Faculty of Science School of Physiology 9808215t romainh@physiology.wits.ac.za / There is a high prevalence of insulin and leptin resistance and increased cortisol concentrations in sleep apnoea patients, independent of obesity. Chronic intermittent hypoxia is used an experimental animal model to simulate the hypoxia occurring in sleep apnoea patients. The aim of this study was to measure plasma insulin and leptin concentrations and hypothalamic-pituitary-axis activity in rats exposed to either intermittent hypoxia (CIH) or sham hypoxia (SH) for fourteen days. To induce CIH plexiglass cylinders were flushed with 100% nitrogen for nine seconds every 90 seconds, seven hours/day. The rats were weighed each day during the exposure period. Venous blood samples for insulin and leptin were collected on days one, three, five, eight and fifteen. Faecal samples were collected to measure glucocorticoid metabolites. There was no significant difference in the daily change in body weight between the rats exposed to CIH compared to the rats exposed to SH (unpaired t-test). Plasma insulin concentrations were not affected by CIH. In both groups of rats plasma leptin concentrations were significantly higher on day fifteen compared to day five (p=0.03, unpaired t-test). Glucocorticoid metabolites were significantly increased in the intermittent hypoxia group on day two (p=0.003 one-way ANOVA). In conclusion, exposing normal weight rats to CIH for fourteen days resulted in a transient iv increase in HPA axis activity on day two and an elevation in plasma leptin levels, in both groups of rats, at day fifteen.

Many experimental therapies have been used in the search for effective approaches to improve recovery after spinal cord injury (SCI). One of the most promising approaches is the augmentation of spontaneously occurring plasticity in uninjured neural pathways. Acute intermittent hypoxia (AIH-brief exposures to reduced O2 levels alternating with normal O2 levels) elicits plasticity in respiratory and non-respiratory spinal systems in experimental animals. AIH treatment has also been shown to improve walking abilities in persons with chronic incomplete SCI. In this thesis, I first examined the effect of AIH treatment, alone or in combination with motor training, on functional recovery in a rat model of incomplete cervical SCI. Second, I examined the effect of AIH on the expression of plasticity- and hypoxia-related proteins in the spinal cords of SCI rats. In a randomized, blinded, normoxia-controlled study, rats were trained to cross a horizontal ladder and footslip errors were measured before surgery for SCI, 4 wks post-surgery, each day of daily AIH treatment, and 1, 2, 4 and 8 weeks after treatment. dAIH treatment consisted of 10 episodes of AIH: (5 min 11% O2: 5 min 21% O2) for 7 days beginning at 4 wks post-SCI. AIH-treated rats made fewer footslips on the ladder task compared to normoxia-treated control rats after 4 days of treatment and this improvement was sustained for 8 wks post-treatment. Importantly, daily ladder training was required for AIH treatment to facilitate recovery. AIH treatment + motor training also increased the expression of Hypoxia-inducible factor-1α (HIF-1α), Vascular endothelial growth factor (VEGF), Brain-derived neurotrophic factor (BDNF), tyrosine kinase B receptors (trkB) and phospho-trkB in spinal motor neurons in SCI rats compared to normoxia-treated SCI rats. In particular these hypoxia- and plasticity-related proteins were differentially expressed both temporally and spatially in the spinal cord during AIH treatment. These findings demonstrate that AIH + motor training can augment neural plasticity and improve motor recovery in an animal model of SCI. Taken together with the promising findings from human SCI studies, the results of this thesis suggest that AIH has potential as an effective therapy to restore motor function after nervous system injury.

Tumors need the development of new vessels from the pre-existing vasculature to bring nutrients and oxygen to the whole tumor mass. The tumor vascular network is known to be poorly functional due to architectural and functional abnormalities. The end result is an inadequate and heterogeneous tumor perfusion leading to the development of tumor hypoxia. From a therapeutic perspective, hypoxia is a source of radioresistance and the dysfunctional perfusion hampers drug delivery. Historically, tumor hypoxia refers to chronic hypoxia (or diffusion-limited hypoxia) that results from the increasing distance between O2-consuming cells and blood vessels due to the high metabolic rate of tumor cells. Many studies have demonstrated the impact of chronic hypoxia on the clonal selection of tumor cells resistant to conventional anti-cancer therapies. Growing evidence for the existence of another form of hypoxia caused by heterogeneities in tumor perfusion, namely acute or perfusion-limited hypoxia, plead however for a non-genetic source of phenotype conversion reaching not only tumor cells but also the tumor vasculature and in particular endothelial cells. In the cardiovascular field, the cyclic exposure to different pO2 levels is known to precondition cardiac myocytes to resist more prolonged ischemic insults. We hypothesized that this concept of myocardium preconditionning to promote the resistance vs pro-apoptotic stresses could be translated in tumors. Indeed, intermittent hypoxia in tumors is nothing else than cyclic changes in pO2 and radio- and chemotherapy can be viewed as pro-apoptotic stresses that the tumor can face. In particular, in the case of the tumor vasculature, the resistance could be a capacity to re-initiate angiogenesis after treatment. Radioresistance would be further potentiated since low pO2 is per se associated to reversibility of the damages. Also, since intermittent hypoxia is thought to be due in part to fluctuations in tumor blood flow (TBF), access of chemotherapy to the tumor could also further participate to chemoresistance. To address the above hypotheses, we first aimed to explore the extent and the origin of TBF fluctuations in tumor mouse models and to determine whether therapeutic modulation of such potential TBF heterogeneities could improve the efficacy of chemotherapy. We then more directly examined whether and how intermittent hypoxia could influence endothelial cell survival and modulate resistance to radiotherapy. We also took advantage of this study to dissect the molecular mechanisms driving the phenotype conversion of endothelial cells exposed to intermittent hypoxia. Finally, because VEGF plays a major role in hypoxia-mediated angiogenesis but also regulates major pro-survival pathways in endothelial cells, we evaluated the potential role of caveolin as a new therapeutic target to tackle EC resistance. Caveolin is, indeed, a key structural protein recently documented to interact with many downstream targets of VEGF. 1. To explore the extent and the origin of TBF fluctuations in tumor mouse models and to determine whether therapeutic modulation of such potential TBF heterogeneities could improve chemotherapy. We focused this part of the work on the vascular tone modulator endothelin-1. Indeed, this peptide is over-expressed in many mouse and human tumors where it is documented to act as a mitogenic factor in both para- and autocrine manners. Endothelin-1 is also a potent vasoconstrictor acting through the ETA receptors located on VSMCs. In our lab, we previously showed that over-expression of endothelin-1 in tumors accounted for the development of a myogenic tone within the tumor vasculature. We have now documented that an ETA receptor antagonist induces the relaxation of microdissected tumor arterioles and selectively and quantitatively increases tumor blood flow in experimental tumor models. We also combined dye staining of functional vessels, fluorescent microsphere-based mapping, and magnetic resonance imaging to identify heterogeneities in tumor blood flow and to examine the reversibility of such phenomena. We showed that administration of an ETA receptor antagonist reduces the extent of underperfused tumor areas, proving the key role of vessel tone variations in tumor blood flow heterogeneity. We also provided evidence that ETA antagonist could improve the access of cyclophosphamide to the tumor compartment and thereby induces a significant tumor growth delay. 2. To examine whether and how intermittent hypoxia could influence endothelial cell survival and modulate resistance to radiotherapy. To dissect the mechanisms driving the phenotype conversion of endothelial cells exposed to intermittent hypoxia. This second part of our work, is a comprehensive investigation of the consequences of intermittent hypoxia, as caused by TBF heterogeneities, on the endothelial cell phenotype. First, we postulated that intermittent hypoxia (IH) favors endothelial cell (EC) survival, thereby extending the concept of hypoxia-driven resistance to the tumor vasculature. We showed that exposing EC to cycles of hypoxia/re-oxygenation reduces radiation-induced cell death and promotes angiogenesis. In contrast, prolonged hypoxia failed to achieve such protection and even appeared deleterious. We also observed that although HIF-1£ is completely degraded during each re-oxygenation, its abundance is paradoxically found higher at each new hypoxic challenge. Moreover, the use of siRNA targeting HIF-1£ pointed out that HIF-1ƓÑ accumulation account for the increased resistance of EC to radiotherapy. Finally, we extended this concept in vivo by forcing IH in tumor-bearing mice and found that it is associated with less radiation-induced apoptosis within both the vascular and the tumor cell compartments (vs normoxia or prolonged hypoxia). Next, we focused our work on the underlying mechanisms of EC phenotype conversion exposed to IH and particularly on potential actors that may favor HIF-1£ accumulation during IH. Prolylhydroxylases (PHD), MAPK and PI3K/Akt pathways as well as eNOS are known to regulate HIF-1£ abundance and transcriptional activity. We documented that PHD2 and PHD3 abundance are slightly decreased during IH, whereas prolonged hypoxia increases PHD3 expression in EC. We then showed that, ERK, Akt as well as eNOS were phosphorylated during reoxygenation periods of the IH protocol. We also used specific inhibitors of these cascades (i.e. PD98059, LY294002 and L-NAME, respectively), to evaluate their specific impact on HIF-1£ abundance and performed clonogenic assays to evaluate their consequences on EC survival. We showed that although, PD98059 and LY294002 sensitizes EC to pro-apoptotic stresses, only the PI3K/Akt inhibitor abrogates the HIF-1£ signal during IH. Conversely, L-NAME, a non-specific NOS-inhibitor, appears to potentiate the expression of HIF-1£ and to favor the EC survival. 3. To identify new therapeutic targets to prevent endothelial cell resistance by studying VEGF signaling, the major pro-survival and pro-angiogenic growth factor in endothelial cells. Because VEGF plays a central role in hypoxia-mediated angiogenesis and cell survival, the VEGF signaling cascade is a an obvious therapeutic target. To more specifically identify the pathways leading to cell survival and the resistance phenomena that we observed in response to intermittent hypoxia, a careful dissection of the downstream VEGF signaling cascades was performed. In this part of the work, we focused our attention on caveolin since it modulates the activity of eNOS, ERK and Akt that are major effectors acting downstream VEGF stimulation. We demonstrated the paradoxical role of caveolin-1 preventing signaling in basal conditions and ensuring the coupling between VEGFR2 and the downstream cascades upon VEGF stimulation. We used mice deficient for the caveolin-1 gene (Cav-/-) to examine the impact of caveolae suppression in a model of adaptive angiogenesis obtained after femoral artery resection. Evaluation of the ischemic tissue perfusion and histochemical analyses revealed that contrary to Cav+/+ mice, Cav-/- mice fails to recover a functional vasculature and actually loose part of the ligated limbs. We also isolated endothelial cells (ECs) from Cav-/- aorta and showed that on VEGF stimulation, endothelial tube formation is dramatically abrogated when compared with Cav +/+ ECs. The Ser1177 eNOS phosphorylation and Thr495 dephosphorylation but also the ERK phosphorylation were similarly altered in VEGF-treated Cav-/- ECs. Interestingly, caveolin transfection in Cav-/- ECs redirected the VEGFR-2 in caveolar membranes and restored the VEGF-induced ERK and eNOS activation. However, when high levels of recombinant caveolin are reached, VEGF exposure fails to activate ERK and eNOS. Altogether, these data identify caveolin as a new therapeutic target to alter VEGF signaling, in particular the cascades leading to angiogenesis and resistance to stresses.

No description available.

Background: Obstructive sleep apnea (OSA) consists of repeated, involuntary breathing suspension during sleep. These events induce rapid depletion/repletion of blood/tissue oxygen content, a phenomenon known as intermittent hypoxia. Aside from causing daytime sleepiness, the most important health consequence of OSA is a 2-fold increase in cardiovascular (CVD) risk. Animal studies provide evidence that intermittent hypoxia, a simulating model of OSA, causes important rise in plasma TG, especially in the postprandial state. However, the underpinning mechanisms linking intermittent hypoxia to altered postprandial TG levels remain unknown. As such, the objective of this study was to characterize the effects of acute intermittent hypoxia on postprandial TG levels in 2 distinct lipoprotein subtypes in humans: chylomicrons which are secreted by the intestine and carry dietary lipids, and denser TG carriers (mainly VLDL) which are secreted by the liver and carry endogenous lipids. Methods: The research consisted of a randomized crossover design. In collaboration with the Sleep laboratory at Montfort Hospital, 7 individuals diagnosed with moderate sleep apnea were recruited through phone calls as well as 8 healthy individuals without OSA from the University of Ottawa. While lying on a bed, participants were given a meal after which they were exposed for 6 hours to normoxia or intermittent hypoxia corresponding to moderate OSA, e.g. 15 hypoxic events per hour. Blood lipid levels were measured hourly.  Results: Plasma TG levels increased over time in both experimental conditions and tended to be greater under 6-h exposure to intermittent hypoxia (p=0.093, effect size ηp2= 0.383.). This trend toward higher total plasma TG under intermittent hypoxia was attributable to increased levels in denser TG carrying lipoproteins such as VLDL and CM remnants (p= 0.009, ηp2 = 0.173).  Conclusion: Acute intermittent hypoxia, a simulating model of obstructive sleep apnea, tends to negatively affect postprandial TG levels, which is attributable to an increase in denser TG carrying lipoprotein levels such as VLDL and CM remnants. These results lend support to the increase in blood lipid levels in animal studies observing the effect of acute hypoxia in mice.  Contribution to advancement of knowledge: This proposed research will allow a better understanding of the mechanisms by which obstructive sleep apnea may alter blood lipid profile. This information will be beneficial to the treatment of obstructive sleep apnea related dyslipidemia and contribute to reduce CVD risk in the large proportion of obstructive sleep apnea patients who are reluctant to current treatment avenues.

A hipóxia crônica intermitente (HCI) promove hipertensão arterial e aumento da atividade simpática em ratos jovens. Nestes animais, o aumento da atividade simpática ocorre durante uma fase específica da respiração e está correlacionada com o aumento da atividade expiratória. Estas evidências mostram que, após a HCI, os ratos jovens desenvolvem alterações no acoplamento simpático-respiratório, um mecanismo que pode contribuir, pelo menos em parte, para o desenvolvimento da hipertensão observada nestes animais. Em diversos modelos experimentais de hipertensão, os níveis de pressão arterial são menores nas fêmeas do que nos machos, portanto elas são resistentes ao desenvolvimento da hipertensão. Levando em conta essas informações, a hipótese do presente projeto foi a seguinte: fêmeas submetidas à HCI seriam resistentes ao desenvolvimento da hipertensão arterial. Para tanto, ratas jovens e adultas foram submetidas à HCI e seus parâmetros cardiovasculares e respiratórios foram avaliados na condição dos animais acordados e com livre movimentação. Além disso, as alterações na atividade simpática e respiratória também foram avaliadas na preparação coração tronco-cerebral isolados. Os resultados mostram que as fêmeas desenvolvem hipertensão semelhante aos machos submetidos ao mesmo protocolo, contudo as alterações no acoplamento entre a atividade simpática e respiratória são diferentes entre os sexos. Estes dados sugerem que embora os machos e fêmeas desenvolvam um nível similar de hipertensão após a HCI, os mecanismos envolvidos na geração da atividade simpática podem ser diferentes. / Chronic intermittent hypoxia (CIH) produces hypertension and sympathetic overactivity in juvenile male rats. The increase in sympathetic activity occurs during the expiratory phase of respiration, which is correlated with an augmented expiratory activity. This information indicates that after CIH, juvenile rats develop changes in the respiratory sympathetic-coupling, which could explain, at least in part, the hypertension observed in these animals. Female rats are known to be more resistant to the development of hypertension in several experimental models of this pathology. Take in consideration these facts, we hypothesized that female rats exposed to CIH were resistant to the development of hypertension. To test this hypothesis, we studied the cardiovascular and respiratory changes in female rats after the CIH in awake freely moving condition. In another group of animals, we studied the sympathetic and respiratory activity in female rats after the CIH, using for this, the working heartbrainstem preparation. The results show that juvenile female rats develop hypertension similarly to that observed in juvenile male rats submitted to the same protocol. However, juvenile female rats presented changes in the respiratory-sympathetic coupling different from that observed in juvenile male rats. Together, these results suggest that even if the level of hypertension after CIH is similar between males and females, the mechanisms underlying the generation of sympathetic overactivity are different.

Obstructive sleep apnea is a potentially life-threatening condition characterized by repetitive episodes of upper airway obstruction that occur during sleep, usually associated with a reduction in blood oxygen saturation. In US population, 9% of women, 24% of men, and 2% of children have been diagnosed with obstructive sleep apnea, suggesting that 18 million people may suffer from the consequences of nightly episodes of apnea. One of the most significant symptoms of obstructive sleep apnea is profound and repeated hypoxia. The analysis of the interaction between cardiovascular and respiratory signals has been a widely-explored area of research due to the significance of the results in describing a functional relationship between the underlying physiologic systems; however, statistical and analytical approaches to analyze the changes in these signals before and after hypoxia are still in their early stages of evolution. A major motivation for this research has been the lack of methodologies to detect mean and/or variance shifts and identify root sources of variation in time-frequency characteristics of multichannel data. The contributions of this thesis are twofold. First, multiscale energy distributions based on wavelet transformations of the analyzed physiological signs are analyzed. This is followed by the development of an online multichannel monitoring approach based on principal curves that detects changes in the wavelet coefficients extracted from the analyzed signals.

L'apnée du sommeil (AS) induit des variations constantes d'oxygénation artérielle (hypoxie intermittente – HI) qui affectent environ 5 à 7 % de la population. Il se produit une augmentation du stress oxydatif (production d'espèces réactives de l'oxygène - ROS), augmentant les risques cardiovasculaires, neurologiques et métaboliques. Les études épidémiologiques démontrent que la prévalence d'AS est inférieure chez les femmes que chez les hommes, mais après la ménopause la prévalence augmente pour atteindre le même niveau que chez les hommes. L'oestradiol (E2) est un puissant agent antioxydant, mais son rôle éventuel dans le traitement ou la prévention de l'AS n'est pas exploité. Toutefois, l'oestradiol (associé ou non à la progestérone) permet de réduire l'AS chez les femmes ménopausées. Les ROS peuvent être produites par les mitochondries, la NADPH oxydase et/ou la Xanthine oxydase. La mitochondrie est le plus important producteur de ROS (90% de l'oxygène consommé) et son dysfonctionnement est très préjudiciable. L'oestradiol est une cible de la mitochondrie à travers ses récepteurs nucléaires alpha et bêta (ERα et ERβ) qui sont capables de moduler le fonctionnement de la mitochondrie et diminuer la production de ROS. Nous avons testé l'hypothèse dans un modèle animal ovariectomisé exposé à une HI, que l'estradiol et ses agonistes spécifiques ERα et Erβ sont capables de limiter le stress oxydatif cérébral, la dysfonction mitochondriale et l'apparition des désordres systémiques. Nos résultats ont permis de montrer que l'estradiol est capable d'éviter l'augmentation de la pression artérielle et la survenue de désordres respiratoires causés par l'HI. De plus, l'HI augmente le stress oxydatif cérébral en augmentant l'activité d'enzymes pro-oxydantes et en diminuant l'activité d'enzymes antioxydantes. L'estradiol permet de prévenir l'augmentation du stress oxydatif. On retrouve également une dysfonction de la chaine respiratoire mitochondriale dans le cortex en HI qui est préservée de manière différente par le traitement avec les modulateurs sélectifs des récepteurs ERα et ERβ (SERMs). Nous avons montré que ERβ joue un rôle dans le contrôle cardio-respiratoire et la fonction mitochondriale dans le cerveau. Nos résultats apportent une meilleure compréhension du rôle de l'estradiol comme agent protecteur contre l'apnée du sommeil et ses conséquences associées. L'utilisation d'agonistes spécifiques nous renseigne sur le rôle que tient chaque récepteur dans la protection induite par l'estradiol contre la dysfonction mitochondriale. L'utilisation du remplacement hormonal avec de l'estradiol ou des SERMs peut constituer une thérapie efficace contre l'apnée du sommeil et ses conséquences / Sleep apnea (SA) induces constant changes of arterial oxygenation (Intermittent hypoxia - IH) that affect about 5 to 7% of the general population. IH increases oxidative stress (production of reactive oxygen species – ROS) and lead to cardiovascular, neurological and metabolic risks. Epidemiological studies show that the prevalence of SA is lower in women than in men, but after menopause the prevalence increases to the same level that in men. Estradiol (E2) is a potent antioxidant, but its potential role in the treatment or prevention of SA is not exploited. However, estradiol (with or without progesterone) can reduce SA in postmenopausal women. ROS can be produced by mitochondria, NADPH oxidase and/or Xanthine oxidase. Mitochondria is the most important producer of ROS (90% of oxygen consumed) and its dysfunction is very detrimental. Estradiol is a target of mitochondria through its mitochondria alpha and beta (ERα et ERβ) that are able to modulate mitochondrial function and decrease ROS production. We tested the hypothesis in ovariectomized animal model exposed to IH, that estradiol and its specific receptor ERα and ERβ agonists are able to limit cerebral oxidative stress, mitochondrial dysfunction and the appearance of systemic disorders. Our results have shown that estradiol is able to avoid the increase of blood pressure and the occurrence of respiratory disorders caused by IH. Furthermore, IH increases cerebral oxidative stress by increasing activity of pro-oxidant enzymes and decreasing activity of antioxidant enzymes. Estradiol prevents against the increase of oxidative stress. There is also a mitochondrial respiratory chain dysfunction in the cortex by IH, that is preserved differently by treatment with selective ERα and ERβ receptor modulators (SERMs). We have shown that ERβ plays an important role in cardiorespiratory control and mitochondrial function in the brain. Our results provide a better understanding of the role of estradiol as a protective agent against sleep apnea and its associated consequences. The use of specific agonists informs us on the role of each receptors in estradiol-induced protection against mitochondrial dysfunction. The use of hormone replacement with estradiol or SERMs may be an effective therapy against sleep apnea and its consequences

Introdução: Apneia do sono é uma doença respiratória crônica com alta prevalência que causa múltiplas interrupções respiratórias, levando à hipóxia intermitente (HI). A HI culmina com a geração de radicais livres, estresse oxidativo, inflamação e esteatose hepática. A Melatonina (MEL) e N-acetilcisteína (NAC), são potentes antioxidantes, capazes de inibir esses radicais livres e o estresse oxidativo. Objetivos: Investigar o mecanismo de inflamação em um modelo de hipóxia intermitente que simule a apneia do sono, avaliando-se o fígado e o pulmão, as respostas aos tratamentos com MEL e NAC frente às alterações oxidativas e inflamatórias no fígado de camundongos. Métodos: Utilizamos 120 camundongos machos, adultos, divididos em três experimentos: avaliação do modelo experimental (n=36), avaliação inflamatória molecular em fígado e pulmão (n=12) e avaliação molecular em camundongos com uso de antioxidantes (n=72). Para a hipóxia intermitente foi utilizado o sistema de câmaras que mantêm os roedores em um equipamento que simula a apneia do sono, durante oito horas diárias. No primeiro experimento avaliaram-se as alterações hepáticas em dois momentos, com 21 dias de exposição e 35 dias de exposição à HI. Nos demais experimentos foram utilizados o mesmo sistema durante 35 dias de exposição. No terceiro experimento, a partir do 21° dia iniciaram-se a administração intraperitoneal dos antioxidantes (MEL-200uL/Kg) e NAC-10mg/Kg). Resultados: Foi verificado que o tempo de 21 dias de exposição, não foi encontrado alterações nos fígados dos camundongos. Nos animais expostos durante 35 dias à HI, contatou-se a presença de estresse oxidativo, com aumento de dano oxidativo a lipídios e ao DNA e a redução das defesas antioxidantes, e aumento significativo de metabólitos de óxido nítrico (NO), além da presença de lesão tecidual na histologia hepática. Nos pulmões e nos fígados dos camundongos submetidos à HI, contatou-se a presença de estresse oxidativo, e aumento de expressão de fatores de transcrição: hipóxia induzível (HIF-1α), nuclear (NF-κB) e necrose tumoral (TNF-α), como mediadores inflamatórios, bem como elevação da expressão da óxido nítrico sintase induzível (iNOS) e fator de crescimento vascular endotelial (VEGF), como mediadores de resposta vascular, e Caspase 3 clivada, como enzima responsável pela apoptose. Nos animais que foram tratados com MEL e NAC, houve redução significativa, nos fígados, de todas proteínas que apresentaram-se elevadas expressões do grupo exposto sem tratamento, assemelhando-se aos controles. Conclusão: Sugerimos que o tempo necessário de hipóxia intermitente, que simule a apneia do sono, para lesão hepática e estresse oxidativo seja de 35 dias, nesse tempo de exposição sabemos que tanto o pulmão quanto o fígado possuem estresse oxidativo, inflamação e apoptose, e que o uso de Melatonina e N-acetilcisteína foram capazes de proteger os fígados dessas agressões. / Introduction: Sleep apnea is a chronic respiratory disease with high prevalence causing multiple interruptions in breathing, leading to intermittent hypoxia (IH). The IH culminates with the generation of free radicals, oxidative stress, inflammation and hepatic steatosis. Melatonin (MEL) and N-acetylcysteine (NAC) are potent antioxidants, capable of inhibiting these free radicals and oxidative stress. Objectives: To investigate the mechanism of inflammation in a model of intermittent hypoxia that simulates sleep apnea, evaluating the liver and lung, responses to treatment with MEL and NAC front of oxidative and inflammatory changes in the liver of mice. Methods: We used 120 male mice, adults, divided into three experiments: evaluation of the experimental model (n = 36), inflammatory molecular assessment in liver and lung (n = 12) and molecular evaluation in mice with antioxidants (n = 72) . For intermittent hypoxia was used to maintain camera system rodents in a device that simulates sleep apnea during eight hours. The first experiment evaluated the hepatic changes in two stages, with 21 days of exposure and 35 days of exposure to IH. In other experiments we used the same system for 35 days of exposure. In the third experiment, from day 21 began intraperitoneally administration of antioxidants (MEL-200uL/Kg and NAC-10mg/Kg). Results: It was found that the time of exposure of 21 days, no changes were found in the livers of mice. In animals exposed for 35 days to IH, contacted the presence of oxidative stress, with increased oxidative damage to lipids and DNA and reduction of antioxidant defenses, and a significant increase of metabolites of nitric oxide (NO), and the presence of tissue injury in liver histology. In the lungs and livers of mice subjected to IH, contacted the presence of oxidative stress, and increased expression of transcription factors: hypoxia inducible (HIF-1α), nuclear (NF-κB) and tumor necrosis factor (TNF-α), such as inflammatory mediators and increase the expression of inducible nitric oxide synthase (iNOS) and vascular endothelial growth factor (VEGF), vascular response mediators, and cleaved Caspase 3 as enzyme responsible for apoptosis. In animals treated with NAC and MEL, a significant reduction in the livers of all proteins that were elevated expression of the exposed untreated, similarly to controls. Conclusion: We suggest that the time required for intermittent hypoxia, simulating sleep apnea, to liver damage and oxidative stress is 35 days exposure at this time we know that both the lungs and the liver have oxidative stress, inflammation and apoptosis, and the use of Melatonin and N-acetylcysteine were able to protect the livers of these aggressions.

Introdução: Apneia do sono é uma doença respiratória crônica com alta prevalência que causa múltiplas interrupções respiratórias, levando à hipóxia intermitente (HI). A HI culmina com a geração de radicais livres, estresse oxidativo, inflamação e esteatose hepática. A Melatonina (MEL) e N-acetilcisteína (NAC), são potentes antioxidantes, capazes de inibir esses radicais livres e o estresse oxidativo. Objetivos: Investigar o mecanismo de inflamação em um modelo de hipóxia intermitente que simule a apneia do sono, avaliando-se o fígado e o pulmão, as respostas aos tratamentos com MEL e NAC frente às alterações oxidativas e inflamatórias no fígado de camundongos. Métodos: Utilizamos 120 camundongos machos, adultos, divididos em três experimentos: avaliação do modelo experimental (n=36), avaliação inflamatória molecular em fígado e pulmão (n=12) e avaliação molecular em camundongos com uso de antioxidantes (n=72). Para a hipóxia intermitente foi utilizado o sistema de câmaras que mantêm os roedores em um equipamento que simula a apneia do sono, durante oito horas diárias. No primeiro experimento avaliaram-se as alterações hepáticas em dois momentos, com 21 dias de exposição e 35 dias de exposição à HI. Nos demais experimentos foram utilizados o mesmo sistema durante 35 dias de exposição. No terceiro experimento, a partir do 21° dia iniciaram-se a administração intraperitoneal dos antioxidantes (MEL-200uL/Kg) e NAC-10mg/Kg). Resultados: Foi verificado que o tempo de 21 dias de exposição, não foi encontrado alterações nos fígados dos camundongos. Nos animais expostos durante 35 dias à HI, contatou-se a presença de estresse oxidativo, com aumento de dano oxidativo a lipídios e ao DNA e a redução das defesas antioxidantes, e aumento significativo de metabólitos de óxido nítrico (NO), além da presença de lesão tecidual na histologia hepática. Nos pulmões e nos fígados dos camundongos submetidos à HI, contatou-se a presença de estresse oxidativo, e aumento de expressão de fatores de transcrição: hipóxia induzível (HIF-1α), nuclear (NF-κB) e necrose tumoral (TNF-α), como mediadores inflamatórios, bem como elevação da expressão da óxido nítrico sintase induzível (iNOS) e fator de crescimento vascular endotelial (VEGF), como mediadores de resposta vascular, e Caspase 3 clivada, como enzima responsável pela apoptose. Nos animais que foram tratados com MEL e NAC, houve redução significativa, nos fígados, de todas proteínas que apresentaram-se elevadas expressões do grupo exposto sem tratamento, assemelhando-se aos controles. Conclusão: Sugerimos que o tempo necessário de hipóxia intermitente, que simule a apneia do sono, para lesão hepática e estresse oxidativo seja de 35 dias, nesse tempo de exposição sabemos que tanto o pulmão quanto o fígado possuem estresse oxidativo, inflamação e apoptose, e que o uso de Melatonina e N-acetilcisteína foram capazes de proteger os fígados dessas agressões. / Introduction: Sleep apnea is a chronic respiratory disease with high prevalence causing multiple interruptions in breathing, leading to intermittent hypoxia (IH). The IH culminates with the generation of free radicals, oxidative stress, inflammation and hepatic steatosis. Melatonin (MEL) and N-acetylcysteine (NAC) are potent antioxidants, capable of inhibiting these free radicals and oxidative stress. Objectives: To investigate the mechanism of inflammation in a model of intermittent hypoxia that simulates sleep apnea, evaluating the liver and lung, responses to treatment with MEL and NAC front of oxidative and inflammatory changes in the liver of mice. Methods: We used 120 male mice, adults, divided into three experiments: evaluation of the experimental model (n = 36), inflammatory molecular assessment in liver and lung (n = 12) and molecular evaluation in mice with antioxidants (n = 72) . For intermittent hypoxia was used to maintain camera system rodents in a device that simulates sleep apnea during eight hours. The first experiment evaluated the hepatic changes in two stages, with 21 days of exposure and 35 days of exposure to IH. In other experiments we used the same system for 35 days of exposure. In the third experiment, from day 21 began intraperitoneally administration of antioxidants (MEL-200uL/Kg and NAC-10mg/Kg). Results: It was found that the time of exposure of 21 days, no changes were found in the livers of mice. In animals exposed for 35 days to IH, contacted the presence of oxidative stress, with increased oxidative damage to lipids and DNA and reduction of antioxidant defenses, and a significant increase of metabolites of nitric oxide (NO), and the presence of tissue injury in liver histology. In the lungs and livers of mice subjected to IH, contacted the presence of oxidative stress, and increased expression of transcription factors: hypoxia inducible (HIF-1α), nuclear (NF-κB) and tumor necrosis factor (TNF-α), such as inflammatory mediators and increase the expression of inducible nitric oxide synthase (iNOS) and vascular endothelial growth factor (VEGF), vascular response mediators, and cleaved Caspase 3 as enzyme responsible for apoptosis. In animals treated with NAC and MEL, a significant reduction in the livers of all proteins that were elevated expression of the exposed untreated, similarly to controls. Conclusion: We suggest that the time required for intermittent hypoxia, simulating sleep apnea, to liver damage and oxidative stress is 35 days exposure at this time we know that both the lungs and the liver have oxidative stress, inflammation and apoptosis, and the use of Melatonin and N-acetylcysteine were able to protect the livers of these aggressions.