Estudos recentes sobre disfunção ventricular demonstram o potencial terapêutico da modulação da matriz extracelular. Isso se dá pela influência que a referida matriz tem sobre a função sistólica e a diastólica do coração. Outros estudos demonstram a influência do digital sobre os sistemas neurohormonais desbalanceados no cenário de disfunção ventricular levantando uma questão acerca do potencial do digital como modulador da deposição do colágeno intersticial e perivascular miocárdico. Sabendo-se da importância prognóstica que a concentração do colágeno no referido cenário tem, a literatura apresenta uma lacuna de conhecimento. Objetivo: Avaliar o papel do digital no remodelamento miocárdico em um modelo experimental. Material e Métodos: 60 ratos Wistar foram separados em 3 grupos de 20. Um grupo controle (GC); outro grupo submetido ao modelo experimental de uninefrectomia, administração de água de beber com 1% de NaCl e de aldosterona subcutânea (GA); e outro grupo submetido ao mesmo modelo experimental, mas também recebendo digitoxina na ração de comer na dose de 100 g/Kg/dia (GAD). Resultados: A fração de volume de colágeno intersticial e perivascular mostrou-se maior no GA comparado aos outros dois grupos (GC e GAD). O índice de desempenho miocárdico mostrou diferença estatisticamente significativa entre o GA (0,49 ± 0,08) e o GC (0,32 ± 0,06) e o GAD (0,4 ± 0,13) (p=0,001). Os níveis séricos de BNP mostraram diferença estatisticamente significativa entre o GA (1,07 ± 0,32 ng/ml) e o GC (0,75 ± 0,19 ng/ml) e o GAD (0,84 ± 0,21 ng/ml) (p=0,01). Os níveis de metaloproteinases não diferiram entre os grupos. Houve uma correlação positiva entre uma maior fração de encurtamento e menores níveis séricos de BNP no GAD. Conclusões: Esses dados demonstram que a digitoxina teve efeito reduzindo a deposição de colágeno intersticial e perivascular e melhorando a função cardíaca avaliada pelo BNP e IDM nesse modelo experimental / Recent studies on myocardial dysfunction are highlighting the therapeutic potential of the myocardial extracellular matrix management. Its interesting to highlight the importance of the dynamics of the cardiac extracellular matrix, because even the systolic and diastolic functions are implicated on it. Other studies showed that digital compounds may regulates the neuroendocrin misbalance due to myocardial impairment and influencing these systems the digital compounds may regulates the interstitial collagen deposition. Objective: To evaluate the role of the digital on a myocardial fibrosis in an experimental model, examining if the digital is able to prevent the collagen deposition. Methods: The sample was divided in 20 rats from the control group (CG); 20 rats submitted to a fibrosis experimental model in which the rats are uninefrectomized, drink water with 1% NaCl during the protocol and receive aldosterone through an osmotic minipump (AG); and 20 rats submitted to the same experimental model treated with digitoxin in a daily dose of 100 g/Kg (DAG). Results: The interstitial and perivascular collagen volume fraction showed a significant difference between the AG and the other 2 groups (CG and DAG). The myocardial performance index showed a significant difference between the AG (0.49 ± 0.08) and the CG (0.32 ± 0.06) and the DAG (0.40 ± 0.13) (p=0.001). The BNP levels showed a significant difference between the AG (1.07 ± 0.32 ng/ml) and the CG (0.75 ± 0.19 ng/ml) and the DAG (0.84 ± 0.21 ng/ml) (p=0.01). The metalloproteinases levels did not differ among the groups and there was a positive correlation between the shortening fraction and the BNP levels among the GAD animals. Conclusion: These data demonstrate the digitoxin positive effect on the myocardial collagen deposition in this experimental model of interstitial fibrosis and could have a new therapeutic target previously unexplored

Metastatic and primary bone cancers are devastating diseases of paediatric and adult humans because of the morbidity associated with bone pain. Controlling bone pain from multiple metastatic sites can be difficult in end-stage cancers using conventional therapies. Bone-seeking radiopharmaceuticals have been successful in this area as radiation can be delivered with moderate selectivity to the target. Unfortunately, targeted radiotherapy using radiopharmaceuticals have been relegated to palliative therapy as myelosuppression largely limits the radiation dose to sub-therapeutic levels. Efforts to overcome this therapeutic limitation include autologous bone marrow transplants in combination with chemotherapy-radiosensitization and the development of new radiopharmaceuticals. Development work using laboratory rodent models has been complicated by dosimetric limitations because of size and inherent problems with human xenografted tumour models in rodents. To address this need we studied naturally occurring canine osteosarcoma as a translational model for human bone cancer. Central to our hypothesis was that naturally occurring canine osteosarcoma would serve as an investigational model for comparing the pharmacokinetics (biodistribution), dosimetry, toxicity, and therapeutic effect of 153Sm-EDTMP, 188Re-HEDP, 186Re-HEDP, and a novel ligand, polyethyleneiminomethyl phosphonic acid (PEI-MP). Data collected from existing radiopharmaceuticals was then compared to PEI-MP labelled with 99mTc, 153Sm, and 186Re. This innovative and unique study allowed for the evaluation of various radiopharmaceuticals in a naturally occurring animal model of bone cancer, documenting the pharmacokinetics and dosimetry of a novel radiolabelled-ligand (PEI-MP). Benefits resulting from the successful completion of the study would allow more rapid transfer of rodent preclinical data into a naturally occurring cancer model more resembling to the human diseases and would thus more likely identify problems with pharmacokinetics and toxicity before proceeding to expensive clinical trials. The expected outcomes of the study were originally formulated based on limited previous published data in dogs. For instance, no data exists describing the pharmacokinetics or toxicity of 188Re-HEDP and 186Re-HEDP in the dog. The study was conducted in two phases. The first phase deals with the evaluation of 153Sm-EDTMP, 188Re-HEDP, and 186Re-HEDP in the dog model. Phase-two was the development of a novel ligand (PEI-MP) in the dog model of osteosarcoma, which has the characteristics of an ideal ligand. Pharmacokinetic results for 153Sm-EDTMP in normal dogs (n=4) for blood were similar to published reports for dogs and human. When compared statistically to human data the majority of results were the same, lending credence to the hypothesis that dogs could serve as models for human radiopharmaceutical research. Normal dogs and the osteosarcoma dog did differ from human pharmacokinetics in the urine elimination phase (t½-â). This can most likely be explained by the tumour burden in the human research populations or due to the fact that most humans were aged and likely to have some renal disease. Certainly, the trend in dogs with osteosarcoma was to have a prolonged urine elimination phase (t½-â) compared to normal dogs which supported the hypothesis that the biodistribution and pharmacokinetics results from dogs were similar to human published data. Statistical comparisons were also made between normal dogs receiving 188Re-HEDP and 153Sm-EDTMP. The prolonged urine elimination phase (t½-â) and increased blood retention of 188Re-HEDP was most likely a reflection of prolonged bone washout and soft issue retention. This could be attributed to the differences between the antiresorptive capability of bisphosphonate ligands e.g., EDTMP (lexidronam) with a greater than 100-fold antiresorptive capability than HEDP (etidronate). Additional observational biodistribution studies using macro- and micro-autoradiography techniques were also performed in canine tissue and Sprague-Dawley rats. Results from the studies showed heterogeneous uptake within tumours in dogs. In rats, localization of 153Sm-EDTMP in red marrow areas would lead to a high radiation dose to blood producing elements. In addition, high uptake was documented at the metaphyseal growth plate confirming the likelihood of a delay or cessation of growth if 153Sm-EDTMP were used in growing children. Phase-one of the clinical trial in dogs with naturally occurring osteosarcoma identified only mild toxicity at the dosage rate of 37 MBq/kg (1 mCi/kg) for both 153Sm-EDTMP and188Re-HEDP. In addition, a pilot trial was conducted in dogs receiving 153Sm-EDTMP which also received a carboplatin infusion at the time of the radiopharmaceutical administration followed by another 3 cycles of carboplatin at 3 weekly intervals. No differences in toxicity were noted between the carboplatin group and dogs receiving only 153Sm-EDTMP. As a part of the 188Re-HEDP clinical trial, 3 dogs with osteosarcomas received weekly dose of 188Re-HEDP at 37MBq/kg for 4 weeks in which only mild toxicity was noted. Unfortunately, there was no cessation in growth of the tumours, with all dogs showing progression. The median survival time for both radiopharmaceuticals was 4 months, significantly shorter than the 10-month median survival time for amputation and chemotherapy. Interestingly six dogs that had 99mTc-MDP and 153Sm-EDTMP showed scans of tumours that had consistently lower 99mTc-MDP uptake ratios (normal bone compared to cancerous bone) compared to solely 153Sm-EDTMP. In contrast, this was not evident for uptake ratios between 188Re-HEDP and 99mTc-MDP scans. Once again, this finding highlights the differences between the antiresorptive capabilities of the bisphosphonates ligands. Interestingly, another three dogs were scanned with 99mTc-MDP , 153Sm-EDTMP, and 99mTc-PEI-MP (10-30 kDa) showed a variation in uptake between scans of the same tumours. More importantly, the uptake ratios of 99mTc-MDP and 153Sm-EDTMP scans showed wide variation with a coefficient of variance of 52% and 39% respectively. However, the range in uptake from the 99mTc-PEI-MP (10-30 kDa) scan was narrow with a coefficient of variance of only 6%. This could be attributed to more consistent uptake ratio of the unique ligand PEI-MP and its hypothesized mechanism of action: enhanced permeability and retention (EPR) in tumour vasculature. This requires further investigation with larger groups. In phase-two, the pharmacokinetic result for the novel ligand PEI-MP was initially studied labelled with 99mTc. Various molecular weights were tested in normal dogs and compared to previously published results in baboons. Results from the dog studies were found to be similar to those from the primate study. As in the primate study, molecular weight and charge played a significant role in 99mTc-PEI-MP pharmacokinetics. Increasing the size of the macromolecules and altering their charge resulted in marked changes in their pharmacokinetics and biodistribution. The PEI-MP molecular weight of 10-30 kDa and 20-30 kDa were the most promising and fulfilled the hypothesized criteria of an ideal radiopharmaceutical. In keeping with the aims of the study, the 20-30kDa polymer was considered more desirable because of its faster clearance. However, because of the limitations imposed by the percentage yield of the different molecular weights of the ligand during filtration, we decided to label the 10-30kDa molecular weight MW-fraction with 153Sm. Unexpectedly, the 153Sm-PEI-MP 10-30 kDa had a prolonged urine elimination phase (t½-â) associated with increased liver uptake when compared to 99mTc-PEI-MP10-30 kDa. To explain this, computer modelling for blood plasma (ECCLES) was done which predicted that there would be some chemical dissociation of the 153Sm from the PEI-MP polymer in blood. This is due to interaction between the radiopharmaceutical and citrate, forming 153Sm-citrate. The ECCLES model for blood plasma also predicted that the anionic MW-fraction, PEI-MP 10-30kDa, would be a poor ligand complexed to 166Ho, 212Pb, 213Pb, and 89Sr, but was expected to be effective when complexed to 186Re or 188Re, based on their close proximity to 99mTc on the periodic table. As a preliminary study 186Re was complexed to 20-30 kDa (n=2) and 30-50 kDa (n=1) MW-fractions and tested in dogs. The results were similar to 99mTc-PEI-MP 10-30 kDa. The biodistribution data and pharmacokinetic data were also used to do comparative dosimetry between radiopharmaceuticals. Not surprisingly, the dosimetry data confirmed the high red marrow dose for 153Sm-EDTMP and the increased soft-tissue dose of the radionuclides complexed to HEDP. The radiation dose to the tumour for all radiopharmaceuticals fell within the range of 26Gy to 44Gy. This is well within the range used to treat canine osteosarcoma using external beam radiotherapy. When compared to external beam radiotherapy, the probable lack of tumour response in our clinical trial relates to the heterogenous distribution of the radiopharmaceutical in the tumour and the inherent resistance of osteosarcoma cells to continuous low-dose radiation delivery (CLDR) inherent in radionuclide -particle decay. The study met the majority of outcomes with the exception of labelling PEI-MP with 153Sm. This was due to the interaction of the 153Sm-PEI-MP complex with citrate ions in blood. Rapid deterioration of the Rhenium-188 generator also led to earlier than expected curtailment of the 188Re-HEDP therapeutic trial although sufficient data was available to be used in a comparative study. / Thesis (PhD)--University of Pretoria, 2013. / Internal Medicine / unrestricted

This investigation was concerned with measuring aspects of cardiac function in conscious control, diabetic, hypertensive control, and hypertensive diabetic rats. Preliminary studies were conducted to determine catheter suitability and acute responses to atropine and angiotensin II in conscious animals. The catheter-manometer was tested using a square wave impact and was shown to accurately reproduce a left ventricular pressure pulse. Intravenous atropine caused both heart rate and left ventricular +dP/dt to rise. Intravenously administered angiotensin II caused systolic blood pressure to increase dramatically. In this case heart rate fell and +dP/dt was elevated. Hypertension was induced with deoxycorticosterone acetate (DOCA) and saline drinking water. Rats were first made diabetic with streptozotocin (60 mg/kg; i.v.). One week following this, subcutaneous DOCA (25 mg/kg) was administered twice weekly and all animals received saline drinking water. Following 2 and 5 weeks of DOCA treatment rats were catheterized and resting cardiovascular function was measured. DOCA treatment caused increased systolic and diastolic blood pressures to occur in control and diabetic rats at 2 and 5 weeks. Bradycardia was also observed in DOCA-diabetic and DOCA-control rats at 2 and 5 weeks of treatment. Two and 5 week hypertensive diabetic and control rats exhibited elevated -dP/dt and +dP/dt. The rate of contraction was shown to be proportional to the magnitude of systolic blood pressure in all treatment groups. It is concluded that diabetic rats and control rats did not differ in their response to hypertension after 5 weeks of DOCA treatment. / Medicine, Faculty of / Anesthesiology, Pharmacology and Therapeutics, Department of / Graduate

It is well established that a cardiomyopathy independent of atherosclerosis develops in both humans and animals with diabetes mellitus. The etiology of diabetic cardiomyopathy is very complex involving many different processes, one of which may be the increased fatty acid utilization, and/or the concomitant decrease in glucose utilization, by the diabetic heart. We compared control and 6-week streptozotocin(STZ)-induced diabetic isolated working rat hearts and were able to demonstrate cardiac dysfunction in the diabetic as assessed by depressed heart rate (HR), heart rate peak systolic pressure product (HR X PSP), left ventricular developed pressure (LVDP), rate of pressure rise (+dP/dt) and rate of pressure decline (-dP/dt). Paralleling depressed cardiac function in the diabetic were hyperglycemia, hyperlipidemia and decreased body weight gain as compared to age-matched controls. The addition of free fatty acids, in the form of 1.2 mM palmitate, to the isolated working heart perfusate had no effect on either control or diabetic heart function, with the exception of a depressive effect on +dP/dt of diabetic hearts and -dP/dt of control hearts. But, diabetic hearts perfused with palmitate-containing perfusate plus the glucose oxidation stimulator dichloroacetate (DCA) showed a marked improvement in function. Heart rate, HR X PSP, LVDP and +/-dP/dt were all restored to control heart values in diabetic hearts perfused in the presence of DCA. Creatine phosphate and adenosine 5'-triphosphate (ATP) levels were similar under all perfusion conditions, therefore eliminating energy stores as the limiting factor in heart function. Results indicate that DCA-induced stimulation of glucose oxidation acutely reversed diabetic cardiac function depression. Therefore, depression of glucose oxidation in the diabetic heart may be contributing to diabetic cardiomyopathy. / Pharmaceutical Sciences, Faculty of / Graduate

A miostatina é um agente regulador negativo do crescimento muscular. Na terapia de suplementação com testosterona observou-se diminuição na expressão da miostatina. Este trabalho tem como objetivo determinar a influência do esteróides anabolizantes na expressão do gene da miostatina em camundongos normais, C57BL e no modelo distrófico Largemyd. Utilizamos a técnica de PCR em tempo real, para determinarmos a expressão relativa dos genes. Os animais tratados apenas com esteróide apresentaram aumento significativo em sua massa corpórea, com melhora de desempenho nas avaliações funcionais no Largemyd. Não foram observadas diferenças significativas na expressão do genes da miostatina no músculo normal e distrófico. Concluímos que o uso do esteróide anabolizante foi benéfico para o aumento na força do modelo Largemyd, mas o aumento de massa corpórea nestes animais, como no camundongo normal, não deveu-se a inibição da expressão da miostatina. / Myostatin is a negative regulator agent of muscle growth. In the testosterone supplementation therapy we observed decreased myostatin expression. The aim of this project is to determine the influence of anabolic steroids in the expression of myostatin gene in normal C57BL mice and in the dystrophic model Largemyd. We used the Real Time PCR assay to determine the relative expression of genes. Animals treated only with steroids presented significant increase in body mass and Largemyd showed improvement in the functional evaluations. There werent significant differences in the myostatin gene expression in the normal and dystrophic muscle. We concluded that the use of anabolic steroid was benefic to the increase of the strength in the Largemyd model, but the increase of body mass in these animals, as in the normal mice, is not related to the inhibition of myostatin expression.

Estudos anteriores em nosso laboratório demonstraram que pacientes portadores de DAC apresentam diferenças no metabolismo do CL e CE de uma nanoemulsão artificial rica em colesterol (LDE), nos quais o CL apresentou maior remoção plasmática e depósito arterial. Dando continuidade a esta linha de pesquisa, neste trabalho foram avaliadas a cinética plasmática, representada pela taxa fracional de remoção (TFR), e a captação do 3H-colesterol livre (3H - CL) e 14C - colesterol esterificado (14C - CE) da LDE por segmentos arteriais e por órgãos de coelhos normais (n=17) e coelhos submetidos à indução de aterosclerose por dieta rica em colesterol (1%) (n=13). Além disso, avaliou-se a captação in vitro do 3H CL e do 14C CE da LDE por células endoteliais aórticas de coelhos. Por último, foi avaliada a influência da inibição da enzima lecitina-colesterol aciltransferase (LCAT), e indiretamente, a esterificação do CL em ratos normais (n=9) e tratados com diazepam (n=9). Em coelhos que receberam dieta normal, não houve diferença entre a remoção plasmática do 3H - CL e do 14C - CE. Em coelhos que desenvolveram hiperlipidemia e aterosclerose através de dieta rica em colesterol, o 3H - CL foi removido mais rapidamente da circulação do que o 14C - CE (p<0,05), entretanto houve maior captação de 14C - CE do que de 3H - CL no arco aórtico (p<0,05). Em ambos os grupos, os principais órgãos captadores de colesterol da LDE foram fígado, pulmão, adrenais e baço (p<0,05). Tanto a TRF quanto a captação hepática de 3H - CL e 14C - CE foram menores no grupo que recebeu a dieta rica em colesterol. Em células endoteliais aórticas de coelhos, a captação de 3H - CL foi maior que a de 14C CE independente da massa de LDE incubada (p<0,01). Em ratos, não houve diferença entre a captação das duas formas de colesterol da LDE pela aorta no grupo controle, entretanto, quando a atividade da LCAT foi diminuída pelo tratamento com diazepam, a captação arterial de 3H - CL foi maior do que a de 14C - CE (p< 0,01). A hiperlipidemia e distúrbios no processo de estabilização do colesterol, favorecem a dissociação entre o CL e o CE das lipoproteínas, e podem elevar o risco de desenvolvimento da aterosclerose, assim como agravar o processo de aterogênese. / I n previously studies, it was shown that free cholesterol (FC) and cholesterol ester (CE) of a cholesterol-rich nanoemulsion (LDE) behaves differently in patients with coronary artery disease (CAD). The FC plasma clearance and arterial deposition is greater than CE. In the present study we evaluate the plasma kinetics, estimated by the fractional clearance rate (FCR), and the tissue uptake of 3H-free cholesterol (3H FC) and of 14C cholesterol ester (14C - CE) of LDE by arterial segments and organs of rabbits with (n=13) and without atherosclerosis (n=17). Furthermore, it was evaluated the in vitro uptake of 3H FC and 14C - CE by rabbit aortic endothelial cells. Finally, it was evaluated the inhibition of the enzyme lecithin-cholesterol acyltransferase (LCAT), and indirectly, the FC esterification in rats non-treated (n=9) and treated with diazepam (n=9). In rabbits without atherosclerosis that received an standard diet there was no difference between the plasma clearance of 3H FC and 14C CE. In rabbits with hyperlipidemia and atherosclerosis induced by the cholesterol-rich diet the 3H - FC was removed faster than 14C - CE (p<0.05), however the arch aortic uptake of 14C CE was greater than of 3H - FC (0p<0.05). In both groups, liver, lungs, adrenals and spleen were the principal sites of LDE cholesterol uptake. The FCR and tissue uptake were smaller in rabbits with than those without atherosclerosis. In rabbit aortic endothelial cells the 3H - FC uptake was greater than 14C CE independently of incubated LDE mass (p<0.01). In control rats there was no difference on the arterial uptake of both cholesterol forms of LDE, but when the LCAT activity was diminished by the diazepam treatment, the arterial uptake of 3H FC were greater than 14C CE (p< 0.01). The hyperlipidemia and cholesterol stability alterations may lead to dissociation between lipoproteins FC and CE. This dissociation may increase the risk for atherosclerosis and likewise enhance the severity of atherosclerosis.

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It was hypothesized that 192 IgG-saporin lesions of the basal forebrain cholinergic system (BFCS) would disrupt differential reinforcement of low rate (DRL) learning in an uncued DRL task, but would not impair acquisition and performance in the cued version of the task. Results suggest that BFCS lesions impair vigilance to the external cues despite continued practice in the cued DRL, whereas continuous attention to internally produced cues recovers with extended practice in the uncued DRL.