Η νευρολογικές διαταραχές και κυρίως η παραπληγία, αποτελούν τις πιο καταστροφικές επιπλοκές των επεμβάσεων στη θωρακο-κοιλιακή αορτή. Η διαδικασία ισχαιμίας/επαναιμάτωσης κατά τις επεμβάσεις αυτές λόγω του αποκλεισμού της αορτής, προκαλεί την ανάπτυξη τοξικών ελεύθερων ριζών οξυγόνου φαινόμενο που ορίζει το οξειδωτικό stress. Ο σκοπός της παρούσας μελέτης είναι ο καθορισμός και η ανίχνευση των ελευθέρων ριζών, αλλά και η επίδραση της Αμιφοστίνης, ενός αντιοξειδωτικού παράγοντα της κατηγορίας των θειολών. Μέθοδος: Η μέθοδος αφορά δεκαοκτώ αρσενικούς κονίκλους που υποβάλλονται σε ισχαιμία του νωτιαίου μυελού με αποκλεισμό της αορτής με τη χρήση ενδο-αορτικού ασκού. Ο ασκός προωθείται μέσω της μηριαίας αρτηρίας σε επίπεδο αμέσως κάτωθεν της αριστεράς υποκλειδίου αρτηρίας. Τα ζώα αποτελούσαν τρείς ομάδες. Η Ομάδα Ι αποτέλεσε την ομάδα ελέγχου. Στην Ομάδα ΙΙ η αορτή αποκλείσθηκε για 30΄και ακολούθησε επαναιμάτωση για 75΄. Στην Ομάδα ΙΙΙ χορηγήθηκε Αμιφοστίνη μέσω του καθετήρα αποκλεισμού κατά το δεύτερο ήμισυ της περιόδου αποκλεισμού (των 30΄). Στο τέλος της επαναιμάτωσης δείγματα νωτιαίου μυελού υποβλήθηκαν σε ανάλογη επεξεργασία για την ανίχνευση ελευθέρων ριζών οξυγόνου με τη χρήση υδροεθιδίνης και παραγώγων λιπιδικής υπεροξείδωσης με ιδιαίτερα ευαίσθητη μέθοδο φθορισμού. Αποτελέσματα: Τα αποτελέσματα των μετρήσεων έδειξαν αύξηση του υπεροξειδίου του οξυγόνου στην Ομάδα ΙΙ κατά 27.43% σχετικά με την Ομάδα Ι για να ακολουθήσει μείωση στην Ομάδα ΙΙΙ κατά 42.55% σε σχέση με την Ομάδα ΙΙ και κατά 15.25% από την Ομάδα ΙΙΙ. Η μέτρηση ενώσεων λιπιδικής υπεροξείδωσης που αντιδρούν με θειοβαρβιτουρικό οξύ (TBARSassay) έδειξε αύξηση κατά 55.3% στην Ομάδα ΙΙ σε σχέση με την Ομάδα Ι και μείωση κατά 30.3% στην Ομάδα ΙΙΙ σε σχέση με την Ομάδα ΙΙ. Η στατιστική ανάλυση και των δύο μεθόδων ανέδειξε σημαντική διαφορά με ( p<0.05). Συμπεράσματα: Ο αποκλεισμός της κατιούσας αορτής σε επίπεδο αμέσως κάτωθεν της αριστεράς υποκλειδίου αρτηρίας, προκαλεί αναμφισβήτητα ισχαιμία του νωτιαίου μυελού. Η ανάπτυξη οξειδωτικού stressως αποτέλεσμα της διαδικασίας ισχαιμία/επαναιμάτωση ανιχνεύεται μέσω των ριζών υπεροξειδίου και παραγώγων λιπιδικής υπεροξείδωσης. Η έγχυση Αμιφοστίνης προτείνεται ως αντιοξειδωτικός παράγων που μπορεί να ανιχνεύσει και να δεσμεύσει τις ελεύθερες ρίζες οξυγόνου κατά το οξειδωτικό stress που προκαλεί η ισχαιμία /επαναιμάτωση του νωτιαίου μυελού. / Paraplegia is the most devastating complication of thoraco-abdominal aortic procedures. An ischemia-reperfusion procedure is known to elevate free radicals causing oxidative stress. The aim of this study is to determine and to detect the free radical products and to examine the influence of Amifostine, a triphosphate agent, on oxidative stress of spinal cord ischemia-reperfusion in rabbits. Methods: Eighteen male, New Zealand white rabbits were anesthetized and spinal cord ischemia was induced by inflation of a coronary artery balloon catheter, advanced to descending thoracic aorta through the femoral artery. The animals were randomly divided into 3 groups. Group I functioned as control. In group II the aorta was occluded for 30 minutes and then re-perfused for 75 min. In group III, 500mg Amifostine was infused into the distal aorta during the second half-time of ischemia period. At the end of reperfusion all animals were sacrificed and spinal cord specimens were examined for superoxide radicals by an ultra sensitive fluorescent assay. Results: Superoxide radical levels ranged, in group I between 1.52 and 1.76 (1.64±0.10), in group II between 1.96 and 2.50 (2.10±0.21), and in group III (amifostine) between 1.21 and 1.60 (1.40±0.13) (p=0.00), showing a decrease of 43% in the Group of Amifostine. A lipid peroxidation marker measurement ranged, in group I between 0.28 and 0.31 (0.30±0.01), in group II between 0.427 and 0.497 (0.466±0.024), and in group III (amifostine) between 0.343 and 0.357 (0.36±0.005) (p<0.00), showing a decrease of 38% after Amifostine administration. Conclusions: Occlusion of aorta below left subclavian artery causes spinal cause ischemia without the interference of collateral perfusion. Modified use of hydrο-ethidine is a useful assay for the detection of superoxide radicals. By direct and indirect methods of measuring the oxidative stress of spinal cord after ischemia/reperfusion, it is suggested that intra-aortic Amifostine infusion significantly attenuated the spinal cord oxidative injury.

Le syndrome d'ischémie-reperfusion (IR), inhérent à la transplantation rénale, est caractérisé par un infiltrat leucocytaire important et des lésions tissulaires graves dont les signaux initiateurs restent à ce jour peu décrits. Postulant que la libération d'alarmines par les cellules en nécrose est décisive dans ce processus, l'objectif principal du présent travail a été d'étudier la contribution de l'alarmine IL-33 dans la genèse des lésions tissulaires dans un modèle murin d'IR rénale. Nos résultats montrent que l'IL-33 est rapidement libérée du rein après IR comme protéine circulante, dès une heure de reperfusion. Les souris IL-33gt/gt, déficientes en IL-33, sont moins sensibles aux lésions induites par l’IR, comme l'attestent le maintien de la fonction rénale et des lésions histologiques atténuées avec un recrutement de polynucléaires neutrophiles (PNN) diminué par rapport aux souris contrôles. Ceci est associé à la perte du recrutement de cellules iNKT productrices d'IFN-γ/IL-17A. Parallèlement, les souris Jα18KO, déficientes en cellules iNKT et protégées contre les lésions d'IR, possèdent également des niveaux élevés d'IL-33 circulante. Nous proposons donc que l'IL-33 endogène contribue aux lésions d'IR en favorisant le recrutement de cellules iNKT, conduisant ainsi à un recrutement amplifié de PNN au niveau du rein lésé. Notre étude, en identifiant l'alarmine IL-33 comme un médiateur précoce de la réponse immunitaire innée induite par l'IR rénale, mettant en jeu les cellules iNKT, contribue à la compréhension des mécanismes impliqués dans la genèse des lésions associées à la greffe rénale et permet de proposer de nouvelles stratégies thérapeutiques. / Ischemia-reperfusion (IR) injury in renal transplantation is characterized by leukocyte infiltration and tissue damage. However, the signals that initiate these events remain poorly understood. Assuming that alarmin release by necrotic cells during IRI is critical, the main objective of the present study was to investigate the role of alarmin IL-33 in kidney injury using a mouse model of renal IR. We observed release of IL-33 shortly after kidney IR concomitantly with an increase in plasma levels of IL-33 within one hour of reperfusion. IL-33 deficient mice (IL-33gt/gt) exhibited reduced renal IR-induced injury, as attested by function preservation, reduced histological change and attenuation of neutrophil recruitment compared to control mice. This was associated with the loss of IFN-γ/IL-17A-producing iNKT cell recruitment. In the meantime, iNKT cell-deficient (Jα18KO) mice, also protected against IRI, have increased levels of circulating IL-33.These findings lead us to propose that endogenous IL-33 contributes to kidney IRI by promoting iNKT cell recruitment and cytokine production, thereby promoting amplified neutrophil recruitment to the injured kidney. The present study identifies the nuclear alarmin interleukin (IL)-33 as an important and early mediator of innate immune response, involving iNKT cells, following experimental kidney ischemia-reperfusion in mice. Our findings contribute to a better understanding of IR-induced injury and may lead to new therapeutic insights into renal-induced injury associated with renal transplantation.

Thesis submitted in fulfilment of the requirements for the degree of Master of Technology: Biomedical Technology In the Faculty of Health and Wellness Sciences At the Cape Peninsula University of Technology Supervisors: Prof. Adriaan J Esterhuyse Dr Dirk J Bester Bellville January 2014 / Background: Oxidative stress and chronic inflammation contributes significantly to the pathogenesis of several ischaemic heart diseases, including atherosclerotic plaque rupture and myocardial infarction. It is widely demonstrated that ischaemia, followed by reperfusion, results in alterations of the mitochondrial and endothelial function through uncontrolled cascades of events characterized by free radical release and inflammation. Recent experimental evidence shows that modulation of inflammatory and antioxidant signaling mediators may determine the host outcome following myocardial ischaemia-reperfusion injury. Investigations from the past decade indicate that food supplements may play an important role in the prevention and management of chronic inflammatory diseases. Garcinia kola seeds are flavonoid rich nut from a tropical flowering, non-timber plant of the Guttiferae family. This plant is highly valued in several African cultures for its use in herbal medicine. Recently, the majority of experimental research has linked phytochemicals found in Garcinia kola nut, to its proposed beneficial effects in treatment and management of oxidative stress related-chronic diseases. Research performed in our laboratory demonstrated that kolaviron, a prominent Garcinia kola flavonoid extract, reduces myocardial apoptosis during ischaemia-reperfusion injury. Therefore, the aim of our current study was to determine the effects of Garcinia kola supplementation on cardiac inflammatory and antioxidant signaling pathways during ischaemia-reperfusion using a Wistar rat heart model. Materials and Methods: Male wistar rats were randomly divided into two groups: a control group receiving 2ml/kg corn oil and the experimental group receiving 100mg/kg Garcinia kola dissolved in corn oil, daily for 4 weeks. After the feeding period, blood samples were collected and lymphocytic DNA damage was analyzed using the alkaline comet assay. Furthermore, rat hearts were isolated and perfused with Krebs-Henseleit buffer on a working heart perfusion apparatus to measure myocardial functional parameters. Myocardial functional recovery was measured after 15 minutes global ischaemia followed by 25 minutes reperfusion. Hearts were freeze clamped at three different time points for myocardial cytokine concentration determinations using multiplex electrochemilunescent immunoassay. Nuclear factor kappa beta (NF- kβ), p38 mitogen activated protein kinases (p38 MAPK), protein kinase B/Akt (PKB/Akt), nitro-tyrosine, inducible nitric oxide (iNOS), cyclooxygenase-2 (COX-2), poly (adenosine-di-phosphate) ribose polymerase-1 (PARP-1) and caspase-3 expression and their phosphorylated forms (where applicable) were analyzed using the Western blot technique. Results: Dietary Garcinia kola supplementation significantly improved functional recovery when compared to the control group as reflected by the improved aortic output recovery (68.47 ± 6.16% versus 44.96 ± 7.00%; p<0.05). Our biochemical results supports the hypothesis that, dietary Garcinia kola supplementation modulates different cardiac proteins in terms of expression and activation at different time points when compared to the control group. We show that, before induction of ischaemia, Garcinia kola supplementation attenuates expression of inflammatory mediators and pro-apoptotic proteins when compared to the control group. The improved functional recovery was associated with a prompt inflammatory response, activation of PKB/Akt and attenuation of protein nitrosylation after 10 minutes of reperfusion. Modulation of NF-kβ and the p38 MAPK family proteins expression could have also played a significant role in myocardial functional recovery. Conclusion: We have shown that a 4 week period of dietary Garcinia kola supplementation at 100mg/kg daily improves cardiac functional recovery following ischaemia-reperfusion injury. We propose that dietary Garcinia kola supplementation protects cardiac myocytes from ischaemia-reperfusion induced oxidative stress through the induction of a prompt inflammatory response and controlled expression and/or activation of the, NF-kβ, PKB/Akt and p38 MAPK protein signaling pathways PARP-1 and caspase. Finally, we demonstrated that dietary Garcinia kola supplementation did not induce rat lymphocytic DNA damage when compared to the control group.

Thesis submitted in fulfilment of the requirement for the degree Doctor of Technologiae (Biomedical Technology) in the Faculty of Health and Wellness Sciences at the Cape Peninsula University of Technology Supervisor: Prof J van Rooyen Co-supervisor: Prof JL Marnewick Bellville October 2013 / Cardiovascular disease (CVD) is without a doubt one of the most challenging health issues of our time and accounts for the highest number of deaths in both developed and developing countries. Despite the huge strides that have been achieved in the diagnosis and therapeutic intervention of CVD, the disease burden still remains enormous. Therefore, this calls for novel and innovative interventions to curb the surge of CVD. The use of plant based food with bioactive phytochemicals,has a great potential to reduce the incidence of CVD, specifically in resource-strained countries. Red palm oil (RPO) and the indigenous herbal tea, rooibos have previously been shown to exhibit potential cardioprotective effects. Their health promoting properties have largely been attributed to their antioxidant and anti-inflammatory activities and emerging evidence also showed that they have the potential to modulate cell signalling events. Substancial scientific evidence proposes oxidative stress and inflammation to play an important role in the pathogenesis of cardiovascular disease. Hence, natural plant extracts such as RPO and rooibos could be recommended as adjuvants to clinical therapy to reduce the morbidity and mortality associated with CVD. This thesis reports on three studies investigating the cardiovascular protective effects that chronic feeding of either RPO, rooibos or their combination have on 1) antioxidant enzymes and the NO-cGMP pathway in myocardial tissue of spontaneous hypertensive rats, 2) the modulation of systemic and myocardial inflammation and 3) the myocardial ischaemic/reperfusion tolerance in a rat model of lypopolysaccharide induced inflammation. The aim of the first study was to investigate the effect of RPO on cardiac function in sponteneously hypertensive rats. The role of the nitric oxide cyclic-guanosine monophosphate(NO-cGMP) pathway, (as determined by the nitric oxide (NOS) activity) and the antioxidant defence system (selected antioxidant enzymes) were also investigated. Cardiac function was monitored at stabilization and reperfusion using the Langendorff perfusion system. Antioxidant enzymes were determined from left ventricular tissue, while total NOS activity was determined in the aorta and left ventricular tissue. The results show that RPO offered cardiac protection as evidenced by improved left ventricular developed pressure (LVDevP), maximum velocity of pressure rise (+dp/dt) max and fall (-dp/dt) max during reperfusion in sponteneously hypertensive rats (SHR) compared to their control counterparts. Improved function in SHR was associated with increased myocardial superoxide dismutase 2 (SOD2) protein expression compared to the normotensive rats. There was differential modulation of the NOS activity by RPO, an increase in NOS activity was observed in the aorta while a reduction in the activity of NOS was observed in the left ventricular tissue of both RPO supplemented normotensive and hypertensive rats compared to their respective control groups. These results argue a role for elevated NO production in the aorta for endothelial function maintenance. Increased SOD2 protein might lead to reduced oxidative stress. Thus, NO-cGMP pathway and antioxidant defense systems synergistically acted to restore cardiovascular function in SHR. The aim of the second study was to investigate the effect of RPO and rooibos supplementation on the modulation of systemic and myocardial inflammation in a rat model. As RPO and rooibos contain different types of antioxidants which reside and exert their biological effects in different cellular compartments, the combination of these two natural food compounds has the potential to enhance the spectrum of available dietary antioxidants in different cellular compartments, which could result in a better protection against certain pathological conditions such as inflammation. The Langendorff system and the lypopolysaccharide (LPS)-induced inflammatory model were used to determine if RPO and rooibos could protect against the negative effect of LPS-induced inflammation on baseline cardiac function. Both inflammation and dietary supplementation did not have any effect on baseline cardiac functional parameters. Our results show that administration of LPS resulted in elevated plasma levels of IL-1β in supplemented and non-supplemented rats indicating that an inflammatory response was triggered in the LPS-treated rats. However, this increase in IL-1β was counteracted by concurrent elevation of plasma IL-10 in LPS-induced rats consuming either rooibos or RPO alone. Furthermore the combination of RPO and rooibos enhanced myocardial IL-10 levels in LPS-induced rats. This data shows a difference in response to LPS injection between the myocardium and the systemic circulation. The results indicate that the combination of these two natural food substances exhibit potential anti-inflammatory properties which could be beneficial in clinically relevant conditions where inflammation plays a role. Having shown that dietary intervention with RPO and rooibos had the potential to modulate the inflammatory response in the model of inflammation at basal conditions, we then proceeded to the third study to specifically establish if dietary RPO when supplemented alone will improve functional recovery and reduce infarct size in LPS-treated hearts. The Langendorff perfusion system was employed for determination of cardiac function and infarct size. The roles of NFkB, p38 MAPK and the myocardial antioxidant defence systems were investigated as potential mechanisms of protection. LPS-treatment caused significant increases in myocardial IL-1 β indicating that inflammation was induced. However, the levels of myocardial IL-10 was reduced in LPS-treated hearts compared to the non-treated hearts. Intervention with dietary RPO resulted in improved functional recovery and reduced infarct size, in both healthy hearts and in the LPS-treatment group. The RPO-induced cardio-protection was associated with increases in myocardial protein expression of the antioxidant enzymes, SOD1, SOD2, GPX1 as well as increased p38 phosphorylation during reperfusion. LPS treatment increased myocardial protein expression of NFkB p65 which was reversed by RPO supplementation. Reduction of myocardial NFkB protein expression, increased p38 phosphorylation and elevated mitochondrial antioxidant (SOD2 and GPX1) as well as cytosolic enzymes (SOD 1) are proposed as potential mechanisms underlying the RPO-induced cardio-protection in this model. Based on these study results, for the first time, having included vasculature aspects in the cardio-protective effects of RPO we have shown that the NO-cGMP pathway and antioxidant defense systems may act synergistically to restore cardiovascular function in spontaneously hypertensive rats. Results from the second study also provide the first scientific evidence that RPO in combination with rooibos (a flavonoid rich endemic herbal tea) could have potential anti-inflammatory activities at systemic as well as myocardial level, which may be beneficial in clinically relevant conditions where inflammation plays a role. From the third study it can be concluded that dietary RPO improved myocardial tolerance to ischaemia-reperfusion injury in a model of inflammation.

(English) Perfluorocarbons (PFC) are hydrocarbons in which some or all of the hydrogen atoms are replaced with fluorine. PFC have a very high capacity for dissolving oxygen. They are chemically and biologically inert. The most successful clinical application of PFC is the "two-layer method" for pancreas preservation before islet isolation. The two-layer organ preservation method (TLM) is based on oxygenated perfluorocarbon overlaid with University of Wisconsin (UW) solution. In experiment it has been successfully used for heart and intestine transplantation. We tested whether this technique would prevent tissue damage and improve results of kidney, pancreas and islets of Langerhans transplantation with prolonged ischemia time in an experimental model of syngenic rats. In kidney and islets of Langerhanse transplantation model we used TLM preservation method. In pancreas transplantation model we used perfluorohexyloctane (PFH) as a new generation of less lipophilic PFC. 1. Kidneys were stored for 24 hours either in UW solution (n = 16), with TLM (n = 16) or transplanted immediately (control group, n = 12). In half of the animals, survival was observed and in the other animals grafts were procured for semiquantitative histological scoring and TUNEL apoptosis assessment 24 h after transplantation....

INTRODUÇÃO: A lesão medular isquêmica continua sendo uma complicação devastadora das intervenções cirúrgicas na aorta torácica descendente e aorta toracoabdominal. Relatos das diferenças de desfechos clínicos neurológicos entre os gêneros após lesões cerebrais isquêmicas e traumáticas têm levantado o interesse nas influências hormonais, bem como gerado outros estudos buscando a comprovação dos efeitos neuroprotetores do estradiol e da progesterona. Nossa hipótese foi a de que a administração aguda de 17beta-estradiol ou de progesterona seria capaz de prevenir ou atenuar a lesão medular isquêmica causada pela oclusão transitória da aorta torácica descendente proximal. OBJETIVO: Analisar os efeitos na medula espinhal da administração aguda de 17?-estradiol ou de progesterona em modelo experimental de isquemia-reperfusão medular por oclusão transitória da aorta torácica descente proximal de ratos machos. MÉTODOS: Ratos machos, da linhagem wistar, foram divididos aleatoriamente em 3 grupos para a administração de 280ug/Kg de 17beta-estradiol (n=12) ou de 4mg/Kg de progesterona (n=8) ou do veículo de infusão (grupo controle) (n=12), 30 minutos antes da oclusão transitória da aorta torácica descendente por 12 minutos. A confirmação da oclusão efetiva aórtica deu-se por meio da monitorização contínua da pressão arterial média distal com o uso de cateter colocado na artéria caudal dos animais (mantida em 10mmHg). A oclusão da aorta torácica descendente deu-se por meio do posicionamento de um cateter de Fogarty no. 2, passado no sentido caudal, via dissecção da artéria carótida comum esquerda do animal. A função locomotora dos animais foi avaliada no 1o, 3o, 5o, 7o, e 14o dia pós-operatório. No 14o dia pós-operatório, os animais, após anestesia profunda, foram sacrificados e tiveram suas medulas espinhais retiradas para análise histológica e imunohistoquímica. RESULTADOS: Houve comprometimento significativo da função locomotora inicialmente nos 3 grupos de estudo, com recuperação parcial da mesma ao longo do período de observação, não havendo diferença entre os grupos durante o período de observação. A análise histológica da substância cinzenta evidenciou escassos neurônios viáveis e importante vacuolização celular nos 3 grupos de estudo no 14o dia. A análise imunohistoquímica da substância cinzenta medular com anticorpos anti-Bcl2 e anti-anexina V foi similar nos 3 grupos. Houve marcação positiva de necrose celular com o iodeto de propídio, sendo a mesma semelhante nos 3 grupos estudados. CONCLUSÃO: A administração aguda de estradiol ou de progesterona, 30 minutos antes da oclusão transitória da aorta descendente proximal de ratos machos não foi capaz de prevenir ou atenuar a lesão medular isquêmica, até o 14o dia de observação, do ponto vista funcional ou histológico / BACKGROUND: Spinal cord ischemic injury remains a dreadful complication following thoracic and thoracoabdominal aortic interventions. Reports on gender-related neurological outcomes after ischemic and traumatic brain injuries have raised interest in hormonal influences, and have generated studies into neuroprotective effects of estrogen and progesterone. We hypothesized that the acute pre-operative administration of estradiol or of progesterone would prevent or attenuate spinal cord ischemic injury induced by transitory occlusion of the proximal descending thoracic aorta. OBJECTIVE: Evaluate the spinal cord effects of the acute administration of 17beta-estradiol or of progesterone in a spinal cord ischemia-reperfusion model. METHODS: Male rats were divided to receive 280ug/Kg of 17beta-estradiol (n=12) or 4mg/Kg of progesterone (n=8) or vehicle (control group) (n=12) 30 minutes before transitory occlusion of the proximal descending thoracic aorta, mean distal arterial blood pressure was maintained at 10mmHg during 12 minutes. Hind limb motor function was assessed at 1, 3, 5, 7 and 14 days after reperfusion. At the 14th day, a segment of the thoracolumbar spinal cord was harvested and prepared to histological and imunohistochemical analyses. RESULTS: There was an important hind limb motor function impairment initially in the 3 study groups, with partial improvement along time, but no difference was detected between groups during de observational period. Gray matter analysis showed scarce viable neurons and a marked cellular vacuolation in all three groups, but the number of viable neurons per section areas was not different between study groups at day 14th. Immunostaining of the spinal cord gray matter with antibodies anti-Bcl2 and anti-annexin V was similar among the 3 study groups. There was positive staining for the necrotic marker propidium iodide, with all groups presenting a similar staining pattern. CONCLUSION: We found that a single-dose administration of estradiol or of progesterone, 30 minutes before transitory occlusion of the proximal descending thoracic aorta of male rats, was not able to prevent spinal cord ischemic injury through analysis of functional and histological outcomes at 14 days of observation

A maior parte dos casos de doença renal policística autossômica dominante (DRPAD) é causada por mutações no gene PKD1 (Polycystic Kidney Disease 1). O insulto por isquemia/reperfusão (IR) constitui-se em uma causa freqüente de lesão renal aguda, incluindo a população de pacientes com DRPAD, mas a relação entre policistina-1 e IR é essencialmente desconhecida. Uma vez que a policistina-1 modula proliferação, diferenciação celular e apoptose em sistemas de cultura de células, sua menor atividade biológica na DRPAD poderia favorecer um maior grau de lesão renal. Utilizamos uma linhagem endogâmica de camundongos 129Sv com uma mutação nula em Pkd1 para testar esta hipótese. Camundongos Pkd1+/- não apresentam cistos renais até 12 semanas de vida, constituindo-se em um modelo puro de haploinsuficiência para este gene. Um insulto IR bilateral de 32 min foi induzido em camundongos machos de 10-12 semanas de idade, heterozigotos e selvagens, por meio do clampeamento reversível de ambos os pedículos renais. Os animais foram analisados 48 h, 7 dias (d) e 14 d após o insulto. Camundongos Pkd1+/- apresentaram FENa, FEK e SCr mais elevadas que animais Pkd1+/+ 48 h após IR. O dano cortical residual foi mais severo em heterozigotos que em selvagens em todos os tempos avaliados. A marcação para PCNA também foi mais alta em camundongos Pkd1+/- que Pkd1+/+ 48 h e 7 d pós-IR, enquanto a taxa de apoptose e a infiltração inflamatória intersticial foram maiores em heterozigotos que em selvagens nos seguimentos de 48 h, 7 d e 14 d pós-IR. A expressão renal de p21 foi menor nos camundongos Pkd1+/- que Pkd1+/+ no tempo de 48 h pós-insulto, tanto no nível transcricional como traducional. Análises adicionais realizadas 6 semanas após o insulto IR revelaram dilatação tubular e formação de microcistos nos camundongos haploinsuficientes para Pkd1, assim como fibrose renal aumentada nesses animais, comparados aos camundongos selvagens. Por fim, um insulto de 35 min de isquemia/reperfusão acompanhou-se de uma mortalidade precoce substancialmente maior nos animais Pkd1+/-. Esses achados sugerem que isquemia/reperfusão induza uma lesão mais severa em rins de camundongos haploinsuficientes para Pkd1, um processo aparentemente dependente de uma deficiência relativa da atividade de p21, assim como dilatação tubular e formação de microcistos. Em conjunto, nossos resultados sugerem que a heterozigose para mutação nula em Pkd1 em camundongo (e talvez em humanos) esteja associada a um risco aumentado para lesão renal por isquemia/reperfusão e a um pior impacto desse insulto sobre a progressão da doença renal. / The majority of autosomal dominant polycystic kidney disease (ADPKD) cases are caused by mutations in the PKD1 gene. Ischemia/reperfusion is a frequent cause of acute kidney injury, including the ADPKD patient population, but the relationship between polycystin-1 and ischemia/reperfusion is essentially unknown. Since polycystin-1 modulates cell proliferation, cell differentiation and apoptosis in cell culture systems, its lower biological activity in ADPKD might amplify the degree of renal injury. Using an inbred 129Sv mouse line with a Pkd1-null mutation, 32-min renal ischemia/reperfusion was induced in 10-12 week-old male non-cystic mice, heterozygotes and wild types. The animals were analyzed at 48h, 7 days (d) and 14d after the insult. Pkd1+/- mice showed higher FENa, FEK and SCr than Pkd1+/+ animals at 48h of follow-up. The residual cortical damage was more severe in heterozygotes than wild types at all evaluated time points. The PCNA staining was also higher in Pkd1+/- than Pkd1+/+ mice at 48h and 7d, while cell apoptotic rates and the interstitial inflammatory infiltration were higher in heterozygotes than wild types at 48h, 7d and 14d postischemia/ reperfusion. The expression of p21 was lower in Pkd1+/- than Pkd1+/+ kidneys at 48h, both at the transcriptional and translational levels. Additional analyses performed 6 weeks after the insult showed tubular dilatation and microcyst formation in the haploinsufficient mice, and increased renal fibrosis in these animals compared to wild types. Thirty-fivemin ischemia/reperfusion, at last, was accompanied by a substantially higher early mortality of Pkd1+/- animals. These findings suggest that ischemia/reperfusion induces a more severe injury in kidneys of Pkd1- haploinsufficient mice, a process that is apparently dependent on a relative deficiency of p21 activity, as well as tubular dilatation and microcyst formation. Altogether, our results suggest that mouse Pkd1-null heterozygosity (and maybe human) is associated with a higher risk for renal ischemia/reperfusion injury and with a worse impact of this insult upon renal disease progression.

INTRODUÇÃO: A lesão de isquemia-reperfusão continua sendo considerada a maior causa de mortalidade relacionada ao transplante de pulmão e sua gravidade é influenciada por diversos fatores, dentre eles, a preservação pulmonar. OBJETIVO: Comparar duas soluções de preservação pulmonar, Perfadex® e Celsior®, quanto a capacidade de preservação de tecido pulmonar isquêmico. MÉTODOS: Sessenta pulmões de ratos preservados com Perfadex®, Celsior® ou solução salina após períodos de isquemia hipotérmica de 6 ou 12 horas, foram reperfundidos com sangue homólogo em modelo experimental ex-vivo durante 60 minutos consecutivos. A cada 10 minutos os dados de gasometria, hematócrito, mecânica ventilatória, hemodinâmica e peso do bloco cardiopulmonar foram registrados. Ao final da reperfusão o pulmão esquerdo foi pesado e acondicionado por 48h a 70oC para obtenção da razão peso úmido/peso seco, bem como amostras de tecido pulmonar foram retiradas para histopatologia, microscopia eletrônica e TUNEL. A análise estatística incluiu a comparação entre as soluções e os tempos de isquemia, utilizando ANOVA e Kruskall-Wallis. O nível de significância foi de 5%. RESULTADOS: A comparação entre as complacências de pulmões preservados com Celsior® e Perfadex® nos tempos de isquemia de 6 e 12 horas não apresentou significância estatística (p=0,161 e p=0,316, respectivamente). Os pulmões submetidos a 6 horas de isquemia apresentaram complacência pulmonar superior aos de 12 horas (Perfadex® p=0,02; Celsior® p=0,019; Salina p=0,016). Os valores de pressão arterial pulmonar foram semelhantes entre as três soluções nos dois tempos de isquemia, bem como na comparação entre os tempos de 6 e 12 horas, independente da solução. A Capacidade Relativa de Oxigenação não demonstrou diferença estatística entre as três soluções, independentemente do tempo de isquemia. Na comparação entre os dois tempos de isquemia, o desempenho da oxigenação foi significativamente pior nos pulmões preservados com salina por 12 horas (p=0,001). A razão peso úmido/peso seco não apresentou diferença estatística significante entre as três soluções nos dois tempos de isquemia, porém na comparação entre os tempos de isquemia, os pulmões preservados com Perfadex® apresentaram uma relação peso úmido/peso seco maior no tempo de isquemia mais longo (p=0,001). À microscopia óptica, pulmões preservados com salina apresentaram mais edema que os demais, independentemente do tempo de isquemia. A avaliação da apoptose celular através do método de TUNEL não mostrou diferença estatisticamente significativa na comparação entre os grupos. CONCLUSÃO: Os pulmões preservados com Perfadex® e Celsior® apresentaram desempenho similar em relação às trocas gasosas e parâmetros hemodinâmicos e de mecânica ventilatória. Os pulmões preservados com Perfadex® por 12 horas apresentaram mais edema. Os achados histopatológicos não diferiram entre os grupos estudados / INTRODUCTION: Ischemia-reperfusion injury remaisn the leading cause of mortality related to lung transplantation. Its severity is influenced by several factors including lung preservation. OBJECTIVE: To compare two lung preservation solutions, Perfadex® and Celsior® and its ability to preserve ischemic lung tissue. METHODS: Sixty rat lungs were preserved with Perfadex®, Celsior® or saline after a cold ischemic period of 6 or 12 hours and were then reperfused with homologous blood in an ex vivo experimental model for 60 consecutive minutes. At 10-minute intervals during reperfusion of the heart-lung blocks, data were collected for blood gases, hematocrit, mechanical ventilation, hemodynamic and the heart-lung block weight was recorded. At the end of reperfusion, the left lung was weighed and packaged kept at 70oC for 48h to obtain the wet-to-dry weight ratio. Lung tissue samples were processed for histology, electron microscopy and TUNEL. Statistical analysis included a comparison of the solutions and ischemic times, using ANOVA and Kruskal-Wallis. The significance level was set at 5%. RESULTS: The comparison between the compliance of lungs preserved with Celsior® and Perfadex® in ischemic times of 6 and 12 hours was not statistically significant (p=0.161 and p=0.316, respectively). The lungs subjected to 6 hours of ischemia showed higher lung compliance compared to 12 hours (p=0.02 Perfadex®; Celsior® p=0.019; saline p=0.016). The pulmonary artery pressure values were similar between the three solutions in two stages of ischemia and comparing the times of 6 and 12 hours, regardless of the solution. The Relative Oxygenation Capacity showed no significant difference between the three solutions tested, regardless of the ischemic time. The comparison between the two ischemic times showed that oxygenation capacity was significantly worse in lungs preserved with saline for 12 hours (p=0.001). The wet-to-dry weight ratio showed no statistically significant difference between the three solutions in both ischemic times. However, when ischemic times were compared, Perfadex® showed greater wet-to-dry weight ratio in lungs submitted to 12 hours of ischemia (p=0.001). Light microscopy showed that lungs preserved with saline had more edema than the others, regardless of the ischemic time. Assessment of apoptosis by the TUNEL assay showed no statistically significant difference in the comparison between the groups. CONCLUSIONS: The lungs preserved with Celsior® and Perfadex® performed evenly in regards to gas exchange, hemodynamics and ventilatory mechanics. The lungs preserved with Perfadex® for 12 hours were more edematous. Histopathology findings did not differ between the groups

Introdução: A lesão de isquemia/reperfusão do fígado é caracterizada pelo agravamento da lesão isquêmica hepatocelular quando o órgão é revascularizado, podendo originar, nos casos mais graves, uma reação inflamatória sistêmica com lesão de órgãos à distância. O controle desse fenômeno é importante no transplante de fígado, nas cirurgias de ressecção hepática e no choque hemorrágico. A administração de soluções salinas hipertônicas têm se mostrado eficaz no tratamento do choque hemorrágico, pois melhora as alterações hemodinâmicas e, possivelmente, apresenta uma ação antiinflamatória. Neste trabalho foram avaliados os efeitos locais e sistêmicos da administração da solução salina hipertônica na lesão de isquemia/reperfusão hepática em ratos. Métodos: Enquanto 14 ratos Wistar machos, dos 56 utilizados no estudo, compuseram o grupo controle, grupo C; os demais, que foram submetidos à uma hora de isquemia hepática e 4 horas de reperfusão, compuseram outros grupos de 14 animais: o grupo ST, animais que não receberam tratamento; o grupo SSF, animais que receberam 34 mL/kg de NaCl 0,9%, por via endovenosa, 15 minutos antes da reperfusão; o grupo SSH, animais que receberam 4 mL/kg de NaCl 7,5%, 15 minutos antes da reperfusão. Após 4 horas de reperfusão, os materiais foram coletados para análise. Foram realizadas as dosagens das transaminases AST e ALT, a avaliação das funções oxidativas e fosforilativas mitocondriais, a dosagem das interleucinas IL-6 e IL-10, as análises teciduais pulmonares e a análise histológica do fígado isquêmico e não isquêmico. Resultados: Quando comparado aos grupos ST e SSF, o grupo SSH apresentou elevação dos níveis de AST e ALT significantemente menores; preservação da função mitocondrial tanto dos lobos isquêmicos, como dos não isquêmicos, significantemente melhor; elevação dos níveis de IL-6 e IL-10 sem significância estatística; aumento da permeabilidade vascular pulmonar significantemente menor; e elevação da atividade da mieloperoxidase pulmonar sem significância estatística. Em relação à análise histológica da lesão de isquemia/reperfusão hepática, o escore da lesão do grupo SSH foi significantemente menor que o da lesão do grupo C; entretanto, quando comparados os três grupos submetidos à isquemia hepática ST, SSH e SSF não se observaram diferenças significantes estatisticamente. Conclusão: A administração da solução salina hipertônica a 7,5% na isquemia/reperfusão hepática normotérmica melhorou as lesões hepáticas locais e as lesões à distância, principalmente pulmonares / Introduction: During liver ischemia, the drop in mitochondrial energy production leads to cellular damage, which is aggravated during restoration of blood supply. Besides local hepatic injury, the ischemia/reperfusion process can trigger a systemic inflammatory syndrome producing remote organ damage. To control these alterations in clinical conditions like liver transplantation, liver resections and hypovolemic shock, is crucial to achieve proper patient management. Aim: To evaluate the effect of the sodium chloride hypertonic solution on prevention of local and systemic injury during partial liver ischemia/reperfusion. Methods: Animals underwent partial warm liver ischemia/reperfusion. Fity six Wistar male rats were randomly allocated into four groups. Fourteen animals were submitted to sham operation and allocated to C group; 42, submitted to one hour of liver ischemia followed by 4 hours of reperfusion, were allocated in three additional groups: ST group, 14 animals that received no treatment; SSF group, 14 animals that received NaCl 0.9%, 34 mL/kg, intravenously; SSH group, animals that received NaCl 7.5%, 4 mL/kg, intravenously. Blood and tissue samples were collected four hours after reperfusion, when animals were killed. Blood samples were collected to determinate AST, ALT, IL-6 and IL-10 levels. Liver and pulmonary tissues were assembled for liver histology and for liver mitochondrial phosphorylation, pulmonary vascular permeability and myeloperoxidase analyzes. Results: Hypertonic saline solution showed beneficial effects in the treatment of liver ischemia/reperfusion injury. SSH group presented elevation of AST and ALT plasma levels significantly lower than ST and SSF groups. A significant reduction on mitochondrial dysfunction was observed in SSH group compared with ST and SSH groups. Elevation in serum IL-6 and IL-10 was similar among ST, SSF and SSH groups. Pulmonary vascular permeability was significantly lower in group SSH compared with ST and SSF groups. No differences in myeloperoxidase activity were observed among these three groups. Histological score for liver ischemia/reperfusion injury was significantly lower in SSH group compared to ST group, however no differences were observed between SSH and SSF groups. Conclusion: Administration of hypertonic saline solution in an experimental rat model of liver ischemia-reperfusion ameliorated local and systemic injuries

In dieser Dissertation wurden am Ferkelmodell (8-15 kg, drei Gruppen: „Kontrolle“ n=7, „Herz-Lungen-Maschine (HLM)“ n=10, „(-)-Epigallocatechin-3-Gallat (EGCG)“ n=6, die Kontrollgruppe wurde thorakotomiert, die HLM- und die EGCG-Gruppe wurden thorakotomiert und für 90 Minuten an eine HLM angeschlossen, die EGCG-Gruppe erhielt vor und nach der HLM-Zeit EGCG) drei Fragestellungen behandelt: Erstens wurde untersucht, ob die Verwendung einer HLM während eines kardiochirurgischen Eingriffes unter hypothermen Bedingungen mit nicht-pulsatilem Blutfluss und Kardioplegie die Niere schädigte. Dafür wurden Paraffinschnitte der Niere aus der Kontroll- und der HLM-Gruppe mit Hämatoxylin-Eosin (HE) angefärbt und unterschiedliche Strukturen betrachtet, wobei histopathologische Veränderungen in der HLM-Gruppe auffielen. Paraklinisch fanden sich erhöhte nierenspezifische Blutwerte (Serumkreatinin und -harnstoff) in der HLM-Gruppe. Diese Ergebnisse waren hinweisend für eine funktionell relevante Schädigung der Niere durch die HLM. Unterstützend kam ein Absinken des Gesamteiweißes im Serum der HLM-Gruppe hinzu, was auf eine generelle Schädigung des Organismus durch die HLM hindeutete. Zweitens wurde betrachtet, ob die gesetzten Schäden die Merkmale eines Ischämie-Reperfusionsschadens aufwiesen. Hierzu wurden Paraffinschnitte der Niere aus der Kontroll- und der HLM-Gruppe immunhistochemisch (Hypoxie-induzierter-Faktor-1-alpha-Tyramide- Signal-Amplification (HIF-1-alpha-TSA)-, Nitrotyrosin-3-Amino-9-Ethylcarbazol (Nitrotyrosin-AEC)- und Apoptose-induzierender-Faktor-Tyramide-Signal-Amplification (AIF-TSA)-Färbung) angefärbt. Dabei zeigte sich, dass sich die HLM-Gruppe in einer hypoxischen Situation befand (HIF-1-alpha Akkumulation in den Zellkernen), nitrosativem Stress ausgesetzt war (Nitrotyrosin in den Tubuli) und dass sie teilweise so stark geschädigt wurde, dass Apoptose induziert wurde (AIF in Zellkernen) – alle drei Färbungsergebnisse waren hinweisend für einen ischämischen Zustand, in dem sich die HLM-Gruppe befunden hat. Auch die Ergebnisse der durchgeführten renalen Reversed Phase High Performance Liquid Chromatography (RP-HPLC) deuteten auf ebendies hin. Unterstützend wirkten die Ergebnisse des arteriellen Laktats – die HLM-Gruppe zeigte eine Hyperlaktämie – und die Tatsache, dass einige der histologischen Merkmale für eine frühe Schockniere (welche ischämischen Ursprungs sein kann) in der HLM-Gruppe gefunden wurden. Dies alles zeigte, dass der HLM-assoziierte Nierenschaden vorrangig die Natur eines Ischämie-Reperfusionsschadens aufwies. Drittens wurde untersucht, ob EGCG diese HLM-assoziierte Schädigung abmildern konnte. Dafür wurden bei der EGCG-Gruppe alle oben genannten Untersuchungen durchgeführt. Die Ergebnisse zeigten, dass EGCG in der Dosierung 10 mg/kg eine renoprotektive Wirkung gegen die HLM-assoziierten Schäden hatte, und diese abmildern bzw. ihnen entgegenwirken konnte. Diese Ergebnisse sind für die pädiatrische Kardiochirurgie interessant, welche zum Beispiel bei der Korrektur angeborener Herzdefekte auf die Verwendung der HLM angewiesen ist. Komplikationen wie eine Nierenschädigung post operationem sind nicht selten und verkomplizieren den Verlauf. Die vorliegende Dissertation zeigt das renoprotektive Potential des in grünem Tee vorkommenden Katechins EGCG im Umfeld eines kardiochirurgischen Eingriffes mit Verwendung einer HLM. Die Wirksamkeit dieser Substanz ist wahrscheinlich darin begründet, dass sie mehr als ein Antioxidans ist. Neben seiner Radikalfänger- und Stickstoffmonoxidscavenger-Fähigkeiten ist EGCG außerdem antiapoptotisch wirksam. Derzeit wird die Kardiochirurgie mit Verwendung einer HLM in der Veterinärmedizin nur in wenigen Zentren angewendet. Es besteht für die Zukunft jedoch die Hoffnung, dass gerade für Kleintierbesitzer, die ihre Tiere als Familienmitglied betrachten, und auch für zoologische Einrichtungen bei der Diagnose eines Herzfehlers die Kardiochirurgie mit Verwendung einer HLM als Therapiemöglichkeit eine interessante und realistische Alternative zur bislang angewandten palliativen medikamentösen Therapie darstellen kann. / In this dissertation a piglet model (8-15 kg, three groups: “control” n=7, “extracorporeal circulation (EC)” n=10, “EGCG” n=6, the control-group was thoracotomized, the EC- and the EGCG-group were thoracotomized and underwent cardiopulmonary bypass (CPB) for 90 minutes, and the EGCG-group received EGCG before and after the CPB) is presented. Three questions were raised and answered: Firstly, it was investigated if the use of a CPB during cardiac surgery with hypothermia, non-pulsatile blood flow and cardioplegia caused damage to the kidney. In order to answer this question, paraffin slices of the kidney of the control- and the EC-group were stained with hematoxylin-eosin (HE), and different structures were evaluated – this staining showed histopathological changes in the EC-group. Paraclinical, the EC-group showed elevated kidney-specific blood parameters (serumcreatinine and -urea). These findings indicated a functionally relevant impairment of the kidney caused by the CPB. Supporting this, the EC-group also showed a decline of the total amount of proteins in the serum, which was suggestive of a generalized injury of the body by the CPB. Secondly, it was investigated whether the injury of the kidney might have been caused by an ischemia/reperfusion injury. Therefore, paraffin slices of the kidney of the control- and the EC-group were immunhistochemically stained (hypoxia-induced-factor-1-alpha-tyramidesignal-amplification (HIF-1-alpha-TSA)-, nitrotyrosine-3-amino-9-ethylcarbazole (nitrotyrosine-AEC)- and apoptosis-inducing-factor-tyramide-signal-amplification (AIF-TSA)-staining). These stainings revealed, that the EC-group had suffered from a hypoxemic situation (accumulation of HIF-1-alpha in the nuclei), from nitrosative stress (presence of nitrotyrosine in the tubuli), and that the kidney was partly damaged to the point of an induction of apoptosis (presence of AIF in the nuclei) – all three of these findings indicated, that the kidneys of the EC-group were put into an ischemic situation. The findings of the renal reversed phase high performance liquid chromatography (RP-HPLC) indicated the same thing. This was also supported by the blood parameter of lactate – the EC-group showed a hyperlactemia – and by some histological findings in the EC-group, which were characteristical for an early shock-kidney (which may be caused by ischemia). Taken together, these findings showed that the CPB-associated kidney injury was primarily caused by an ischemia/reperfusion injury. Thirdly, it was investigated, whether EGCG might attenuate the CPB-associated kidney injury. For that purpose, all of the investigation methods mentioned above were carried out with the samples of the EGCG-group. The findings showed that EGCG (dose: 10 mg/kg) had a protective effect on the kidney, protecting it against the damage caused by the CPB, and was able to partly attenuate this damage and partly even fully counteract it. These findings are of interest for pediatric cardiac surgery, which for example for the correction of innate heart defects depends on the use of CPB. Complications – like acute renal injury post operationem – occur frequently and complicate the recovery. This dissertation demonstrates the renoprotective potential of the natural compound EGCG in the setting of cardiac surgery with the use of CPB. The reason for the effectiveness of EGCG in this situation probably is that EGCG is more than an antioxidant. EGCG not only works as a radical- and nitric-oxide-scavenger, but also is antiapoptotic. In veterinary medicine cardiac surgery with CPB is done by few centers only. However for the future there is hope that people – especially pet owners who view their companion animals as family members, and zoos – become more and more willing to and interested in having an animal diagnosed with a heart defect treated with cardiac surgery including the use of an CPB, instead of – like its usually done nowadays – only giving palliative medication to the animal.