Intermittent hypoxia (HPX), defined as repeated episodes of hypoxia interspersed with episodes of normoxia, has been observed in different types of sleep-disordered breathing, including obstructive sleep apnea syndrome (OSA) (Lévy et al., 2008).

Repeated episodes of hypoxia elicit changes in a range of physiological responses (Lévy et al., 2008; Foster et al., 2010; Gilmartin et al., 2010; Tamisier et al., 2011). The cumulative effect of intermittent HPX and the chronicity of intermittent HPX may determine whether the response crosses the threshold from having protective value to pathology. Furthermore, the presence of clinical diseases and environmental conditions may interfere with the response to this challenge.

It is not known if acute intermittent HPX–reoxygenation are able to increase polymorphonuclear and peripheral blood mononuclear cell mobilization, enhance superoxide generation in neutrophils, and impair sympato-vagal control and vascular function in healthy volunteers. Indeed, the threshold from physiological to pathophysiological condition may be triggered by inflammation and oxidative stress. It has already been found that the continuum release of a number of pro-inflammatory cytokines by macrophages sensitizes neutrophils and macrophages, thus producing superoxide (Laskin et al., 2011). On the other hand, while the generation of active oxygen species by neutrophils is one of the defense mechanisms against foreign pathogens, in chronic condition it may lead to inflammation and diseases.

In a chronic state, autonomic and hemodynamic changes stimulate the intravascular production of superoxide (Schultz, 2009), promoting inflammation, and damage to the vascular integrity and endothelial cell function (Laskin et al., 2011). Moreover, among subjects with untreated OSA, the release of superoxide from circulating neutrophils, was markedly enhanced when compared to control subjects (Schulz et al., 2000). However, the effect of acute intermittent HPX on immune cells mobilization and activation in healthy humans has not been fully investigated.

OSA has been consistently associated with increased cardiovascular morbidity/mortality (Fu et al., 2016). There is growing evidence that OSA may be directly involved in multiple pathways associated with cardiovascular risk. In this context, autonomic dysfunction seems to play a major role (Trombetta et al., 2013). Recent studies have suggested that chronic and acute intermittent HPX increases sympathetic activation via peripheral chemoreceptors stimulation (Trombetta et al., 2013), acutely increasing arterial blood pressure (BP) (Foster et al., 2010; Gilmartin et al., 2010; Tamisier et al., 2011). Indeed, in OSA patients, regardless of other cardiovascular risk factors, the cumulative effect of the repetitive hypoxia/hypercapnia episodes has been found to lead to an autonomic imbalance shift toward sympathetic tone (Trombetta et al., 2010, 2013; Cepeda et al., 2015), proinflammatory response, (Yokoe et al., 2003; Ryan et al., 2005) endothelial dysfunction (Kato et al., 2000), and chronic oxidative stress (Schulz et al., 2000).

To avoid the effect of pathological chronicity interference and isolate the acute effect of intermittent HPX–reoxygenation in neutrophil-generated superoxide and sympathovagal balance, we chose to investigate the acute intermittent HPX–reoxygenation in in healthy subjects, which was to identify the implication of intermittent HPX as mediator in the genesis of oxidative stress, even in the absence of other pathophysiological changes.

In the present study, we tested the hypothesis that brief cycles of intermittent HPX–reoxygenation are able to increase polymorphonuclear and peripheral blood mononuclear cell mobilization, enhance superoxide generation in neutrophils, and impair sympato-vagal control and vascular function in healthy volunteers.

Baseline characteristics are shown in Table 1. Fifteen healthy subjects (predominantly women) participated in the present study. The volunteers had normal BMI, BP, glucose, lipid profiles and CRP. In addition, all participants were within the normal range of the AHI, based on the polysomnography records (Table 1).

The present investigation explores the role of intermittent HPX, a major component of OSA, in hemodynamic and autonomic responses and in the mobilization and activation of immune cells even in acute response in healthy subjects. The novel finding of our study is that acute exposure to intermittent HPX promoted not only hemodynamic and autonomic impairment in healthy volunteers but also increased superoxide generation in neutrophils.

The major implication of these findings is that intermittent HPX, in the absence of other pathophysiological changes, may be an important mediator in the genesis of oxidative stress in healthy individuals. In fact, in the present study, we found association between O₂ saturation and polymorphonuclear in relative luminescence units (PMNs RLU) and the association between PMNs RLU and peripheral resistance (PR) in response to acute intermittent HPX. However, our results have to be interpreted with caution, because other potential mechanisms could be involved. In addition, a simple correlation between oxygen reactive species and a biological outcome does not always could be interpreted to causality.

The effects of intermittent HPX on several pathways have mainly been explored in animal models of OSA. Several studies have indicated that HPX is related to increased sympathetic activation and BP (Fletcher et al., 1992; Trombetta et al., 2013), as well as to inducing inflammation (Tam et al., 2007), oxidative stress (Jun et al., 2008), insulin resistance (Iiyori et al., 2007), dyslipidemia (Li et al., 2005; Drager et al., 2011a), and atherosclerosis (Savransky et al., 2007). The harmful effects of intermittent HPX have been demonstrated in autonomic and hemodynamic impairment (Foster et al., 2010; Gilmartin et al., 2010; Tamisier et al., 2011) and in insulin resistance (Iiyori et al., 2007). Our findings extend the previous evidence in humans, suggesting that intermittent HPX could trigger superoxide production even in the absence of diseases.

It is well-established that oxidative stress is implicated in vascular dysfunction and atherosclerosis. Several cardiovascular risk factors, such as hypertension, hypercholesterolemia, and diabetes mellitus, stimulate the production of superoxide in the vascular wall (Tousoulis et al., 2011). Accumulated evidence suggests that OSA is associated with increased oxidative stress and endothelial dysfunction. Lavie (2003) and Monneret et al. (2010). Indeed, OSA seems to directly affect the vascular endothelium by promoting inflammation and oxidative stress, while decreasing NO availability and repair capacity (Jelic et al., 2008), which may ultimately contribute to atherosclerosis (Drager et al., 2011b). Our results seems to corroborate these previous investigations. Although responses between health and the chronic “disease” state may be very different, the present study brings to the light the role of intermittent HPX, a hallmark of OSA, in healthy individuals. We found that acute intermittent HPX not only significantly increases the number of polymorphonuclear and peripheral blood mononuclear cells but also augments superoxide generation in polymorphonuclear cells. The precise mechanisms by which intermittent HPX induces oxidative stress through polymorphonuclear cells are yet to be determined. It has been postulated that the episodic hypoxia in OSA leads to the increased production of O₂ and other reactive oxygen species molecules via several enzymatic pathways. For instance, upon stimulation, activation of NADPH oxidase complex in the endothelium and in neutrophils produces a burst of superoxide anions, contributing to oxidative stress and the onset of inflammatory processes (Lavie, 2003). We also cannot rule out the effect of the adrenergic activation triggered by intermittent HPX, as observed in our study on polymorphonuclear cells and oxidative stress. Indeed, several models of stress induction have demonstrated the onset of leukocytosis in response to catecholamines. Iversen et al. (1994) have found an increase in lymphocytes (220%) and neutrophils (160%) in response to epinephrine injection in rats. This effect was quite rapid (5 min or less) and brief, returning to normal 25 min after injection (Iversen et al., 1994). An early rise in leukocyte count is also a feature of ischemia reperfusion injury, as previously demonstrated in experimental studies (Hallenbeck et al., 1986; Grøgaard et al., 1989). Regarding oxidative stress, the role of epinephrine and adrenergic agonists in triggering oxidative stress in cell culture models (Costa Rosa et al., 1992) and animal models has been reported (Zhang et al., 2005). Therefore, the experimental model of acute hypoxia/reoxygenation presently described is capable of inducing a change in autonomic balance resulting in a predominance of sympathetic activity and an increase in white blood cell counts and oxidative stress, resembling several phenomena related to ischemia-reperfusion injury.

Despite these observations, we cannot exclude the possibility that oxidative stress induced by intermittent HPX may, indeed, be a potential trigger for sympathetic activation and increased BP. Evidence in animal models has suggested that increased oxidative stress in the brain, possibly via the activation of NADPH oxidase, may contribute to the progression of hypertension through central sympathoexcitation (Chan et al., 2009; Nagae et al., 2009). These important issues should be further explored in future experiments.

The specific contributions for each authors are: GA, EK, FC-C. Conception design of the work, the acquisition, analysis, interpretation of data for the work, drafting the work and revising it critically for important intellectual content. IT, FC, EH, RC, CM, MI, JB. Interpretation of data for the work; drafting the work, revising it critically for important intellectual content. All above authors read and approved the final version to be published; and all authors agree with all aspects of the work in order to ensure the accuracy and the integrity of the work.

This was not an industry-supported study. The work was performed at the Heart Institute (InCor), University of São Paulo Medical School, São Paulo, Brazil and was supported by Fundação Zerbini. This study was supported by Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP # 04/0222). FXC (FAPESP#2015/17533-6 and #2016/16831-7), RC (FAPESP 2010/02963-2, CNPq 303853/2015-3 and Guggenheim Foundation), CM (CNPq 442374/2014-3 and FAPEMA UNIVERSAL-00358/15 - Edital 40/2014).