Cardiovascular diseases (CVD) are the leading cause of morbidity and mortality in the United States. It is also well recognized that the incidence of CVD is substantially increased in postmenopausal women due to the loss of estrogen. Experimental and clinical data support vascular protective effects of estrogen by various mechanisms. However, administration of estrogen is also associated with an increased incidence of heart disease which limits its therapeutic potential. Given the demonstrated risks of conventional estrogen therapy, a search for novel, cost-effective, alternative vasoactive agents for prevention of CVD is of major importance in the effort to decrease the burden of CVD morbidity. Genistein, a major soy isoflavone, may be one of those alternative agents because of its selective affinity to estrogen receptor-beta and various beneficial effects on CVD. However, the mechanism of the cardioprotective effects of genistein is still unclear. The objectives of this study were (1) to investigate the effect of genistein on the expression of endothelial nitric oxide synthase (eNOS) both in vitro and in vivo; (2) to define the mechanism by which genistein regulates eNOS expression; and, (3) to examine whether genistein protects against tumor necrosis factor-alpha (TNF-α)-induced apoptosis in human aortic endothelial cells (HAECs). The results demonstrated that genistein, at physiologically achievable concentrations (1-10 μM) in individuals consuming soy products, enhanced the expression of eNOS protein and subsequently elevated nitric oxie (NO) synthesis in both HAECs and human umbilical vein endothelial cells, concomitant with the increased eNOS mRNA expression (2.6-fold of control) and eNOS promoter activity, suggesting that genistein activates eNOS transcription. Furthermore, dietary supplementation of genistein to spontaneously hypertensive rats restored aortic eNOS levels, improved aortic wall thickness, and alleviated hypertension, confirming the biological relevance of the in vitro findings. However, the effects of genistein on eNOS and NO were not mediated by activation of estrogen signaling, mitogen-activated protein kinase, phosphatidylinositol 3-kinase/Akt kinase, protein kinase C or inhibition of typrosine kinases, but possibly through activating the cAMP/protein kinase A/cAMP responsive elemant binding protein pathway. These data suggest that genistein has direct genomic effects on the vascular wall that are unrelated to its known actions, leading to increase in eNOS expression and NO synthesis, thereby improving vascular homeostasis. We also found that genistein (5-10 μM) significantly inhibited TNF-α-induced apoptosis in HAECs as determined by caspase-3 activation, apoptotic cell detection and DNA laddering. The anti-apoptotic effect of genistein was associated with an enhanced expression of anti-apoptotic Bcl-2 protein and its promoter activity that was ablated by TNF-α. Moreover, this anti-apoptotic effect of genistein was not mediated by extracellular signal-regulated kinase 1/2, protein kinase A, or estrogen receptor. However, inhibition of p38 mitogen-activated protein kinase (p38) by SB203580 completely abolished the cytoprotective effect of genistein, suggesting that genistein acted through the p38-dependent pathway. Accordingly, stimulation of HAECs with genistein resulted in rapid and dose-dependent activation of p38. Unlike TNF-α which specifically activated p38α, genistein selectively induced phosphorylation of p38β, suggesting that p38β, but not p38α, is essential for the cytoprotective effect of genistein. These findings provide the evidence that genistein acts as a survival factor for vascular ECs to protect cells against apoptosis via activation of p38β. Taken together, the resuls of the present study suggest that genistein can act directly on vascular ECs, improves endothelium homeostasis by promoting eNOS expression and endothelial-derived NO synthesis through activating the cAMP/PKA/CREB cascade, and protects against TNF-α-induced apoptosis via activation of p38 β. These data potentially provide a basic mechanism underlying the physiological effects of genistein in the vasculature. / Ph. D.

Diabetes mellitus is a complex disease characterised by chronic hyperglycaemia due to compromised insulin synthesis and secretion, or decreased tissue sensitivity to insulin, if not all three conditions. Endothelial dysfunction is a common complication in diabetes in which endothelium-dependent vasodilation is impaired. The aim of this study was to examine the involvement of TRPV4 in diabetes endothelial dysfunction. Male Charles River Wistar rats (350-450 g) were injected with 65mg/kg streptozotocin (STZ) intraperitoneally. STZ-injected rats were compared with naïve rats (not injected with STZ) or control rats (injected with 10ml/kg of 20mM citrate buffer, pH 4.0-4.5), if not both. Rats with blood glucose concentrations greater than 16mmol/L were considered to be diabetic. As the results revealed, STZ-diabetic rats showed significant endothelial dysfunction characterised by impaired muscarinic-induced vasodilation, as well as significant impairment in TRPV4-induced vasodilation in aortic rings and mesenteric arteries. Furthermore, STZ-diabetic primary aortic endothelial cells (ECs) showed a significant reduction in TRPV4-induced intracellular calcium ([Ca2+]i) elevation. TRPV4, endothelial nitric oxide synthase (eNOS), and caveolin-1 (CAV-1) were also significantly downregulated in STZ-diabetic primary aortic ECs and were later significantly restored by in vitro insulin treatment. Methylglyoxal (MGO) was significantly elevated in STZ-diabetic rat serum, and nondiabetic aortic rings incubated with MGO (100μM) for 12 hours showed significant endothelial dysfunction. Moreover, nondiabetic primary aortic ECs treated with MGO (100μM) for 5 days showed significant TRPV4 downregulation and significant suppression of 4-α-PDD-induced [Ca2+]i elevation, which was later restored by L-arginine (100μM) co-incubation. Incubating nondiabetic aortic rings with MGO (100μM) for 2 hours induced a spontaneous loss of noradrenaline-induced contractility persistence. Moreover, MGO induced significant [Ca2+]i elevation in Chinese hamster ovary cells expressing rat TRPM8 channels (rTRPM8), which was significantly inhibited by AMTB (1-5μM). Taken together, TRPV4, CAV-1, and eNOS can form a functional complex that is downregulated in STZ-diabetic aortic ECs and restored by insulin treatment. MGO elevation might furthermore contribute to diabetes endothelial dysfunction and TRPV4 downregulation. By contrast, MGO induced the loss of contractility persistence, possibly due to MGO's acting as a TRPM8 agonist.

Le travail présenté ici porte sur l’étude de la production et du transport du monoxyde d’azote (NO) dans le poumon humain. Le NO est une molécule dont l’implication dans des processus physiologiques n’a été mis en évidence qu’en 1987. Depuis, il a été démontré que le NO joue de nombreux rôles dans le corps humain. Le NO est un gaz labile (instable) dans les conditions physiologiques, il diffuse très facilement au travers des parois et il a une grande affinité pour l’hémoglobine. La production du NO est liée à 3 isoformes différentes de la protéine appelées synthases du NO ou NO synthases.<p>\ / Doctorat en sciences, Spécialisation physique / info:eu-repo/semantics/nonPublished

The synergistic effect of nitric oxide toward dephosphorylation reactions involving phosphate esters was the subject of investigation in this research. Sodium nitroprusside under UV irradiations at 254nm, 365nm and white light was utilized as nitric oxide donor in solutions. The effects of cobalt trimethylenediamine and nitroprusside towards dephosphorylation of nitrophenylphosphate and pyrophosphate which were modeled as organophosphate ester substrates were also investigated. The activated substrate models showed more rate enhancement than the unactivated models in all cases. The direct interaction of nitric oxide with the phosphorus centre is presumed to be the reason for enhanced hydrolysis. This study demonstrates the possible role of nitric oxide in decontamination reactions of poorly biodegradable phosphate esters in the biosphere. / Chemistry / M. Sc. (Chemistry)

Thesis (MScMedSc (Biomedical Sciences. Medical Physiology))--University of Stellenbosch, 2009. / In the heart, endothelial nitric oxide synthase (eNOS) is regarded as the most important constitutively expressed enzymatic source of nitric oxide (NO), a major cardiac signalling molecule. On the whole, NO is regarded as a cardioprotective molecule. The role of eNOS during ischaemia / hypoxia is controversial; however, it is generally accepted that ischaemia / hypoxia results in increased cardiac NO production. Most studies focus either on the whole heart or isolated cell models. As yet, no study has compared findings with regard to NO metabolism in these two distinct models, in a single study. We hypothesise that observations in a whole heart model with regard to increased NO production and eNOS involvement in ischaemia are the result of events on cellular level and that the increase in NO production observed during hypoxia in cardiomyocytes and endothelial cells is at least in part due to the increase in expression and / or activation of eNOS. Furthermore, we hypothesize that these effects are mediated via the PI3-K / PKB pathway. We aimed to measure and compare NO-production and eNOS expression and activation in the whole heart and isolated cardiac cells and measure PKB expression and activation in the cells under normoxic and ischaemic / hypoxic conditions. We also aimed to determine the effects of PI3-K / PKB pathway inhibition on NO production and eNOS expression and activation in isolated cardiac cells under normoxic and hypoxic conditions. Adult rat hearts were perfused and global ischaemia induced for 15 and 20 min. Tissue homogenates of perfused hearts were used for the measurement of nitrites and determination of expression and activation of eNOS. Expression of eNOS in the heart was also determined by immunohistochemical (IHC) analysis. Cardiomyocytes were isolated from adult rat hearts by collagenase-perfusion, and adult rat cardiac microvascular endothelial cells (CMEC) purchased commercially. In the cells, hypoxia was induced by covering cell pellets with mineral oil for 60 min. Cell viability was determined by trypan blue and propidium iodide (PI) staining and intracellular NO production measured by FACS analysis of the NO-specific probe, DAF-2/DA and by measurement of nitrite levels (Griess reagent). Results show that in ischaemic hearts, nitrite production increased by 12 % after 15 min ischaemia and 7 % after 20 min ischaemia. Total eNOS expression remained unchanged (Western Blot and IHC) and activated eNOS (phospho-eNOS Ser1177) increased by 38 % after 15 min ischaemia and decreased by 43% after 20 min ischaemia. In the cells, both viability techniques verified that the hypoxia-protocol induced significant damage. In isolated cardiomyocytes, NO-production increased 1.2-fold (by DAF-2/DA fluorescence), total eNOS expression increased 2-fold and activated eNOS increased 1.8-fold over control. In CMECs, NO-production increased 1.6-fold (by DAF-2/DA fluorescence), total eNOS increased by 1.8- fold and activated eNOS by 3-fold. With regards to our PI3-K / PKB investigations, results showed an increase of 84 % and 88 % in expression vii and activation of PKB (phospho Ser473) in hypoxic cardiomyocytes, respectively. In hypoxic CMECs, there was no change in PKB expression but there was a 69 % increase in phosphorylated PKB. NO production in wortmannin-treated hypoxic cardiomyocytes decreased by 12 % as compared to untreated hypoxic cells. In treated hypoxic CMECs, NO production decreased by 58 % as compared to untreated hypoxic cells. Treatment with wortmannin did not change the expression of eNOS protein in the cardiomyocytes, however, activated eNOS decreased by 41 % and 23 % under baseline and hypoxic conditions in treated cells respectively. There was a significant increase in NO production after exposure to O2 deficient conditions in all models investigated, a trend similar to what previous studies in literature found. However, the source of this NO is not fully understood although it has been discovered that NOS plays a role. Our data reveals similar trends in 15 min ischaemia in whole hearts and 60 min hypoxia in the cells; however, the trends observed at 20 min ischaemia are in conflict with our cell data (i.e. decrease in activated eNOS). This may be due to the severity of the ischaemic insult in whole hearts and/or the presence of other cell types and paracrine factors in the whole heart. Hypoxia increased the activation of PKB in the isolated cardiac cells. Inhibition of the PI3-K / PKB pathway reduced NO production and hypoxia-induced eNOS activation in cardiomyocytes. In conclusion, we have, for the first time, demonstrated that the increase in NO production during hypoxia is due (at least in part) to an increase in eNOS phosphorylation at Ser1177 and that this is mediated via the PI3-K / PKB pathway.

Oxidative stress (OS) plays a critical role in the pathogenesis of atherosclerosis potentially through interaction with nitric oxide (NO) generated by the inducible nitric oxide synthase (iNOS) pathway. Although considerable literature supports a pro-atherogenic role for iNOS-induced NO, recent evidence suggest an anti-atherogenic property for this enzyme where iNOS-induced NO attenuates atherosclerotic lesions after immune injury, enhancing endothelial integrity, survival, protecting against OS-induced apoptosis and necrosis. We therefore hypothesize that iNOS may have a cardio-protective role in the atherosclerotic vessel and that under conditions of OS, expression and function of this enzyme may be impaired, thus contributing to the deleterious consequences of OS. Experiments have therefore been conducted to establish whether pro-oxidants regulate iNOS expression/function in rat cultured aortic smooth muscle cells (RASMCs). These cells were induced for 24 hours with LPS and IFN-γ to mimic inflammatory conditions. Oxidative stress inducers may modulate iNOS-induced NO production through alteration of the expression and/or function of the inducible L-arginine-NO pathway. We examined the effects of hydrogen peroxide (H2O2), antimycin A and diethyl maleate (DEM) on this pathway in vascular smooth muscle cells. H2O2 had little effect on NO production or L-arginine transport while antimycin A and DEM independently caused a concentration dependent inhibition of both processes. Only DEM induced hemeoxygenase-1 (HO-1) expression, monitored by western blotting as a marker of OS. The effects of statins on NO synthesis and L-arginine transport in the presence and absence of OS were also investigated. The benefits of statins therapy in cardiovascular medicine are ascribed in part to their lipid-lowering effect by inhibiting 3-hydroxy-3-methoxyglutaryl coenzyme A (HMG-CoA) reductase, the rate limiting enzyme for cholesterol synthesis. However, statins may possess anti-inflammatory properties and are able to improve endothelial function, stabilize atherosclerotic plaque, and inhibit platelet aggregation, vascular smooth muscle cells proliferation and vessel wall inflammation. These effects may be exerted through novel actions of statins that include interaction with specific signalling pathways in cells which may be associated with the induction of iNOS and/or cationic amino acid transporters (CATs). Thus, we have extended our investigations to include an examination of the effects of statins on both iNOS and CAT function and expression under control conditions and following exposure of cells to OS. Atorvastatin caused a bell shaped response on NO production and iNOS expression and also enhanced L-arginine transport but in a non-concentration dependent manner. Simvastatin only affected NO synthesis without altering transporter activity. Pravastatin was without effect on either system. Further studies demonstrated that that atorvastatin was able to reverse the effects of antimycin A and DEM but only on NO production. These findings confirm that the inducible L-arginine-NO pathway can be downregulated by pro-oxidants. This mechanism may therefore contribute to the deleterious effects observed in disease states associated with OS. Moreover, statins (in particular atorvastatin) appear to be effective in reversing the inhibition of NO production caused by inducers of OS. This, together with the fact that atorvastatin and simvastatin can potentiate iNOS-induced NO production and indeed L-arginine transport (with atorvastatin), highlights a potential novel mechanism through which the cardio-protective actions of these compounds could be mediated.

There are gaps in the fundamental understanding of the expression of nitric oxide synthases (NOS) in the microvasculature. We examined co-localization of NOS1 (nNOS), NOS2 (iNOS) and NOS3 (eNOS) in the spinotrapezius muscle of young adult male Wistar-Kyoto (WKY) and Spontaneously Hypertensive (SHR) rats according to fiber type using immunohistochemistry and brightfield microscopy. Data regarding fiber distribution, population and morphology data were collected. Alkaline phosphatase staining was used to determine capillary density and average number of capillaries around a fiber. Gel electrophoresis and Western blot techniques were used to compare myosin heavy chain (MHC) protein expression with fiber type population data and to determine NOS1-3 protein expression in whole muscle homogenate. This study should provide a more accurate understanding of differences in NOS expression between these two strains of rats.

Há décadas a exposição ao mercúrio é alvo de estudos toxicológicos devido ao alto potencial de danos a saúde humana. Na região amazônica os primeiros estudos reportavam a exposição ocupacional pelo uso nos garimpos de ouro, entretanto recentemente destacam-se os estudos relacionados a exposição ambiental que ocorre na região decorrente do consumo de peixes contaminados com mercúrio. Muitos estudos se concentram em populações ribeirinhas residentes na região do rio Tapajós, onde o consumo de peixes é frequente e o metil-mercúrio (MeHg) contido nos peixes é o responsável pela exposição dessas pessoas ao metal. O MeHg apresenta efeitos tóxicos relevantes sobre o sistema cardiovascular, e muitos grupos de pesquisa buscam elucidar os mecanismos que expliquem tais efeitos. Alguns estudos apontam uma diminuição significativa na disponibilidade do óxido nítrico (NO) após exposição ao organometal, o que poderia contribuir para uma alteração da fisiologia cardiovascular uma vez que o NO é um modulados desse sistema. O NO é sintetizado pela óxido nítrico sintase endotelial (eNOS) e sua atividade pode ser alterada por vários fatores, dentre eles, os polimorfismos nos genes que codificam essa proteína, são eles: T-786C na região promotora, 27-pb VNTR no intron 4 e Glu298Asp no exon 7. Neste sentido, o presente estudo teve por objetivo avaliar os efeitos dos polimorfismos da eNOS sobre a síntese de NO entre os indivíduos expostos a metilmercúrio. Foram analisadas amostras de sangue de 214 voluntários com idade entre 15 e 84 anos, dos quais 103 homens e 111 mulheres. A concentração de mercúrio no sangue (Hg sangue) total variou de 1,7 a 179,3 µg/L e a concentração plasmática de nitrito variou entre 85,7 e 695,8 M. Foram determinados os valores de pressão arterial sistólica (PAS), pressão arterial diastólica (PAD), índice de massa corporal (IMC) e freqüência cardíaca (FC) de todos os voluntários. A PAS média foi de 119,8 mmHg e a média da PAD foi 71,8 mmHg. O IMC médio foi de 24,5 Kg/m2 e a FC média foi 70,4 batimentos por minuto (bpm). Não foram observadas diferenças entre os grupos, segundo genótipos dos três polimorfismos, quanto às características dos voluntários: idade, PAS, PAD, IMC, FC, Hg sangue e as concentrações plasmáticas de nitrito. Quando os polimorfismos foram estudados isoladamente foi observado que o alelo C na região promotora, o alelo 4b no intron 4 e o alelo Glu no exon 7 apresentaram-se associados a concentrações reduzidas e nitrito plasmático. Quando a população foi estratificada com base na concentração de Hg essa associação desapareceu, provavelmente mascarada pelas altas concentrações do metal. Entretanto quando foram estudados os haplótipos pode ser observada novamente a associação desses mesmos alelos com a diminuição da concentração do nitrito, confirmando os achados iniciais. O haplótipo mais frequente na população combina os alelos selvagens para todos os polimorfismos (T, 4b e G) e o haplótipo menos freqüente combina os alelos variantes. O haplótipo associado à menor concentração plasmática de nitrito combina os alelos selvagens (C, 4b e G), confirmando os primeiros resultados. Essa abordagem haplotípica é muito útil na observação de efeitos mais discretos uma vez que é possível observar os efeitos dos três polimorfismos agindo simultaneamente sobre uma variável, nesse caso o óxido nítrico. O presente estudo sugere que os fatores genéticos exercem grande influência sobre a produção e biodisponibilidade de NO e que esses fatores combinados com a exposição ambiental ao Hg podem agir de maneira sinérgica, aumentando a suscetibilidade aos efeitos cardiotóxicos do metal através da modulação da atividade da eNOS. / The mercury (Hg) exposure has been target of toxicological studies due the high potential of damage to human health. In the Amazon region the first studies reported the occupational exposure due the use in gold mining, however, recently become relevant the studies about the environment exposure due the fish intake in the riparian population. Several studies have been concentrated in the riparian community in the Tapajós river region, where the fish consumption is frequent and the methylmercury content in fish is responsible to exposure of this people. The MeHg presents toxic effects in the cardiovascular system and many researches groups try to elucidate the mechanisms that explain this effects. Some studies report a significant reducing in nitric oxide (NO) production after the Hg exposure, which could contribute to an altered physiology of the cardiovascular system, once the NO is a modulating factor of this system. The NO is produced by the endothelial nitric oxide synthase (eNOS) and its activity can be altered by many factors like polymorphisms in gene that codify this protein, among this: : T-786C in the promoter region, 27-pb VNTR in intron 4 and Glu298Asp in exon 7. In this regard, the present study mean to evaluate the effects of the eNOS polymorphisms over the NO synthesis among the Hg exposed subjects. In this work, the whole blood samples of 214 volunteers were analyzed for determination of Hg concentration, nitrite plasma concentration and genotyping. The age of the volunteers varied between 15 and 84 years old, including 103 men and 111 women. The blood mercury concentration varied between 1.7 and 179.3 µg/L and the nitrite plasma concentration varied between 85.7 and 695.8 M. Was determinate the systolic arterial pressure (SAP), diastolic arterial pressure (DAP), body mass index (BMI) and heart rate (HR). The SAP mean was 119.8 mmHg and the DAP mean was 71.8 mmHg. The BMI mean was 24.5 Kg/m2 and the HR mean was 70.4 beats per minute. There was no difference among the groups of the three polymorphisms according the volunteers characteristics: age, DAP, SAP, BMI, HR, blood Hg concentration and nitrite plasma concentration. When the polymorphisms were observed separately the reduced nitrite plasma concentration was associated with the presence of the alleles: C in promoter region, 4b in intron 4 and G in exon 7, however there is lack of association when the volunteers were grouped according the blood Hg concentration, probably due a mask effect of the high Hg concentration. When these three polymorphisms were observed simultaneously, in analysis of the haplotypes, the association between the same alleles and the nitrite plasma concentration was observed again, confirming the initial findings. The commonest haplotype in the volunteers combine the alleles of the three polymorphisms (T, 4b and G) and the less frequent haplotype combine the three variants alleles. There was an association between the haplotype C, 4b and G and reduced nitrite plasma concentration, according the result of the polymorphisms separately. The haplotype analysis is too interesting to observe discrete effects, once is possible to analyze the effects of the three polymorphisms acting simultaneously above one variable, in this case, nitric oxide production. The present study suggest that genetic factors could exert a relevant influence above the NO production and bioavailability and that this factors combined with environmental Hg exposure can acting synergic, increasing the susceptibility to Hg cardiovascular effects, through the modulation of the eNOS activity.

Liu Kin Hang. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 88-98). / Abstracts in English and Chinese. / Abstract (in English) --- p.i / Abstract (in Chinese) --- p.iii / Acknowledgement --- p.v / Table of Contents --- p.vi / List of Tables --- p.ix / List of Figures --- p.x / Glossary of Terms and Abbreviations --- p.xi / Chapter Section I: --- Overview --- p.1 / Chapter Chapter 1: --- Introduction --- p.2 / Chapter 1.1 --- Asthma and Assessment of A irway Inflammation --- p.2 / Chapter 1.1.1 --- Assessment of A irway Inflammation --- p.4 / Chapter 1.1.2 --- Invasive and Noninvasive Methods --- p.4 / Chapter 1.1.3 --- Exhaled Nitric Oxide as a Diagnostic Marker and Its Correlation with Other Markers of Inflammation --- p.6 / Chapter 1.1.4 --- Normal Reference Studies of Exhaled Nitric Oxide --- p.8 / Chapter 1.2 --- Aim of Study --- p.10 / Chapter Chapter 2: --- Plan of Study --- p.11 / Chapter Section II: --- Literature Review --- p.13 / Chapter Chapter 3: --- Nitric Oxide Biology --- p.15 / Chapter 3.1 --- Exhaled Nitric Oxide Production in Airway --- p.15 / Chapter 3.2 --- Nitric Oxide Production and Function --- p.16 / Chapter 3.3 --- Nitric Oxide Synthase Pathway --- p.18 / Chapter 3.4 --- Factors Affecting Exhaled Nitric Oxide Level --- p.21 / Chapter 3.4.1 --- Procedure-related Factors --- p.22 / Chapter 3.4.1.1 --- Nasal Nitric Oxide Contamination --- p.22 / Chapter 3.4.1.2 --- Exhalation Procedure 226}0´ؤؤStarting Lung Volumes --- p.23 / Chapter 3.4.1.3 --- Exhalation Procedure 226}0ؤ Flow --- p.23 / Chapter 3.4.1.4 --- Circadian Rhythm --- p.25 / Chapter 3.4.2 --- Patient Factors --- p.26 / Chapter 3.4.2.1 --- Sex --- p.26 / Chapter 3.4.2.2 --- Upper Respiratory Tract Infection --- p.26 / Chapter 3.4.2.3 --- Diet and Exhaled Nitric Oxide --- p.27 / Chapter 3.4.2.4 --- Effect of Spirometry and Exercise --- p.28 / Chapter 3.4.3 --- Environmental Factors --- p.28 / Chapter Chapter 4: --- Exhaled Nitric Oxide in Asthmatics and Its Relationship to Anti-inflammatory Treatment --- p.31 / Chapter Chapter 5: --- Relationship of Exhaled Nitric Oxide with Other Inflammatory Markers --- p.33 / Chapter 5.1 --- Correlation of Findings from Biopsy and Bronchoalveolar Lavage with Exhaled Nitric Oxide --- p.33 / Chapter 5.2 --- "Exhaled Nitric Oxide, Induced Sputum Analysis and Sputum Eosinophil Cationic Protein" --- p.35 / Chapter Section III: --- Original Study --- p.37 / Chapter Chapter 6: --- Methodology --- p.38 / Chapter 6.1 --- Study Population --- p.38 / Chapter 6.2 --- The International Study of Asthma and Allergies in Childhood --- p.40 / Chapter 6.3 --- ISAAC Questionnaires --- p.42 / Chapter 6.4 --- Standardized Approach for Answering Questions in the Field --- p.44 / Chapter 6.5 --- Anthropometric Measurements --- p.45 / Chapter 6.6 --- Exhaled Nitric Oxide Measurement --- p.46 / Chapter 6.6.1 --- "NIOY® (Aerocrine AB, Stockholm, Sweden)" --- p.46 / Chapter 6.6.2 --- Calibration Procedures --- p.47 / Chapter 6.6.3 --- Exhaled Nitric Oxide Measurement --- p.48 / Chapter 6.7 --- Classification of Subjects --- p.51 / Chapter 6.8 --- Statistical Analysis --- p.53 / Chapter Chapter 7: --- Results --- p.54 / Chapter 7.1 --- Subjects and Demography --- p.54 / Chapter 7.2 --- Exhaled Nitric Oxide in Chinese Children --- p.58 / Chapter 7.3 --- Exhaled Nitric Oxide in Caucasians and Other Ethnic Groups --- p.66 / Chapter Chapter 8: --- Discussion --- p.69 / Chapter Chapter 9: --- Conclusion and Further Studies --- p.76 / Appendix 1 Questionnaires (Chinese Version) --- p.80 / Appendix 2 Questionnaires (English Version) --- p.84 / References --- p.88

Although survival rates are increasing, brain injury continues to be a leading cause of death after cardiac arrest (CA). Permanent brain damage after CA is determined by limited tolerance to ischemia from CA and cardiopulmonary resuscitation (CPR), as well as the unique cerebral response to reperfusion after return of spontaneous circulation (ROSC). A major pathway leading to neurotoxic cascade and neuronal injury after CA involves the increased presence of reactive oxygen and nitrogen species generated during ischemia and reperfusion. The magnitude of cerebral oxidative injury induced by free radicals increased with the duration of CA (Paper I). Nitric oxide (NO), a free radical responsible for the formation of reactive nitrogen species, is increased during global ischemia from CA and reperfusion (Paper IV). Hypothetically, the administration of a drug that counteracts the overproduction of NO and also acts as a scavenger of oxygen free radicals might be warranted in order to reduce the damage caused by nitrosative and oxidative stress. For these purposes we used methylene blue (MB), an old dye that has been used in medicine for almost half a century, and an experimental pig model of 20 min of ventricular fibrillation (VF) to reflect a clinical scenario of ischemia/reperfusion injury. Administration of MB added to a hypertonic-hyperoncotic solution (MBHSD) that was started during CPR and continued for 50 min after ROSC increased short-term survival by decreasing myocardial damage, as well as cerebral peroxidation and inflammatory injury (Paper II). Immunostaining of cerebral tissue collected at different time points after CA and ROSC (Paper IV) provided experimental evidence that cortical blood-brain barrier (BBB) disruption begins as early as  during the initial phase of untreated as well as treated CA. The results indicated that MB administration reduced the neurologic injury and BBB disruption considerably, but did not reverse the ongoing detrimental processes. The demonstrated positive effects of MB were related to a decrease of nitrite/nitrate tissue content, and thus to a decrease of excess NO due to the MB inhibitory effects on NOS isoforms. A mixture of MB in hypertonic sodium lactate (MBL) was investigated to facilitate administration of MB in “the field.” Based on findings that MBL cardio- and neuroprotective properties were similar to those of MBHSD, there is reason to believe that the use of MBL might be extended during ongoing CPR and after ROSC (Paper III). It would therefore make sense to try using MB as a pharmacological neuroprotectant during or after clinical CPR in order to expand the temporal therapeutic window before other measures for neuroprotection such as hypothermia are available.