Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Os PeptÃdeos NatriurÃticos (NPs) tÃm significativa participaÃÃo na regulaÃÃo na homeostasia cardiovascular, renal e endÃcrina, e tÃm sido descritos nos venenos de serpentes peÃonhentas. A Crotalus durissus cascavella, uma serpente terrÃcola caracterÃstica da caatinga do Nordeste do Brasil, possui em seu veneno total um NP (NPCdc), ao qual sÃo relatados efeitos vasculares e renais. O objetivo desse trabalho foi sintetizar em fase sÃlida o NPCdc e avaliar suas aÃÃes renais atravÃs da perfusÃo de rim isolado e em cultura de cÃlulas tubulares renais da linhagem MDCK e LLC-MK2. Rins de ratos Wistar machos, pesando entre 250-300 g, foram excisados cirurgicamente e perfundidos com soluÃÃo de Krebs-Henseleit modificada com 6% p/v de albumina bovina previamente dialisada. Foram investigados os efeitos do NPCdc em quatro concentraÃÃes (0,03 Âg/mL; 0,1 Âg/mL; 0,3 Âg/mL e 1 Âg/mL; n=6). As cÃlulas MDCK e LLC-MK2 foram cultivadas em meio de cultura RPMI 1640 suplementado com 10% v/v de Soro Bovino Fetal e entÃo avaliadas na presenÃa do NPCdc em diversas concentraÃÃes em um perÃodo de incubaÃÃo de 24 horas. ApÃs esse perÃodo, foram realizados ensaios de viabilidade celular. Os dados foram comparados estatisticamente considerando P<0,05. Houve aumento na pressÃo de perfusÃo (PP) em 0,03 Âg/mL e reduÃÃo em 1 Âg/mL. A resistÃncia vascular renal (RVR) apresentou aumento em 0,03 Âg/mL. O fluxo urinÃrio (FU) aumentou em 0,03 Âg/mL e diminuiu em 0,1 Âg/mL e 1 Âg/mL. O ritmo de filtraÃÃo glomerular (RFG) encontrou-se diminuÃdo nas quatro concentraÃÃes testadas. O clearance osmolar (COsm) apresentou-se aumentado em 0,03 &#956;g/mL e reduzido em 0,1 &#956;g/mL e 1 &#956;g/mL. O percentual de transporte tubular total e proximal de sÃdio (%TNa+ e %TpNa+, respectivamente) e cloreto (%TCl- e %TpCl-, respectivamente) apresentaram reduÃÃes nas quatro concentraÃÃes testadas. JÃ o percentual de transporte tubular total e proximal de potÃssio (%TK+ e %TpK+, respectivamente) apresentaram-se reduzidos em 0,03 &#956;g/mL e 0,3 &#956;g/mL. A anÃlise histopatolÃgica mostrou a presenÃa de alteraÃÃes morfolÃgicas significativas, como degeneraÃÃo hidrÃpica concentraÃÃo-dependente em todas as concentraÃÃes, juntamente com discreta a moderada deposiÃÃo de material proteÃceo nos tÃbulos na concentraÃÃo de 0,03 &#956;g/mL. Na cultura de cÃlulas MDCK e LLC-MK2, porÃm, o NPCdc nÃo foi capaz de diminuir a viabilidade celular. Esses resultados demonstram que o NPCdc modificou todos os parÃmetros avaliados na perfusÃo de rim isolado, alÃm de revelar alteraÃÃes de carÃter citotÃxico na anÃlise histopatolÃgica dos mesmos, todavia, nÃo as apresentando em culturas de cÃlulas tubulares renais. / Natriuretic Peptides (NPs) have significant interest in regulating the cardiovascular, renal and endocrine homeostasis, and have been described in the venom of venomous snakes. Crotalus durissus cascavella, a characteristic terrestrial snake of caatinga biome of northeastern Brazil, has in his whole venom an NP (NPCdc), of which are reported vascular and renal effects. The aim of this study was to synthesize NPCdc in solid phase and evaluate its renal actions through isolated kidney perfusion and culture of renal tubular cells of MDCK and LLC-MK2 line. Kidneys from male Wistar rats weighing 250-300 g were surgically excised and perfused with modified Krebs-Henseleit solution with 6 g% of previously dialyzed bovine serum albumin. The effects of NPCdc were investigated in four concentrations (0.03 &#956;g/mL, 0.1 &#956;g/mL, 0.3 &#956;g/mL and 1 &#956;g/mL, n=6). MDCK and LLC-MK2 cells were cultured in RPMI 1640 culture medium supplemented with 10% v/v Fetal Bovine Serum and then evaluated in presence of several concentrations of NPCdc at an incubation period of 24 hours. After this period, cell viability assays were performed. Data were statistically compared considering P<0.05. There was increase in perfusion pressure (PP) in 0.03 &#956;g/mL and reduction in 1 &#956;g/mL. Renal vascular resistance (RVR) was increased at 0.03 &#956;g/mL. Urinary flow (UF) increased in 0.03 &#956;g/mL and decreased in 0.1 &#956;g/mL and 1 &#956;g/mL. The glomerular filtration rate (GFR) was reduced at all the concentrations tested. Osmolar clearance (OsmC) was increased in 0.03 &#956;g/mL and reduced in 0.1 &#956;g/mL and 1 &#956;g/mL. Percentage of the total and proximal tubular transport of sodium (%TNa+ and %pTNa+, respectively) and chloride (TCl-% and pTCl-%, respectively) showed reductions at all concentrations tested. Percentage of the total proximal tubular transport and potassium (%TK+ and pTK+%, respectively) were reduced in 0.03 &#956;g/mL and 0.3 &#956;g/mL. The histopathological analysis revealed the presence of significant morphological changes, such as concentration-dependent hydropic degeneration at all concentrations, along with mild to moderate deposition of proteinaceous material in the tubules in concentration 0.03 &#956;g/mL. In the culture of MDCK and LLC-MK2 cells, however, NPCdc was not able to lower cell viability. These results demonstrate that NPCdc modified all parameters assessed in renal perfusion of isolated kidney, and showed cytotoxic alterations in histopathological analysis, however, not presenting it in cultures of renal tubular cells.

Heat-induced hepatotoxicity accompanying hyperthermic liver perfusion was studied in the isolated, haemoglobin-free perfused rat liver. Trypan blue uptake, a sensitive indicator of cell death, was used to examine the relationship between the efflux of oxidized glutathione (oxidative stress), the appearance of cytosolic enzymes in the perfusate and cell death. Livers were perfused at 37, 42, 42.5 and 43°C. The efflux of total glutathione (GSH) and oxidized glutathione (GSSG) increased with time and temperature. Differences between temperature groups were significant for both parameters for 37 versus 42, 42.5 and 43°C (p < 0.05). Temperature-related differences in GSH levels appeared at 15 min for 37 versus 42 °C and in GSSG levels at 30 min for 37 versus 42 and 42.5°C. Biliary excretion of total GSH increased from 72 nmol at 37°C to 144 nmol at 42°C, 160 nmol at 42.5°C and 124 nmol at 43°C, which was significant for 37 versus 42 and 42.5°C (p < 0.05). The release of allantoin into the perfusate, a measure of purine catabolism and flux through xanthine oxidase, was increased at 42, 42.5 and 43°C compared to 37°C (p < 0.05). Liver injury was assessed by measuring the release of asportate aminotransferase (AST) and lactate dehydrogenase (LDH) and uptake of trypan blue after perfusion at each temperature. There was a pronounced release of LDH and AST into the perfusate after 60 min of perfusion at 42, 42.5 and 43°C, the levels of which were significantly different from the 37°C mean level. There was no uptake of trypan blue after 60 min perfusion at 37°C. Perfusion at 42, 42.5 and 43°C resulted in the uptake of trypan blue in the pericentral areas, but the dye uptake was significant (p < 0.05) compared to 37°C at 42.5 and 43°C only. These data show that heat-induced pericentral cell death is minimal after 60 min at 42-43°C, and that the biochemical processes which occurred during this period suggest 'oxidative stress' as a causative factor in hyperthermic hepatotoxicity. In addition, this liver toxicity is probably related to xanthine oxidase activity or the depletion of GSH as the initiating event which leads to lipid peroxidation and cellular damage.

Four cases of extremity cancers received effective management with cisplatin and etoposide via isolated limb perfusion. They demonstrated minimal, if any, soft tissue damage. This result counters the theory that a caustic response is a prerequisite for successful therapy. This characteristic allows for simultaneous surgical resection with regional, isolated limb perfusion of potent cytotoxic agents without increased morbidity from tissue necrosis, a common consequence of previously used drugs. There is no apparent affect on wound healing, even in cases involving extensive, radical operative procedures.

No description available.

L'imagerie par résonance magnétique dynamique (IRM-dynamique) consiste en l'observation de la distribution dans le temps d'un agent de contraste à l'aide de l'IRM. L'une des approches très répandues est d'analyser les données à l'aide de modèles mathématiques qui décrivent la pharmacocinétique de cet agent dans les tissus. L'une des hypothèses utilisées par l'ensemble des modèles présentés dans la littérature à ce jour est que les images d'IRM-dynamique peuvent être analysées pixel-par-pixel ce qui néglige implicitement la possibilité de diffusion de l'agent à l'intérieur des tissus. Dans ce mémoire, un nouveau modèle est proposé dans lequel la diffusion de l'agent est explicitement incluse dans un modèle à deux compartiments. Les deux paramètres couramment utilisés dans la littérature sont : K[indice supérieur trans] , le taux de transfert transcapillaire, et [nu]e, la fraction de volume extravasculaire extracellulaire. Deux méthodes d'optimisation stochastique ont été évaluées pour le lissage avec le modèle proposé à cause de la très grande taille de l'espace des solutions. Le modèle a été caractérisé avec des données simulées incluant la diffusion de l'agent de contraste et des données expérimentales montrant des signes de diffusion à l'intérieur du tissu tumoral. Les résultats avec les données simulées montrent que le modèle peut retrouver de façon fiable les valeurs des paramètres utilisés pour générer ces données (erreur relative moyenne de 16% pour K trans et 17% pour [nu]e) même lorsqu'un niveau de bruit raisonnable est ajouté. Le modèle standard à deux compartiments négligeant la diffusion retourne des distributions de valeurs de K[indice supérieur trans] erronées (erreur relative moyenne de 43%) qui sont moyennée sur le tissu. Lorsque les données expérimentales sont ajustées avec le modèle standard, les valeurs de K[indice supérieur trans] retournées montrent une perfusion moyennée sur l'ensemble du tissu, ce qui n'est pas en accord avec le rehaussement initial du signal qui est observé. À l'opposé, le modèle proposé retourne des cartes de K[indice supérieur trans] ayant une démarcation franche entre les zones bien perfusées et celles très peu perfusées en accord avec ce qui est observé sur les images d'IRM. De plus, le modèle standard à deux compartiments assigne des valeurs n'ayant pas de sens physique à [nu]e ([nu]e [supérieur à] 1) dans le centre des tumeurs où l'agent parvient par diffusion à partir de la périphérie bien vascularisée. De son côté, le modèle proposé trouve des valeurs plausibles de [nu]e pour l'ensemble du tissu.

Persufflation (PSF; gaseous oxygen perfusion) is an organ preservation technique with a potential for use in donor heart preservation. Improved heart preservation with PSF may improve outcomes by maintaining cardiac tissue quality in the setting of longer cold ischemia times and possibly increasing the number of donor hearts available for allotransplant. Published data suggest that PSF is able to extend the cold storage times for porcine hearts up to 14 hours without compromising viability and function, and has been shown to resuscitate porcine hearts following donation after cardiac death. This review summarizes key published work on heart PSF, including prospective implications and future directions for PSF in heart transplantation. We emphasize the potential impact of extending preservation times and expanding donor selection criteria in heart allotransplant. Additionally, the key issues that need to be addressed before PSF were to become a widely utilized preservation strategy prior to clinical heart transplantation are summarized and discussed.

The aim of this thesis is to replace the intractable problem of using discrete flow models within large vascular networks with a suitably parameterised and tractable continuum perfusion model. Through this work, we directly address the hypothesis that discrete vascular data can be incorporated within continuum perfusion models via spatially-averaged parameterisation techniques. Chapter 1 reviews biological perfusion from both clinical and computational modelling perspectives, with a particular focus on myocardial perfusion. In Chapter 2, a synthetic 3D vascular network was constructed, which was controllable in terms of its size and properties. A multi-compartment static Darcy perfusion model of this discrete system was parameterised via a number of techniques. Permeabilities were derived using: (i) porosity-scaled isotropic (ϕI); (ii) Huyghe and Van Campen (HvC); and (iii) projected-PCA parameterisation methods. It was found that HvC permeabilities and pressure-coupling fields derived from the discrete data produced the best comparison to the spatially-averaged Poiseuille pressure. In Chapter 3, the construction and analysis of high-resolution anatomical arterial vascular models was undertaken. In Chapter 4, various anatomically-derived vascular networks were used to parameterise our perfusion model, including a microCT-derived rat capillary network, a single arterial subtree, and canine and porcine whole-organ arterial models. Allowing for general-connectivity (as opposed to strictly-hierarchical connectivity) yielded a significant improvement on the continuum model pressure. For the whole-organ model however, it was found that the best results were obtained by using porosity-scaled isotropic permeabilities and anatomically-derived pressure-coupling fields. It was also discovered that naturally occurring small length but relatively large radius vessels were not suitable for the HvC method. In Chapter 5, the suitability of derived parameters for use within a dynamic perfusion model was examined. It was found that the parameters derived from the original static network were adequate for application throughout the cardiac cycle. Chapter 6 presents a concluding discussion, highlighting limitations and future directions to be investigated.

No description available.

Hyperpolarization via the Dynamic Nuclear Polarization (DNP) technique has revolutionized our ability to study metabolic changes in real time. The aim of this thesis was to build upon previous work centered around the use of DNP within the isolated perfused rat heart, a well established model system for the study of cardiac metabolism, to enhance the information that can be obtained through the combination of DNP with perfused organs. Initially this was done by using the widely studied DNP probe, [1-¹³C]pyruvate, to generate images of metabolism within the isolated perfused rat heart. The developed technique was then successfully demonstrated in two models of myocardial infarction. The thesis then proceeds to develop an understanding of how the supra-physiological concentrations of [1-¹³C]pyruvate commonly used in DNP experiments can affect metabolism in the isolated perfused rat heart, and the way in which the myocardium responds to those changes if it is not adequately supplied with substrates ordinarily present in vivo, namely fatty acids. New methods of providing the heart with these required substrates were developed, without significant interference to the biochemical information acquired from DNP experiments. As [1-¹³C]pyruvate only provides information on a small subset of carbohydrate metabolism, the next chapter develops new compounds to be used with DNP, which would allow the exploration of short chain fatty acid metabolism (butyrate) as well as ketone body metabolism (β-hydroxybutyrate and acetoacetate), and other aspects of carbohydrate metabolism (lactate and alanine). These compounds were developed and then tested for their potential usefulness in the isolated perfused heart. Finally, as the isolated perfused rat heart lacks the diversity of genetic disease models available in the mouse, the final chapter expanded the use of DNP to the isolated perfused mouse heart with all the size challenges that this entails, and makes the transition from the heart to the liver, in order to provide an alternative metabolic viewpoint on the biochemistry of disease models. This thesis thereby permits studies involving isolated perfused organs to be carried out whilst exploiting all the tools that DNP has to offer and consequentially, allows for a vast array of physiologically derived information to help us better understand metabolic diseases.

Rho Kinase (Rho-K) has been implicated in the pathophysiology of many deleterious conditions and its inhibition was shown to ameliorate these compromising effects. It is unclear; however, whether inhibition of Rho-K would decrease infarct size in hearts after ischemia/reperfusion. Adult ICR mice were randomized to 1 of 4 treatments: saline, fasudil (Rho-K inhibitor (10 mg/kg i.p.), Fasudil+L-NAME (Nitric Oxide synthase inhibitor, 15 mg/kg), and L-NAME. Hearts were isolated, perfused in Langendorff mode and subjected to 30 min stabilization before 30 min ischemia and 60 min reperfusion. Left ventricular (LV) function was monitored. Hearts were stained and infarct size measured. Fasudil reduced infarct size as compared with control hearts; however, this protective effect was abolished by L-NAME. LV function mirrored these trends. The loss of cardioprotection after L-NAME administration indicates that cardioprotection by Rho-K inhibition is mediated through nitric oxide-dependent pathway. Furthermore, Fasudil administration at and throughout reperfusion showed similar cardioprotection.