INTRODUCCIÓN: A pesar de que ductus arterioso persistente (DAP) es una de las cardiopatías congénitas más frecuentes a nivel mundial, sus implicancias patobiológicas continúan siendo un desafío científico mantenido por décadas. Una de las ultimas y más importantes contribuciones han sido los de Echtler y cols,1 quienes proponen una actividad protrombótica fundamental en el cierre funcional del ductus arterioso. OBJETIVO: Determinar si la trombocitopenia es un indicador predictivo de cierre deficitario de ductus arterioso en prematuros del Instituto Nacional Materno Perinatal (INMP) entre enero a julio del 2016. MATERIALES Y METODOS: Se realizó un estudio de tipo retrospectivo, casos y controles. La población estuvo constituida por 250 recién nacidos entre las 22 y 36 semanas de edad gestacional en el periodo de enero a julio del 2016. 87 recién nacidos presentaron DAP y 163 no. Estudiamos el recuento plaquetario del primer día de vida y su capacidad predictiva como prueba diagnóstica, así como el grado de respuesta al tratamiento con AINES con respecto a DAP. Empleamos un análisis estadístico descriptivo e inferencial utilizando el paquete estadístico SPSS v.23. RESULTADOS: La trombocitopenia, encontrada en el primer día de vida estuvo asociada de forma estadísticamente significativa con la presencia de DAP (OR=8.9; IC: 95%, 4.7-16.8; p< 0,05) así como con el DAP hemodinámicamente significativo (OR=8.9; IC: 95%, 4.7-16.8; p< 0,05). Fabricamos una curva ROC que nos reveló que la trombocitopenia es una prueba de baja sensibilidad (59.2%) y de alta especificidad (89.6%). No encontramos asociación entre la respuesta al tratamiento con AINES y la trombocitopenia en sujetos con DAP. CONCLUSIONES: En nuestro estudio pudimos demostrar que la trombocitopenia del primer día de vida y el DAP están asociadas de forma estadísticamente significativa, la curva ROC, demostró que la trombocitopenia tiene capacidad predictiva de baja sensibilidad, pero de alta especificidad. No encontramos con la respuesta al tratamiento con AINES.

Thesis (Ph. D.)--University of North Texas, Dec., 2009. / Title from title page display. Includes bibliographical references.

Thesis (M.S.)--University of North Texas, Dec., 2007. / Title from title page display. Includes bibliographical references.

L'acide rétinoïque (AR), métabolite actif de la vitamine A, est une molécule qui a une fonction pléiotropique au cours du développement embryonnaire dont l'excès ou le déficit entraine diverses pathologies cardiaques. Les mutants perte de fonction pour l'enzyme de synthèse de l'AR, la rétinaldéhyde déshydrogénase 2 Raldh2), possèdent de multiples défauts cardiaques. Ces embryons meurent vers le neuvième jour de développement mais cette létalité peut être sauvée en ajoutant de l'AR dans la nourriturre des femelles en gestation. Les mutants Raldh2-/- sauvés ont un Tronc Artériel Commun (TAC) qui est un vaisseau unique au pôle artériel du coeur à la place de l'AO et du TP. Le but de ma thèse a été de comprendre les mécanismes moléculaires et cellulaires conduisant à ce TAC. Ce TAC est à dominance aortique et les connexions des artères coronaires au TAC sont malpositionnées. Nos résultats montrent que la réduction de la longueur de la VE des mutants Raldh2-/- sauvés est associée à une hypoplasie du mésoderme pharyngé. Les mutants Raldh2-/- sauvés possèdent une altération de l'expression de gènes essentiels pour l'élongation de la VE, la rotation de la VE est aussi affectée . Les cellules de la crête neurale cardiaque (CCNC) sont nécessaires pour la septation de la VE et leur contribution à la VE n'est pas altérée dans les mutants Raldh2-/- sauvés. En revanche, elles sont anormalement localisées à proximité du myocarde et anormalement orientées perpendiculairement au futur plan de septation. Cette délocalisation des CCNC est corrélée à une augmentation du processus de transition endothélio-mésenchymateux. L'AR est donc une molécule essentielle pour la morphogenèse de la VE. / Retinoic acid (RA), the active metabolite of vitamin A, is a molecule with pleiotropic functions during development. An excess or deficiency of RA during development leads to various cardiac pathologies. Loss-of-function of retinaldehyde dehydrogenase 2 (Raldh2), the enzyme that synthesises RA, results in various cardiac defects. Mutant embryos die at around the ninth day of embryonic development (E9), but this lethality can be rescued by supplementing the gestating female's diet with RA. RA-rescued Raldh2-/- displayed a persistent truncus arteriosus (PTA), a unique artery at the arterial pole of the heart, rather than separate aorta and pulmonary trunk. The aim of my thesis was to understand the molecular and cellular mechanisms leading to PTA. This PTA is predominantly an aortic phenotype, associated with malpositioned of the coronary artery connections. Our results show that the reduced length of RA-rescued Raldh2-/- OFTs was linked to pharyngeal mesoderm hypoplasia. RA-rescued Raldh2-/- mutants expressed altered levels of several genes that are essential for OFT elongation and OFT rotation was also affected. Cardiac neural crest (CNC) cells play an essential role in OFT septation and their contribution to the OFT was not altered in RA-rescued Raldh2-/- mutants. However, CNC cells display abnormal characteristics, and are found in close proximity to the myocardium, oriented perpendicularly to the plane of septation. This mis-location of the CNC cells was associated with increased endothelial-to-mesenchymal transition (EMT). Accordingly, an increased contribution of endocardial EMT to valve mesenchyme. In conclusion, RA is essential for OFT morphogenesis.

The ductus arteriosi (DA) are two blood vessels connecting the pulmonary arteries to the descending aorta in the avian embryo. Following hatching, the DA closes, separation of the systemic and pulmonary circulation. I present the morphological changes that occur in the chicken DA during prepipping, internal pipping, external pipping, and hatching. The avian DA consists of two distinct tissue types, a proximal and a distal portion. Histological examination shows developmental differences between the proximal and distal portions of the DA with regard to lumen occlusion, endothelial cells, smooth muscle and elastin. Endothelial cell proliferation begins to occur as early as external pipping, with the lumen almost completely occluded by the 3rd day of post-hatching life. Expression of vascular endothelial growth factor (VEGF) increases in avian endothelial cells during hatching. I provide a morphological timeline of changes in the DA as the chicken develops from embryo to hatchling.

Developing chicken embryos have two ductus arteriosus (DA) that shunt blood away from the lungs and to the chorioallantoic membrane, the embryonic gas exchanger. In mammals, DA closure is stimulated by an increase in blood gas O2 that occurs as the animal begins to breathe with its lungs. The goal of this study was to determine the influence of O2 levels during incubation on the vascular reactivity and morphology of the O2-sensitive DA and to examine the effects of changing O2 levels during late incubation on the morphology of the DA from chicken embryos. In comparison to normoxia, hypoxia (15%) reduced venous O2 levels in day 16 and day 18 embryos and reduced aircell O2 values in day 16, day 18, and internally pipped (IP) embryos, whereas hyperoxia (30%) increased venous O2 levels and aircell O2 level in day 16, day 18, and IP embryos. In comparison to normoxia, hypoxia delayed closure of the DA, whereas hyperoxia accelerated DA closure. In comparison to the left DA from externally pipped (EP) normoxic embryos, the left DA from EP hypoxic embryos exhibited a significantly weaker contractile response to O2. The DA from day 18 hypoxic embryos exhibited a significantly weaker contractile response to norepinephrine and phenylephrine when compared with the DA from day 18 normoxic and hyperoxic embryos. The effect of incubation in hypoxia / hyperoxia during different developmental windows on the DA O2-induced contractile response was observed only in IP embryos that were incubated in normoxia for 16 days and were then moved to hyperoxia. Incubation in hypoxia / hyperoxia resulted in differences in embryo mass, yolk mass, and heart mass. There is an association between the decreased contractile response to O2 and delayed closure in the proximal portion of the DA from hypoxic embryos; as well as an increased contractile response to O2 and accelerated closure in the proximal portion of the DA from hyperoxic embryos.

OBJECTIVES: The objective of this thesis is to explore the effectiveness and safety of common pharmacotherapeutic options used for the management of patent ductus arteriosus (PDA) in preterm infants. METHODS: Following the Preferred Reporting Items for Systematic Review and Meta-Analysis Protocols (PRISMA-P) guidance, article 1 develops the protocol to conduct the systematic review and network meta-analysis to answer the research question. Article 2 details the actual methods implemented to conduct the network meta-analysis and presents the results in the form of network plots, league tables, rank heat maps, rankograms and forest plots. RESULTS: Article 1 suggests the need to conduct a Bayesian random-effects network meta-analysis of randomized controlled trials (RCTs) as the analysis would involve multiple treatments with potentially both direct and indirect comparisons. Article 1 also a priori defines potential effect modifiers and statistical strategies to control for the same. In article 2, the results of the meta-analysis show that in 68 RCTs that included 4802 infants, 14 different variations of indomethacin, ibuprofen or acetaminophen were used. Oral high-dose ibuprofen was associated with a significantly higher odds of PDA closure compared with standard-dose intravenous ibuprofen (Odds Ratio [OR], 3.59; 95% Credible Interval [CrI],1.64-8.17) and intravenous indomethacin (OR, 2.35; 95% CrI,1.08-5.31). Oral high-dose ibuprofen ranked the best option for PDA closure (SUCRA [surface under the cumulative ranking curve],0.89 [SD, 0.12]) and to prevent surgical PDA ligation (SUCRA,0.98 [SD, 0.08]). There was no significant difference in the odds of mortality, necrotizing enterocolitis or intra-ventricular hemorrhage with use of placebo or no treatment compared with any of the other treatment modalities. CONCLUSION: This thesis suggests that oral high-dose ibuprofen could be the best treatment option for closure of a hemodynamically significant PDA. Placebo or no treatment for a hemodynamically significant PDA may not increase morbidity and mortality. / Thesis / Master of Science (MSc) / The following thesis explores the effectiveness and safety of commonly used drugs for the treatment of a heart condition in premature infants called the patent ductus arteriosus (PDA). Article 1 outlines the protocol for the systematic review and network meta-analysis designed to evaluate the effectiveness and safety of indomethacin, ibuprofen and acetaminophen for the treatment of PDA in preterm infants. Article 2 provides in detail the results of the network meta-analysis that examined all eligible randomized controlled trials that compared intravenous or oral formulations of indomethacin, ibuprofen or acetaminophen compared against to other or placebo for the treatment of a PDA that may be harmful for a premature infant based on certain clinical and echocardiographic criteria set by the clinicians and researchers. Overall, this body of work suggests that a higher dose of oral ibuprofen is the best treatment for PDA in premature infants.

Transition of the cardiopulmonary circulation at birth requires functional closure of the ductus arteriosus (DA). The DA is an arterial shunt that is vital in the fetus for diverting the pulmonary circulation away from the uninflated lungs. Failure of the vessel to functionally close after birth is known as patent DA, which is the second most common congenital heart disease. Patent DA may seriously compromise neonatal health and current pharmacological treatments are often limited by serious complications or a significant failure rate, thereby increasing the necessity for surgical intervention. Recently, we were the first to show that genetic or pharmacological inactivation of cyclooxygenase (COX) -2 produces postnatal patent DA in mice. We also demonstrated that the DA expresses high levels of COX-2 during normal closure after birth, suggesting novel contractile actions of COX-2-dependent prostanoids in the DA. In humans, patent DA is more common in preterm infants than those born at full-term, however, mechanism(s) responsible for the reduced DA closure have not been identified. In the current studies, we examined COX-1 and COX-2 expression in the DA at multiple stages of gestation to determine whether alterations in the expression of these enzymes contribute to patent DA in preterm mice. Using real-time PCR, analysis of the time-course of COX-2 mRNA in the fetal mouse DA indicated that COX-2 expression significantly increased with advancing gestational age. The preterm (day 17.5) neonatal mouse DA showed attenuated COX-2 expression, as compared to the full-term (day 19.5) neonatal DA at 3 hours after birth. Furthermore, the DA of preterm neonatal mice showed incomplete closure after 3 hours of birth, a time-point when the DA of full-term neonates was completely remodeled. These data indicate a correlation between reduced DA closure and attenuated COX-2 expression. Additionally, COX-2 expression was significantly attenuated in the DA of mice deficient in the prostanoid receptor EP4, which also show a patent DA phenotype, suggesting the importance of this receptor for the induction of COX-2 required for DA closure. Overall, these studies suggest that attenuated expression of COX-2 may contribute to increased patent DA at preterm gestation.

Os polifenois são normalmente considerados compostos que apresentam atividades biológicas promissoras, em especial pelos seus efeitos antioxidantes e anti-inflamatórios. No entanto, estudos recentes têm demonstrado que o consumo materno de alimentos ricos em polifenois (ARP) durante a gestação interfere na dinâmica de fluxo do ductus arteriosus (DA) no coração fetal de humanos, provavelmente pelo efeito anti-inflamatório dos polifenois, e também tem sido demonstrado que a restrição da ingestão de ARP é capaz de reverter a constrição ductal. Neste trabalho, um estudo experimental foi desenvolvido com ovelhas prenhas, no qual os animais receberam suplementação oral de polifenois durante 14 dias. Realizou-se ecocardiografia fetal e a análise de amostras de sangue e urina para investigar biomarcadores de estresse oxidativo e inflamação além da excreção de polifenois totais na urina. Houve aumento nas velocidades sistólicas (VS) e diastólicas (VD) e uma diminuição no índice de pulsatilidade (IP), o que indica uma constrição prematura do DA após o consumo de polifenois. Houve diminuição da peroxidação lipídica, determinada pelos níveis de TBARS, e nos níveis de tióis reduzidos não proteicos após o tratamento. Houve um aumento das atividades das enzimas catalase (CAT) e glutationa peroxidase (GPx) após o tratamento. Apesar do não envolvimento de dano lipídico na constrição ductal, observou-se um aumento no dano proteico através da dosagem de proteínas carboniladas (PCO). O efeito vasoconstritor e anti-inflamatório foi verificado pela diminuição nos níveis de nitritos/nitratos (NOx) após o consumo de polifenois. O estresse oxidativo estava associado com parâmetros de constrição ductal, através das correlações de dano protéico (PCO) com VS (r=0,629, p=0,028), VD (r=0,905, p=0,0001) e IP (r=-0,772, p= 0,003). Ainda, VS foi correlacionada com catalase (r=0,672, p=0,033) assim como IP com GPx (r=-0,629, p= 0,05). A constrição ductal estava ainda associada com o parâmetro inflamatório, sendo VS e VD correlacionadas com NOx (r=-0,853, p=0,0004 e r=-0,705, p=0,010, respectivamente) além da correlação entre IP e NOx (r=0,599, p=0,039). Além disso, ambos os mecanismos anti-inflamatórios e antioxidantes estavam correlacionados: NOx e GPx (r=-0,755, p=0,004) e entre NOx e catalase (r=-0,812, p=0,001), confirmando a ocorrência de ambos efeitos atribuíveis aos 10 polifenois. Neste estudo, foi possível perceber que um elevado consumo de polifenois induziu constrição ductal em ovelhas prenhas com uma excreção urinária aumentada de polifenois totais e alterações em biomarcadores de estresse oxidativo e inflamação. Estes resultados ressaltam a necessidade de uma orientação dietética ao final da gestação com relação ao consumo de alimentos ricos em polifenois devido à possibilidade de indução de constrição ductal através da ação anti-inflamatória em fetos expostos. / Polyphenols are often referred to as compounds with promising biological activities, especially antioxidant and anti-inflammatory effects. However, it has been recently reported that maternal consumption of polyphenol-rich foods (PRF) interferes with ductus arteriosus (DA) flow in human fetuses’ hearts, probably by an anti-inflammatory effect and it has also been shown that restriction of PRF ingestion reverses ductal constriction. In this work, an experimental study was carried out with pregnant sheep, in which the animals received oral polyphenol supplementation for 14 days. Fetal echocardiography was performed along with blood and urine analysis to investigate antioxidant and anti-inflammatory biomarkers and total polyphenol (TP) urinary excretion. We found a decrease in lipid peroxidation by TBARS levels and a decrease in non-protein reduced thiols after treatment. In addition, an increase in enzymatic activities of catalase (CAT) and glutathione peroxidase (GPx) was observed. Despite that lipid peroxidation was not involved in ductal constriction, protein damage by enhanced protein carbonyls (PCO) were found. Anti-inflammatory and vasoconstrictive effects were observed by a decrease in nitrites/nitrates (NOx) in pregnant sheep after polyphenol consumption. Oxidative stress was associated to ductal constriction parameters, according to the correlations.of protein damage marker PCO to SV (r=0.629, p=0.028), VD (r=0.905, p=0.0001) and IP (r=-0.772, p=0.003). Also, SV was positively correlated to CAT (r=0.672, p=0.033) and IP negatively correlated to GPx (r=-0.629, p=0.05). Ductal constriction was also associated to the inflammatory parameter, due to the correlations of SV and DV to NOx (r=-0.853, p=0.0004 and r=-0.705, p=0.010, respectively) as well as the correlation between IP and NOx (r=0.599, p=0.039). Besides, association of both inflammatory and antioxidant mechanisms were found: NOx vs. GPx (r=-0.755, p=0.004) and NOx vs. CAT (r=-0.812, p=0.001), confirming the presence of both effects attributed to polyphenols. We report that high polyphenol intake induced fetal DA constriction in pregnant sheep followed by an increased TP excretion and alterations in inflammatory and oxidative biomarkers. These results highlight the need for a dietary orientation in late-pregnancy regarding maternal intake of foods with high polyphenol contents in light of the possible induction of ductal constriction through an anti-inflammatory action of polyphenols in exposed fetuses.

Os polifenois são normalmente considerados compostos que apresentam atividades biológicas promissoras, em especial pelos seus efeitos antioxidantes e anti-inflamatórios. No entanto, estudos recentes têm demonstrado que o consumo materno de alimentos ricos em polifenois (ARP) durante a gestação interfere na dinâmica de fluxo do ductus arteriosus (DA) no coração fetal de humanos, provavelmente pelo efeito anti-inflamatório dos polifenois, e também tem sido demonstrado que a restrição da ingestão de ARP é capaz de reverter a constrição ductal. Neste trabalho, um estudo experimental foi desenvolvido com ovelhas prenhas, no qual os animais receberam suplementação oral de polifenois durante 14 dias. Realizou-se ecocardiografia fetal e a análise de amostras de sangue e urina para investigar biomarcadores de estresse oxidativo e inflamação além da excreção de polifenois totais na urina. Houve aumento nas velocidades sistólicas (VS) e diastólicas (VD) e uma diminuição no índice de pulsatilidade (IP), o que indica uma constrição prematura do DA após o consumo de polifenois. Houve diminuição da peroxidação lipídica, determinada pelos níveis de TBARS, e nos níveis de tióis reduzidos não proteicos após o tratamento. Houve um aumento das atividades das enzimas catalase (CAT) e glutationa peroxidase (GPx) após o tratamento. Apesar do não envolvimento de dano lipídico na constrição ductal, observou-se um aumento no dano proteico através da dosagem de proteínas carboniladas (PCO). O efeito vasoconstritor e anti-inflamatório foi verificado pela diminuição nos níveis de nitritos/nitratos (NOx) após o consumo de polifenois. O estresse oxidativo estava associado com parâmetros de constrição ductal, através das correlações de dano protéico (PCO) com VS (r=0,629, p=0,028), VD (r=0,905, p=0,0001) e IP (r=-0,772, p= 0,003). Ainda, VS foi correlacionada com catalase (r=0,672, p=0,033) assim como IP com GPx (r=-0,629, p= 0,05). A constrição ductal estava ainda associada com o parâmetro inflamatório, sendo VS e VD correlacionadas com NOx (r=-0,853, p=0,0004 e r=-0,705, p=0,010, respectivamente) além da correlação entre IP e NOx (r=0,599, p=0,039). Além disso, ambos os mecanismos anti-inflamatórios e antioxidantes estavam correlacionados: NOx e GPx (r=-0,755, p=0,004) e entre NOx e catalase (r=-0,812, p=0,001), confirmando a ocorrência de ambos efeitos atribuíveis aos 10 polifenois. Neste estudo, foi possível perceber que um elevado consumo de polifenois induziu constrição ductal em ovelhas prenhas com uma excreção urinária aumentada de polifenois totais e alterações em biomarcadores de estresse oxidativo e inflamação. Estes resultados ressaltam a necessidade de uma orientação dietética ao final da gestação com relação ao consumo de alimentos ricos em polifenois devido à possibilidade de indução de constrição ductal através da ação anti-inflamatória em fetos expostos. / Polyphenols are often referred to as compounds with promising biological activities, especially antioxidant and anti-inflammatory effects. However, it has been recently reported that maternal consumption of polyphenol-rich foods (PRF) interferes with ductus arteriosus (DA) flow in human fetuses’ hearts, probably by an anti-inflammatory effect and it has also been shown that restriction of PRF ingestion reverses ductal constriction. In this work, an experimental study was carried out with pregnant sheep, in which the animals received oral polyphenol supplementation for 14 days. Fetal echocardiography was performed along with blood and urine analysis to investigate antioxidant and anti-inflammatory biomarkers and total polyphenol (TP) urinary excretion. We found a decrease in lipid peroxidation by TBARS levels and a decrease in non-protein reduced thiols after treatment. In addition, an increase in enzymatic activities of catalase (CAT) and glutathione peroxidase (GPx) was observed. Despite that lipid peroxidation was not involved in ductal constriction, protein damage by enhanced protein carbonyls (PCO) were found. Anti-inflammatory and vasoconstrictive effects were observed by a decrease in nitrites/nitrates (NOx) in pregnant sheep after polyphenol consumption. Oxidative stress was associated to ductal constriction parameters, according to the correlations.of protein damage marker PCO to SV (r=0.629, p=0.028), VD (r=0.905, p=0.0001) and IP (r=-0.772, p=0.003). Also, SV was positively correlated to CAT (r=0.672, p=0.033) and IP negatively correlated to GPx (r=-0.629, p=0.05). Ductal constriction was also associated to the inflammatory parameter, due to the correlations of SV and DV to NOx (r=-0.853, p=0.0004 and r=-0.705, p=0.010, respectively) as well as the correlation between IP and NOx (r=0.599, p=0.039). Besides, association of both inflammatory and antioxidant mechanisms were found: NOx vs. GPx (r=-0.755, p=0.004) and NOx vs. CAT (r=-0.812, p=0.001), confirming the presence of both effects attributed to polyphenols. We report that high polyphenol intake induced fetal DA constriction in pregnant sheep followed by an increased TP excretion and alterations in inflammatory and oxidative biomarkers. These results highlight the need for a dietary orientation in late-pregnancy regarding maternal intake of foods with high polyphenol contents in light of the possible induction of ductal constriction through an anti-inflammatory action of polyphenols in exposed fetuses.