Original articleIntermittent hypoxia causes REM sleep deficits and decreases EEG delta power in NREM sleep in the C57BL/6J mouse
Introduction
Obstructive sleep apnea (OSA) is a common disorder affecting 2% of women and 4% of men in the United States [1] and is associated with increased risk of cardiovascular, cerebrovascular, and endocrine diseases [2], [3], [4], [5], [6], [7]. OSA is characterized by recurrent collapse of the upper airway during sleep, leading to periods of intermittent hypoxia (IH) that are often terminated by arousal from sleep, causing fragmentation of normal sleep architecture [8]. Many of the pathophysiological sequelae of OSA have been attributed to the effects of IH [9], [10].
Several animal models have been developed to study the effects of IH on a variety of cardiovascular, metabolic, and neurocognitive outcomes. The most sophisticated models have utilized either airway obstruction or the delivery of hypoxic gases with the onset of sleep and subsequent removal of the stimulus when arousal or wakefulness occurs [11], [12], [13], [14]. However, the need to constantly assess sleep state has resulted in the majority of studies utilizing rodent models of IH that are not dependent on the presence of sleep. The approach is to expose rodents to periods of hypoxia of fixed duration (usually in the range of 30–120 s) throughout the 12 h of the sleeping or light phase and maintain normoxia during the active or dark phase. Several studies have assessed the effects of IH on various aspects of polysomnography. In adult rats, IH caused an initial overall reduction in non-rapid eye movement sleep (NREMS) and rapid eye movement sleep (REMS) in the light phase that normalized within 2 days [15]. In neonatal rats, mild IH resulted in a decrease in wakefulness and an increase in REMS 3 weeks after exposure [16]. In mice, exposure to IH for 8 weeks resulted in an increase in NREMS, whereas the amount of REMS was unchanged by the end of the exposure and increased 2 weeks after cessation of the exposure [17]. Thus, it has been previously shown that IH has a significant impact on sleep architecture.
However, several questions concerning the relevance of IH models for studying the hypoxic stress of OSA remain unanswered. For example, the hypoxic period of OSA typically terminates in arousal [18], yet, in the animal model, it is not clear how frequently IH results in arousal and how often hypoxic episodes occur during wakefulness. In addition, it is not known whether exposure to more severe short-term IH than previously reported [15], [17] will result in more pronounced disruption of sleep architecture. Most importantly, it is unclear whether the changes in sleep architecture observed in the previous studies [15], [17] were induced by non-specific sleep fragmentation (SF), rather than hypoxia per se.
The major purpose of the current study was to assess and compare the impact of short-term severe IH and non-hypoxic SF on arousal and sleep architecture. Our approach was to continuously record sleep architecture in chronically instrumented mice exposed to 5 days of IH and 5 days of non-hypoxic SF elicited by an auditory/tactile stimulus. We hypothesized that intermittent exposure to both hypoxic and auditory/tactile stimuli throughout the 12-h light phase will (1) produce a pattern of sleep and arousal that simulates human OSA and (2) result in significant disruption of both NREMS and REMS.
Section snippets
Animals
Five male C57BL/6J mice from Jackson Laboratory (Bar Harbor, ME), weighing 27–30 g, were used in the study. The study was approved by the Johns Hopkins University Animal Use and Care Committee and complied with the American Physiological Society guidelines [19]. For all surgical procedures, anesthesia was induced and maintained using isoflurane (1–2%) administered through a face mask. At the completion of experiments, animals were euthanized with pentobarbital sodium (60 mg ip).
Polysomnography and housing chamber
In anesthetized
Event analyses
The number of arousals/h of sleep induced by IH during Days 1–5 of exposure is shown in Table 1. On the first day of IH, the animals were asleep at the beginning of a period of hypoxia for 44±10.3% of the events (Table 1). However, over time there was a significant (P<0.01) increase in the number of hypoxic episodes initiated with the animal asleep, such that by Day 5 of IH 64±6.2% of the episodes occurred during sleep.
During episodes of IH that were initiated with the animal asleep, there was
Discussion
The purpose of our study was to compare the effects of IH and non-hypoxic SF administered during the 12-h light phase on sleep architecture in C57BL/6J mice. Several new findings resulted from the study. First, we demonstrated that during IH the likelihood of being asleep at the onset of a hypoxic episode increased with time of exposure and that the stimulus was almost 100% effective at inducing arousal. Second, IH caused significant disruption in sleep during both the light phase, when the
Conclusion and clinical implications
We have verified that the application of IH and non-hypoxic SF during the light phase in mice leads to a pattern of repetitive disruption of sleep, similar that in human OSA. A normalization of total NREMS during both the light and dark phases occurred over the course of 5 days, but the delta power of NREMS and the amount of REMS remain impaired during the hypoxic exposure. In contrast, non-hypoxic SF did not cause a significant reduction in either EEG delta power or REMS. Our data suggest that
Acknowledgements
This work was supported by National Heart, Lung and Blood Institute Grants K08 HL68715 to V.Y. Polotsky, F32 HL71469 to A.E. Rubin, and R01 HL63767 and R01 HL66324 to C.P. O'Donnell.
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