Magister Scientiae (Medical Bioscience) - MSc(MBS) / Cardiovascular disease is a major health problem and remains the number one cause of death worldwide. For centuries, medicinal plants have been used in different cultures as medicines for the treatment and control of various diseases. Olea africana, also known as the wild olive, is amongst the herbal plants used by people to treat many ailments.Recently, scientific studies on the hypotensive, vasodilatory and antidysarrhythmic effects of O. africana have been reported. Triterpenoids isolated from the O. africana leaves, for example, have antioxidant properties. The aqueous extract from the leaves of O. africana also have angiotensin-converting enzyme (ACE) inhibitory effects. ACE inhibitors and antioxidants protect the heart against ischemic-reperfusion injury. The serine / threonine protein kinase B (PKB) also known as Akt is activated downstream of phosphoinositide 3- (PI-3) kinase (PI-3-Kinase) and is involved in cardioprotection against ischemia-reperfusion injury. Angiotensin II (AII) decreases the intrinsic PI-3-kinase activity. In this study, we hypothesized that ACE inhibitors increase PI-3-kinase activity and thus activates PKB. The aims of this study were: 1) to determine whether treatment with the crude aqueous extract of leaves of O. africana protect the heart against ischemic-reperfusion injury and 2) if so, to determine whether the protection is mediated via the PKB signaling mechanism. Hearts isolated from male Wistar rats were perfused with different concentrations of the plant extract. In one set of experiments, male Wistar rats were treated with the plant extract (1000 mg/kg/day) for 5 weeks for the evaluation of cardiac function before and after ischemia. At the end of the experiments, hearts were freeze-clamped and kept for PKB / Akt determination. In another set of experiments, we determined the effect of O. africana extract (1000 mg/kg/day) or captopril (50 mg/kg/day) on infarct size. Rats fed jelly served as controls for captopril. In a subset of experiments, hearts were frozen immediately after treatment with O. africana extract (1000 mg/kg/day) or captopril (50mg/kg/day) and PKB were determined.Perfusion with the plant extract significantly decreased coronary flow (p<0.05). The heart function was decreased as evidenced by observed decreases in the force of contraction and heart rate, although these were not measured. Chronic treatment with the crude aqueous plant extract had no effect on cardiac function before ischemia, functional recovery (% left ventricular developed pressure and % rate pressure product) and PKB /Akt phosphorylation (p>0.05). Both the aqueous extract of O. africana leaves and captopril had no effect on infarct size compared to the control group (p>0.05). Captopril,however, improved the recovery of the left ventricular developed pressure. Non-perfused hearts isolated from rats treated with O. africana extract and captopril did not show any response to both captopril and the O. africana extract treatment as measured by PKB /Akt phosphorylation. The results of the present study suggest that the crude aqueous extract of O. africana is not cardioprotective against ischemia-reperfusion injury in this system of the isolated perfused rat heart.

Ischemia and reperfusion are natural steps during kidney transplantation, and IRI is considered one of the most important nonspecific factors affecting allograft dysfunction. Transplanted organs experience several episodes of ischemia, in which cold ischemia occurs during allograft storage in preservation solutions. Even though cold ischemia has been studied extensively, all of the studies have been carried out in vitro and ex vivo models. There is no in vivo model available to examine renal IRI induced solely by cold ischemia. In the present study, we developed an in vivo mouse model to study renal IRI induced exclusively by cold ischemia through clamping the renal pedicle for 1 to 5 hours. During the ischemic phase, blood was flushed from the kidney with cold saline through a small opening on the renal vein. The kidney was kept cold in a kidney cup with circulating cooled saline, while the body temperature was maintained at 37℃ during the experiment. The level of kidney injury was evaluated by plasma creatinine, KIM-1, NAGL, GFR, and histology. Plasma creatinine was significantly increased from 0.15±0.04 mg/dl in the sham group to 1.14±0.21 and 2.65±0.14 mg/dl in 4 and 5-hours ischemia groups at 24 hours after cold IRI. The plasma creatinine in mice with ischemic time <3 hours demonstrated no significant increase compared with sham mice. Changes in KIM-1, NAGL, GFR and histology were similar to plasma creatinine. 65 In summary, we developed and characterized a novel in vivo IRI-induced AKI mouse model exclusively produced by cold ischemia.

The miR-17- 92 cluster is necessary for cell proliferation and development of the cardiovascular system. Deletion of this cluster leads to death in neonatal mice. The role of this cluster still needs to be defined following ischemia and reperfusion. Methods and Results: Adult male mice were injected with Tamoxifen- was to induce inducible cardiac-specific miR-17- 92-deficient (miR-17- 92-def: MCM:TG:miR-17- 92 flox/flox ) and wild type (WT: MCM:NTG:miR-17-92 flox/flox ) mice were subjected to 30 minutes of myocardial ischemia via left anterior descending coronary artery ligation followed by reperfusion for 24 hours. Post I/R survival (48%) and ejection fraction were reduced, while myocardial infarct size enlarged in miR-17- 92-deficient mice as compared to WT mice (survival: 71%). Necrosis (trypan blue staining) and apoptosis (TUNEL assay) both were higher in adult cardiomyocytes isolated from miR-17- 92-deficient mice as compared to WT mice subjected to simulated ischemia/reoxygenation with a concomitant reduction of mitochondrial membrane potential (JC1 staining). The electron transport chain was compromised through dysregulation of glutamate+malate as complex I substrate and malate dehydrogenase in the hearts of miR-17- 92-deficient mice compared to WT. After 4 hours of reperfusion, PTEN expression, a downstream target of miR-20A, increased, while phosphorylation of AKT reduced in the hearts of miR-17- 92-deficient mice in comparison to WT. The induced knockdown of cardiac miR-17- 92 increases myocardial I/R injury by ceasing suppression of PTEN, leading to decreased concentrations of AKT and mitochondrial dysfunction. These results suggest that innovative therapeutic strategies can focus on genetic upregulation of miR-17- 92 in patients with coronary artery disease.

Ischemic injury in the heart is followed by an increase in SNS activity and the higher this activity, the poorer patient outcomes. An index of SNS activity in models of ischemia can be achieved by measuring NE, β-AR, and perhaps indirectly PDE4 to give an intracellular aspect on SNS signaling. A 20 minute ischemia-reperfusion was induced in a rat model with physiological measurements at 2-5 weeks post IR. At 3 weeks post IR, rats displayed increased PDE4 expression, decreased β 1-AR expression, increased plasma NE, decreased tissue NE storage, increased Doppler E/A ratio and unchanged LV ejection fraction. PET analysis with FDG revealed no infarct at 2 weeks, while analysis with [13N]NH3 displayed no resting flow defect but revealed trends in flow reserve impairment as early as 2.5 weeks with recovery at 5 weeks post-surgery. Applications of this model could be non-invasive imaging of PDE4 with (R)-[11C]Rolipram PET at early time points for development towards prognostic and therapy guidance in humans.

Studies on the survival of central nervous system of hypoxia-tolerant species under challenges of reduced energy availability have characterised adaptive mechanisms of brain at the cell and tissue level that lead to reduced excitability and protection. However, evidence of hypoxic suppression of retinal activity in these species has not been followed up with mechanistic studies. Microspectrofluorometric monitoring of intracellular free Ca2+ concentration ([Ca2+]i) is useful for identifying cellular mechanisms that may lead to adaptive strategies, as unregulated increases in [Ca2+]i cause toxicity. Horizontal cells (HCs) are second order retinal neurons that receive tonic excitatory input from photoreceptors, and possess voltage-gated Ca2+ conductances and other channels that can facilitate toxic increases in [Ca2+]i under conditions of reduced energy availability (modeled as oxygen-glucose deprivation, OGD). It was demonstrated that isolated HCs of the hypoxia-tolerant goldfish display spontaneous, transient [Ca2+]i activity (SA) which decreased in amplitude and area under the curve following OGD or glucose removal (20 min) without recovery. SA was shown to be dependent on extracellular Ca2+ influx through voltage-gated Ca2+ channels, though mechanisms of SA generation and regulation has yet to be determined. Additionally, glutamate-elicited peak increases in [Ca2+]i were reduced after 20 min of OGD. The removal of O2 during OGD insult seemed to be protective as an increase in baseline [Ca2+]i was seen during and following glucose removal under normoxic conditions. The mechanisms mediating these decreases in spontaneous and elicited [Ca2+]i activity are currently unknown, though candidate pathways are discussed. This thesis contributes a hint towards how HCs may tolerate conditions of low energy availability, which may also inform investigations on their role in situ during these insults.

Implications for the endocannabinoid system in global cerebral ischemia has not been clearly defined. Ischemia produces an excitotoxic environment that is severely damaging to neurons, causing degradation of cell membrane and ultimately cell death. Contradicting research suggests both the benefits and adverse effects of endocannabinoids on neurological injury. Due to the excitotoxic nature of ischemic injury, and the mechanisms at play with endocannabinoid agonists, such as increased transmission of dopamine and glutamate, it is suspected that endocannabinoid antagonists, such as AM251, may a provide cell protection.40 male Wistar rats were separated into 4 groups (n=10/group). The first group of rats were administered AM251 (2 mg/kg, i.p) 30 minutes prior to global cerebral ischemia (four vessel occlusion), while the second group were given AM251, 30 minutes prior to sham surgery. Finally the last two groups were given a vehicle control instead of AM251 and given either ischemia or the sham surgery. Behavioural testing, open field test and elevated plus maze, took place after a five day recovery period following ischemia. Immunohistochemical analyses were performed using to mark tyrosine hydroxylase (TH) and dopamine receptor 1(DRD1) to compare dopamine function amongst groups. Cell survival was also evaluated using thionin staining. Ischemia induced significant reduction in dopamine within the mesolimbic circuit, including: ventral tegmental area, nucleus accumbens, CA3 & CA1 of the hippocampus, and basolateral amygdala. These reductions in dopamine transmission by global ischemia were partially or fully reversed when AM251 was given beforehand. Furthermore, cell survival was increased in the CA1 from treatment of AM251. Behavioural results show similar results that AM251 reversed emotional irregularities associated with ischemia insult. The endocannabinoid antagonist AM251 improves deficits in dopamine function, prevents cell death and regulates emotionality when given prior global cerebral ischemia.

Dans le cadre du développement d’une stratégie clinique de diminution de l’invasivité de l’acte de Chirurgie cardiaque, axée à la fois sur la réduction du traumatisme de la paroi thoracique, de l’ischémie myocardique peropératoire, et de l’agressivité de la CEC, une étude prospective randomisée a été réalisée pour comparer l’impact sur le métabolisme myocardique en peropératoire de l’utilisation de la cardioplégie cristalloïde Custodiol® versus la solution de cardioplégie de St Thomas au cours de la chirurgie coronarienne. L’objectif de cette étude est de comparer les modifications periopératoire de la concentration dans l’espace interstitiel de lactate, pyruvate, glycérol et glucose dans les deux groupes de cardioplégie et ceci depuis le déclampage jusqu'à 24h en post-opératoire. Matériels et méthodes. Vingt-huit patients ont pu être inclus dans l’étude. Le monitorage a été pratiqué avec la technique de microdialyse (cathéter CMA 70, Analyseur CMA 600, CMA Microdialysis,Sweden), avec une mesure toutes les 10 minutes pendant le temps du clampage et la première heure post déclampage, puis toutes les heures, des concentrations interstitielles des métabolites. Les concentrations plasmatiques des troponines à la sortie du bloc opératoire et à H +12 ont été également évaluées dans les deux groupes. Résultats : Des 28 patients inclus et randomisés, 22 ont pu bénéficier d’un monitorage complet (12 pour le groupe Custodiol® et 10 pour le groupe St Thomas). Six ont été exclus pour des raisons techniques (1 arrachement, 3 plicatures, 1 chute du cathéter et 1 dysfonctionnement de l’analyseur). Une analyse comparative entre les patients inclus et exclus de l’étude ne montre pas de différences significatives pour les facteurs de risque cardiovasculaires, la FEVG, l’âge, le genre. Les valeurs moyennes des concentrations +/- écart type de lactate, pyruvate, glucose et glycérol au déclampage (T0,) sont les suivants : groupe Custodiol® : 2.77+/-1.81 mmol l-1 ; 13.74+/-20.87 μmol l-1 ; 0.46+/-0.84 mmol l-1 ; 196.99+/-122.22 mmol l-1 ; groupe St Thomas : 0.89+/-0.64 mmol l-1 ; 6.49+/-9.10 μmol l-1 ; 0.19+/-0.18 mmol l-1 ; 73.17+/-72.11 mmol l-1. Les temps de CEC et de clampage ont été respectivement dans le groupe Custodiol® de : 94.2+/-14 min et 59.8+/-15 min, et, dans le groupe St Thomas de 82.6+/-15.9 min et 55.8+/-16.29 et min (p=ns). Les concentrations post-opératoires en troponine T (sortie de bloc et H+12) ont été respectivement de 2.8+/-1.8 et 7.4+/-5.3 μmol/L pour le groupe Custodiol® et de 3.3+/-4.0 et 5.0+/-3.6 μmol/L (p=ns) pour le groupe Saint Thomas. Aucun évènement clinique ou électrocardiographique n’a eu lieu en post opératoire dans les deux groupes. Conclusion. Le monitorage de l’état redox myocardique interstitiel a été possible dans les deux groupes de façon sûre et efficace et a permis de déceler des variations des concentrations en métabolites dans les deux groupes en l’absence d’évènements cliniques. Les résultats de ces analyses retrouvent, au déclampage, des concentrations significativement plus hautes de lactate et glycérol dans le groupe Custodiol®. Ces différences s’effacent rapidement pendant la phase de reperfusion avec une tendance (non significative) à une concentration de lactates plus basse dans le groupe de patients du groupe Custodiol®. Des études multicentriques ciblées sur des clampages longs supérieurs à 90 min nous semblent nécessaires pour définir si une différence à la fois métabolique et clinique peut exister entre les différentes solutions de protection cardiaque / In our unit, the challenge is to develop a clinical strategy of reduction of the invasiveness of the “On pump procedure” of cardiac surgery: that means a reduction of the chest wall trauma, of the cross clamping perioperative myocardial ischemia, and of the invasiveness of the extra-corporeal circulation. In this background, we organized a randomized perspective study in order to assess the impact of the perioperative myocardial redox metabolism during the on pump coronary surgery protected with Custodiol® versus St Thomas crystalloid cardioplegias. Objectives: To assess the presence and the severity of the perioperative myocardial ischemia in the Custodiol® versus St Thomas group, defined as the interstitial myocardial concentrations of lactate, pyruvate, glycerol and glucose, at the time of the removal of the aortic clamp. Materials and methods : Twenty height patients could be enrolled in the study and were randomized in the Custodiol® and in the St-Thomas group. Monitoring was assessed with the technique of the cardiac microdialysis (CMA 70 probe, CMA 600 analyzer, CMA Microdialysis, Sweden), by dosing every ten minutes during the aortic cross clamping period and every hour out of the operating room, up to 24 hours, the interstitial myocardial concentrations of Lactate, pyruvate, glycerol and glucose. The Lactate/pyruvate ratio and glucose/lactate ratios and 12 hours post-operative troponin plasmatic concentrations were also assessed. Statistical analysis comparing the Custodiol® versus ST Thomas group were performed via a t-test. Results: Out of the 28 enrolled patients, twenty-two (12 of the Custodiol® group and 10 of the St Thomas group) could be successfully monitored with the microdialysis technique. Six were excluded because of technical reasons (one intempestive ablation, 3 iatrogenic plication of the tube, 1 felled out of the table, one due to a dysfunction of the analyzer). The comparative analysis between included and excluded patients did not prove any statistical result in terms of cardiovascular risk factors, EF, age and gender. At declamping time (T0), mean values of concentrations of lactate, pyruvate, glucose and glycerol were the following: Custodiol® group: 2.77+-1.81 mmol l-1;13.74+-20.87 μmol l-1;0.46+-0. mmol l-1;196.99+-122.22 mmol l-1 ; St Thomas : 0.89+-0.64 mmol l-1 ; 6.49+-9.10 μmol l-1; 0.19+-0.18 mmol l-1; 73.17+-72.11 mmol l-1. Cross clamping and CPB times were respectively 94.2+/-14 et 59.8+/-15 min (Custodiol®), and 82.6+/-15.9 et 55.8+/-16.29 et minutes (St-Thomas) (p=ns) . Post operative plasmatic levels of Troponin (arrival in the ICU and 12 H+12) were respectively de 2.8+/-1.8 and 7.4+/-5.3 (pour le groupe Custodiol®) et de 3.3+/-4.0 et 5.0+/-3.6 μmol/L (Saint Thomas) (ns). Conclusion: Monitoring of the interstitial myocardial redox state was safely possible in both groups and allowed to assess metabolic different findings in the two cardioprotective methods that were not enhanced by perioperative clinical ischemic events. Microdialysis assessed, at the time of aorta declamping, significantly higher concentrations of lactate and Glycerol in the Custodiol® group. That difference regressed during the reperfusion phase with a tendency for a lower lactate level in the Custodiol® group. Multicentric studies focused on cross clamping time longer than 90 minutes seem necessary to enhance metabolic interstitial and clinical superiority between cardioprotective solutions

O conteúdo de zinco (Zn) reativo cerebral é importante para o equilíbrio da sinaptofisiologia neural. A prova disto é que um aumento nos seus níveis, após evento hipóxico-isquêmico, resulta em neurotoxicidade, o que tem estimulado o tratamento desta disfunção cerebral com quelantes de Zn, tal como o dietilditiocarabamato (DEDTC). No caso do DEDTC, o uso deste composto sobre esta disfunção deve ser analisado com cuidado, pois ele também apresenta muitos efeitos colaterais sobre o sistema nervoso central. Desta forma, para atender este propósito, é necessário antes obter uma concentração de DEDTC com menores efeitos colaterais. Por esta razão, no presente trabalho, nós decidimos usar um modelo vertebrado mais simples, tal como o peixe-zebra, o qual permitiria a triagem, em larga escala, dos efeitos de DEDTC sobre o Zn reativo. Entretanto, jamais foi mostrada a presença de Zn reativo no cérebro de peixe-zebra. Com isto, através de marcações histológicas, nós conseguimos mostrar pela primeira vez a distribuição citoarquitectônica de Zn reativo em neurônios glutamatérgicos, bem como o número desses neurônios contendo Zn no cérebro de peixe-zebra. Isto nos permitiu avaliar o efeito de diferentes concentrações de DEDTC sobre o conteúdo de Zn cerebral do peixe-zebra, o qual foi intensamente quelado por elevadas quantidades do composto, induzindo comportamentos tipo-crise. Neste mesmo estudo nós obtemos também uma concentração de DEDTC com poucos efeitos colaterais que poderia exercer neuroproteção sobre o aumento de Zn reativo induzido pela hipóxia-isquemia. Assim, após a padronização de um modelo de hipóxia em peixe-zebra, que demonstra danos relacionados à isquemia, nós testamos se essa concentração de DEDTC poderia ser neuroprotetora sobre este modelo. Contudo, DEDTC apresentou efeitos pró-oxidantes, embora ele tenha atenuado o elevado conteúdo de Zn reativo induzido pela hipóxia. Portanto, mesmo que o DEDTC tenha falhado, este modelo, agora, está apto para a triagem de outros fármacos com potencial ação sobre o alterado conteúdo de Zn reativo que ocorre em eventos hipóxicos-isquêmicos. / The content of brain reactive zinc (Zn) is important for the synaptophysiology in the central nervous system (CNS). This is evidenced in hypoxic-ischemic events, when an increase in their levels results in neurotoxicity. Consequently, this has stimulated the treatment of cerebral ischemia with Zn chelators, such as diethyldithiocarbamate (DEDTC). In the case of DEDTC, the use of this compound in this dysfunction should be examined carefully, because it also has many side effects on the (CNS). Thus, to meet this, it is necessary first to obtain a concentration of DEDTC with negligible side effects. Here, we decided to use a simpler vertebrate model, such as zebrafish, which would allow large-scale screening of DEDTC effects on reactive Zn. However, the presence of reactive Zn has never been shown in zebrafish brain. Then, using histological markers, we were able to show for the first time the cytoarchitectonic distribution of reactive Zn in glutamatergic neurons as well as the number of these neurons containing Zn in the zebrafish brain. This allowed us to evaluate the effect of different DEDTC concentrations on the brain content of Zn in zebrafish. As a result, high levels of the compound did strongly chelate the metal, inducing seizure-like behaviors. In this study we also obtained a DEDTC concentration with few side effects that could exert neuroprotection on the increased reactive Zn induced by hypoxia-ischemia. Then, after the standardization of an ischemic-sensitive model of hypoxia in zebrafish, we tested if this DEDTC concentration could be neuroprotective on this model. Nevertheless, DEDTC showed pro-oxidant effects, though it had mitigated the elevated content of reactive Zn induced hypoxia. Therefore, despite the DEDTC have failed as neuroprotective drug, this model enables the screening of other chemical agents with potential action on the increased content of reactive Zn that occurs in hypoxic-ischemic events.

Orientador: Ivan Felizardo Contrera Toro / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-07T09:33:33Z (GMT). No. of bitstreams: 1 Mussi_RicardoKalaf_D.pdf: 7974637 bytes, checksum: 40bf555137ac51738964d4e90337bddf (MD5) Previous issue date: 2006 / Resumo: Objetivos: A proposta deste estudo foi avaliar os efeitos do treinamento físico na resposta inflamatória (permeabilidade vascular e influxo neutrofílico) após isquemia-reperfusão pulmonar (IRP) em ratos. Considerando que o treinamento físico tem propriedades protetoras em determinadas doenças cardiovasculares, hipotetizamos que animais submetidos a treinamento com exercício físico podem ficar protegidos contra a lesão causada pela isquemia e reperfusão pulmonar. Métodos: ratos Wistar machos (pesando entre 350g e 450g) foram divididos em três grupos: sham operados (SHAM); animais sedentários submetidos à isquemia-reperfusão (IR/SD) e animais treinados submetidos à isquemia-reperfusão (IR/TR). Animais treinados foram submetidos a um programa de oito semanas de treinamento em corrida, com sessões de 60min por dia, cinco dias por semana. Os ratos foram anestesiados e ventilados artificialmente. Após a toracotomia, a artéria pulmonar esquerda, o brônquio principal esquerdo e a veia pulmonar esquerda foram clampeados, mantendo o pulmão inflado parcialmente por 90 minutos. O clampe foi então removido e os pulmões voltaram a ventilar, e foram reperfundidos por duas horas. A circulação pulmonar foi perfundida através do tronco arterial pulmonar com solução salina. O extravasamento de proteína plasmática foi medido através do acúmulo de albumina sérica marcada com I. em ambos os pulmões, enquanto que a infiltração neutrofílica foi medida através da atividade de mieloperoxidase (MPO). 125Resultados: Após oito semanas de treinamento em corrida, o peso corpóreo foi significativamente menor em 15% no grupo treinado comparado com o do grupo sedentário (p<0,05). A contagem de células mononucleares e polimorfonucleares em ratos treinados não se altera significativamente quando comparada à dos ratos sedentários. A IRP causou acentuado aumento (p< 0,05) na permeabilidade vascular e na atividade da mieloperoxidase do pulmão direito dos animais sedentários. O precondicionamento físico dos animais atenuou significativamente (p< 0,05) a elevação da permeabilidade vascular, sem afetar o influxo neutrofílico. O protocolo de isquemia e reperfusão também resultou em níveis elevados de IL-1 ß e TNF-a no grupo sedentário, e foram revertidos pelo programa de treinamento em corrida. Os níveis séricos de IL-10 não foram alterados nos três grupos. Conclusões: Os dados deste estudo mostram que o precondicionamento físico atenua a elevação da permeabilidade vascular, mas não o influxo neutrofílico induzido pela isquemia-reperfusão em ratos. Estes dados sugerem que o treinamento físico age no componente vascular mas não no celular da resposta inflamatória, deste modelo, protegendo contra a lesão pulmonar causada pela isquemia-reperfusão / Abstract: Objective: The purpose of this study was to evaluate the effects of exercise training on the inflammatory response (vascular permeability and neutrophil influx) after lung ischemia-reperfusion in rats. Considering that exercise training have protective properties in certain cardiovascular disease, we hypothesized that animals undergoing exercise physical training would be protective against direct lung ischemia-reperfusion injury. Methods: Male Wistar rats (weighing 350g and 450g) were divided into tree groups: Sham operated (SHAM); sedentary animals submitted to ischemia-reperfusion (IR/SD) and trained animals submitted to ischemia-reperfusion (IR/TR). Trained animals underwent 8 weeks of run training program in sessions of 60 min/day, 5 days/week. Rats were anesthetized and artificially ventilated. After thoracotomy, the left pulmonary artery, bronchus and pulmonary vein were occluded, maintaining the lung in a partially inflated state for 90min. The clamp was then removed, and lungs were allowed to ventilate and reperfuse for 2h. The pulmonary circulation was flushed through the main pulmonary artery with salin solution. Protein plasma extravasation was measured as accumulation of human serum albumin labeled with I in the botht lung tissues, whereas neutrophil infiltration was measured as the myeloperoxidase (MPO) activity.125 Results: After 8 weeks of run training, the body weight was significantly 15% lower in trained groups compared with the sedentary group (p<0.05). Counts of mononuclear and polymorphonuclear cells in trained rats did not significantly changed compared with sedentary rats. The lung ischemia-reperfusion caused a marked increase (p<0.05) in vascular permeability and MPO activity in the right lung of sedentary animals. The physical preconditioning of the animals significantly attenuated (p<0.05) the increased vascular permeability, without affecting the neutrophil influx. The ischemia-reperfusion protocols also resulted in increased levels of serum IL-1ß and TNF-a in sedentary group, and that was reversed by run training program. The serum levels IL-10 were not altered in all groups. Conclusions: Our data show that physical preconditioning attenuate the increased vascular permeability but not the neutrophil influx induced by rat lung ischemia-reperfusion. This data suggests that physical training act in the vascular (but not in the cellular) component of the inflammatory response to protect against the injury caused by lung ischemia-reperfusion / Doutorado / Cirurgia / Doutor em Cirurgia

Orientador: Joyce Maria Annichino-Bizzacchi / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-07T17:15:50Z (GMT). No. of bitstreams: 1 Paula_ErichViniciusde_D.pdf: 3311580 bytes, checksum: 0e2725586b5aed078f6a78de1edd1d14 (MD5) Previous issue date: 2006 / Resumo: A doença arterial oclusiva (DAO) é a principal causa de morbidade e mortalidade em países industrializados. Com o aumento da expectativa de vida da população mundial a DAO tornou-se um problema de saúde publica também em países em desenvolvimento. O tratamento de uma das formas graves de DAO, a isquemia crítica de membros inferiores (ICMI), normalmente oferece alívio temporário e muitas vezes requer a amputação do membro afetado. No Brasil, a sobrevida desses pacientes é comparável a algumas formas de câncer. Desta forma, a busca de estratégias terapêuticas alternativas à amputação é fundamental para estes pacientes. Nesse estudo, nós exploramos o papel da angiogênese terapêutica induzida por terapia gênica (TG) em modelos animais ICMI. A angiogênese terapêutica se baseia na utilização de fatores de crescimento vascular sob a forma de proteínas recombinantes ou TG. A reposição de proteína deve ser mantida por períodos prolongados e normalmente requer administração sistêmica. Isto aumenta o risco de formação de vasos em locais indesejáveis como tumores ocultos ou retina. Estudos clínicos anteriores demonstraram que a indução de angiogênese terapêutica utilizando apenas um fator pró-angiogênico é eficaz. No entanto, esses estudos sugerem que integridade destes neovasos é deficiente pela ocorrência de edema de membros inferiores ou proteinúria após a monoterapia angiogênica.Neste trabalho avaliamos a eficácia da TG combinada usando vetores não virais com os genes de dois fatores de crescimento: FGF-2 e PDGF-BB, em comparação com a transferência gênica isolada do VEGF-A. A escolha destes dois fatores deveu-se ao fato de eles atuarem em etapas distintas e complementares do processo de formação vascular: formação e estabilização dos novos vasos, respectivamente. Para este fim utilizamos o modelo de isquemia da pata posterior em ratos, e um novo método não invasivo, o 99mTc-sestamibi para avaliação da neovascularização. A expressão combinada do FGF-2 e PDGF-BB induziu a formação de neovasos com a mesma intensidade e com a mesma relevância funcional que o uso do VEGF-A isolado. No entanto, a estratégia combinada se mostrou mais atraente por induzir a formação de vasos mais íntegros, e necessitar de doses individuais 50% menores de cada gene terapêutico. Em conjunto esses dados demonstram que a TG combinada com FGF-2/PDGF-BB é eficaz e potencialmente mais segura para o tratamento da ICMI. Com o objetivo de definir fatores que afetam a resposta angiogênica pós-natal, nós investigamos o papel da trombina durante a indução de neovasos. Animais receberam diversos inibidores da coagulação imediatamente ou após 72 horas da indução de isquemia periférica. Nós documentamos que a inibição da coagulação sangüínea na fase aguda da isquemia diminui a resposta angiogênica, mas esse efeito não é observado quando a anticoagulação é iniciada após 72 horas. A ativação dos receptores celulares da trombina (e outras proteases) pode oferecer um novo alvo para otimizar a resposta angiogênica pós- natal. Em conjunto, os resultados obtidos formam a base para estudos futuros utilizando a TG combinada com FGF-2/PDGF-BB como uma alternativa potencialmente eficaz e segura paro tratamento da ICMI / Abstract: Arterial occlusive disease (AOD) is the main cause of mortality and morbidity in industrialized countries. Global improvements in life expectancy have turned it into a public health problem in developing countries as well. The treatment of one of the severe presentations of DAO, which is critical limb ischemia (CLI) normally offers temporary relief to symptoms, often requiring major amputations of the affected limb. In Brazil, the prognosis of CLI is similar to that of some forms of cancer. Therefore, new therapeutic strategies alternative to amputations are necessary for these patients. In the present work we evaluated the role of therapeutic angiogenesis induced by gene therapy (GT) in animal models of CLI. Therapeutic angiogenesis consists in the use of vascular growth factors by means of recombinant proteins or GT. When using protein formulations, therapy must be maintained for prolonged periods and normally involves systemic administration. These requirements increase the risk of inducing the formation of neovessels in undesired places such as occult malignant lesions or retina. Previous studies have demonstrated that therapeutic angiogenesis using a single pro-angiogenic factor is effective. However, these studies suggest that the new formed vessels are leaky, as evidenced by the occurrence of lower limb edema and proteinuria after pro-angiogenic monotherapy. In the present work we evaluated efficacy of a combined GT strategy using non-viral vectors expressing two vascular growth factors: FGF-2 and PDGF-BB, and compared this strategy to the gene transfer of VEGF-A alone. The choice of these two growth factors was due to their complementary roles in the process of vascular development: formation and stabilization of new vessels. In order to do so we used a hindlimb ischemia model in rats, and a new non-invasive method based on 99mTc-sestabimi scintigrapy, to evaluate the neovascularization response. Dual gene transfer of FGF-2 and PDGF-BB using non-viral vectors was able to induce a similar revascularization response as VEGF-A alone, both in terms of anatomical and functional parameters. In regard to safety, the dual gene transfer strategy was more attractive because it was able to induce more stable capillaries, and required a 50% lower individual dose of each therapeutic gene. Taken together our data demonstrate that this dual gene transfer strategy is an effective safer strategy for the treatment of CLI by therapeutic angiogenesis. In order to investigate factors that might affect the revascularization post-natal response, we further investigated the role of thrombin during the induction of neovascularization. Animals were treated with different coagulation inhibitors immediately after or 72 hours after the creatin of hindlimb ischemia in rats. We documented that the inhibition of blood coagulation in the acute phase of ischemia impairs the angiogenic response. In contrast, this effect was not observed when anticoagulation was initiated after 72 hours of ischemia. The activation of cellular receptors of thrombin and other proteases might therefore offer an new target to optimize post-natal revascularization. Based on our results, future studies using combined GT with FGF-2 and PDGF-BB are warranted in order to translate these findings into a new and safer strategy for the treatment of CLI / Doutorado / Medicina Experimental / Doutor em Fisiopatologia