase in noradrenaline reactivity was due to supersensitivity and another factor which increased KCl reactivity and was characterized by an elevated maximum response. The noradrenaline response potentiating effects of exogenous angiotensin II were attenuated in the early but not the later stages of hypertension. Vascular reactivity to noradrenaline in tissues from renal hypertensive rats was similar to that in renal/salt hypertensive rats, but with a slower time course. The decay of noradrenaline responses in calcium-free conditions was slower in tissues from early renal/salt hypertensive rats, indicating that an increased availability of activator calcium was the mechanism of supersensitivity. The characteristics of the α-adrenoceptor did not appear to differ in the renal/salt hypertensive rat as the pA(2) value for phentolamine did not change. Differences in the pA(2) value for indorarin were observed in tissues from hypertensive rats but probably resulted from the unusual properties of this drug. The early supersensitivity to noradrenaline was partially attributable to the effects of a positive sodium balance.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1969. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliography.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / According to the Brazilian Ministry of Health, about 8000 cases of scorpion envenomation are reported yearly in Brazil. Most cases are due to the sting of Tityus serrulatus, known popularly as yellow scorpion. It belongs to the Arachnidea, class, Scorpionidae order, Buthidae family, Tityinae subfamily and Tityus genus. It is present in the Brazilian States of Minas Gerais, EspÃrito Santo, Bahia, Rio de Janeiro, SÃo Paulo, ParanÃ, GoiÃs, Mato Grosso do Sul and Ceara and it is the most dangerous scorpion in Brazil, causing severe envenoming and even death. The effects of T. serrulatus venom (TsV) on the renal physiology in humans consist of increased renal parameters such as urea and creatinine. So far, effects had not been tested in the perfused rat kidney. Isolated kidneys from Wistar rats, weighing 250 to 300g, were perfused with Krebs-Henseleit solution containing 6g% of previously dialysed bovine albumin. The effects of T. serrulatus venom in the 1, 3 and 10 &#956;g/mL concentrations, were studied on the perfusion pressure (PP), renal vascular resistance (RVR), urinary flow (UF), glomerular filtration rate (GFR), sodium tubular transport (%TNa+), potassium tubular transport (%TK+) and chloride tubular transport (%TCl-). Tityus serrulatus venom was added to the system after 30 minutes of internal control; it increased PP (PP30â = 112.4  2.0 vs PP60â = 145.8  17.4mmHg*,*p<0.05) and RVR (RVR30â = 5.88  0.33 vs RVR60â = 7.52  0.86mmHg/mL.g-1.min-1*,*p<0.05), decreased GFR (GFR30â = 0.671  0.076 vs GFR120â = 0.467  0.062mL.g-1.min-1*,*p<0.05) and UF (UF30â = 0.147  0.011 vs UF90â = 0.119  0.013mL.g-1.min-1*,*p<0.05). The mesenteric bed was perfused with Krebs solution kept warm at 37ÂC by a constant flow (4mL/min), while the variable perfusion pressure was measured by means of a pressure transducer. The vascular effects of T. serrulatus venom were examined and compared to the infusion of the perfuse solution. The infusion of T. serrulatus venom (10&#956;g/mL/min) increased the basal perfusion pressure of isolated arteriolar mesenteric bed (basal pressure = 74.17  3.42 vs TsV = 151.8  17.82 mmHg*,*p<0.05). The histopathological evaluation revealed deposits of protein in the renal tubules and urinary spaces of kidneys perfused with TsV. This may reveal that there has been a perfusate extravasation to the parenchyma probably due to increase of hydrostatic pressure induced by TsV. T. serrulatus venom affects renal hemodynamics increasing resistance and perfusion pressure, determining extravasation of perfusate and decreasing the glomerular filtration rate and renal flow. / No Brasil, sÃo notificados, anualmente, cerca de 8.000 acidentes escorpiÃnicos. A espÃcie Tityus serrulatus, popularmente conhecido como escorpiÃo amarelo, pertence à classe Arachnidea, ordem Scorpionidae, familia Buthidae, subfamÃlia Tityinae, gÃnero Tityus. à encontrado nos estados de Minas Gerais, EspÃrito Santo, Bahia, Rio de Janeiro, SÃo Paulo, ParanÃ, GoiÃs, Mato Grosso do Sul e CearÃ, sendo responsÃvel pelos acidentes com maior gravidade e letalidade. Os efeitos do veneno de T. serrulatus (VTs), sobre a fisiologia renal humana, mostraram aumentar os parÃmetros renais, urÃia e creatinina. Entretanto, na perfusÃo de rins de ratos, os efeitos nÃo haviam sido testados atà agora. O objetivo desse trabalho foi estudar os efeitos renais e a reatividade vascular induzidos pelo veneno do escorpiÃo Tityus serrulatus. Foram utilizados ratos Wistar machos pesando entre 250 e 300g, cujos rins foram isolados e perfundidos com SoluÃÃo de Krebs-Hanseleit contendo 6g% de albumina bovina previamente dialisada. Os efeitos de veneno de T. serrulatus, nas concentraÃÃes de 1, 3 e 10 &#956;g/mL (n=6), foram estudados sobre a PressÃo de PerfusÃo (PP), ResistÃncia Vascular Renal (RVR), Fluxo UrinÃrio (FU), Ritmo de FiltraÃÃo Glomerular (RFG), Percentual de Transporte Tubular de SÃdio (%TNa+), de PotÃssio (%TK+) e de Cloreto (%TCl-). O veneno de T. serrulatus foi adicionado apÃs 30 minutos de controle interno e aumentou a PP (PP30â = 112,4  2,0 vs PP60â = 145,8  17,4mmHg*,*p<0,05), a RVR (RVR30â = 5,88  0,33 vs RVR60â = 7,52  0,86mmHg/mL.g-1.min-1*,*p<0,05) e diminuiu o RFG (RFG30â = 0,671  0,076 vs RFG120â = 0,467  0,062mL.g-1.min-1*,*p<0,05) e o FU (FU30â = 0,147  0,011 vs FU90â = 0,119  0,013mL.g-1.min-1*,*p<0,05). O leito mesentÃrico foi perfundido, com soluÃÃo de Krebs, sob a temperatura de 37ÂC, a um fluxo constante (4mL/min), enquanto a pressÃo de perfusÃo era mensurada atravÃs de um transdutor de pressÃo. Os efeitos vasculares do veneno de T. serrulatus 10&#956;g/mL/min; n = 6) foram examinados e comparados com a infusÃo apenas da soluÃÃo perfusora. A infusÃo do veneno de T. serrulatus aumentou a pressÃo basal de perfusÃo do leito mesentÃrico isolado (pressÃo basal: 74,17  3,42 vs VTs 151,8  17,82mmHg*, *p<0,05). A avaliaÃÃo histolÃgica revelou depÃsitos de proteÃnas nos tÃbulos renais e nos espaÃos urinÃrios dos rins perfundidos com o veneno de T. serrulatus. Isso pode revelar que houve um extravasamento do perfusato para o parÃnquima, devido, provavelmente, ao aumento da pressÃo hidrostÃtica promovida por VTs. O veneno de T. serrulatus, entÃo, promove efeitos hemodinÃmicos renais que elevam a resistÃncia e a pressÃo de perfusÃo, forÃando a passagem de proteÃnas para os tÃbulos e diminuindo o ritmo de filtraÃÃo glomerular e o fluxo urinÃrio.

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京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21624号 / 医博第4430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 川口 義弥, 教授 福田 和彦, 教授 坂井 義治 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM