Online Issues

Journal Vol 50 (3)

Insight from Animal Models into the Cognitive Consequences of Adult Sleep-Disordered Breathing

Sigrid Veasey

Sigrid Veasey, MD, is an associate professor of medicine in the Department of Medicine at the University of Pennsylvania in Philadelphia.

Address correspondence and reprint requests to Dr. Sigrid Veasey, Translational Research Laboratories, University of Pennsylvania, 125 South 31st Street, Philadelphia, PA 19104 or email veasey@mail.med.upenn.edu.

Abstract

Obstructive sleep apnea (OSA) afflicts tens of millions of Americans and hundreds of millions of people worldwide, and the possibility that the disease may cause permanent neural injury is therefore a significant concern. Numerous comorbidities—including diabetes, cardiovascular disease, and obesity—are associated with the disease, and it is quite difficult, if not impossible, in clinical studies to determine whether they increase the propensity for neural injury or whether OSA alone causes such injury. It is nonetheless clear that the severity of hypoxemia in sleep apnea correlates with the severity of cognitive impairments, and animal models of OSA have been instrumental in elucidating the potential for this disease to elicit neurobehavioral impairment independent of comorbidities. At present, there is no animal model of severe OSA with which to explore mechanisms of neural injury. Because oxyhemoglobin saturation patterns correlate with neural injury, researchers have used rodent models of the oxygenation patterns of severe sleep apnea to study mechanisms of neural injury and cognitive impairment, and these models have provided tremendous insight into the molecular mechanisms by which sleep apnea oxygenation patterns injure neurons. Oxidative, inflammatory, and organelle injury all contribute to neural dysfunction. Moreover, molecular targets of injury have now been identified for many neuronal groups injured in sleep apnea. Researchers are poised to use this knowledge to develop pharmacotherapies that may prevent or partially reverse neural injury from sleep apnea.

Key Words: apoptosis; cognitive impairment; comorbidity; hippocampus; intermittent hypoxia; memory; oxygenation; sleep apnea; wake neurons

Cognitive Impairment in Adults with Obstructive Sleep Apnea

Obstructive sleep apnea (OSA¹) entails recurrent partial or complete obstructions of the upper airway that typically occur 50 to thousands of times per night and are associated with hypoxemia, hypercapnia, dramatic swings in blood pressure and heart rate, and disrupted sleep. Most OSA patients present with one or more of the following neurobehavioral complaints: depressed mood, sleepiness, inattentiveness, fatigue, poor fine motor skills, and impaired memory and/or executive functioning (e.g., planning, prioritizing, organizing, synthesizing facts, managing time) (Saunamaki and Jehkonen 2007), but not procedural skills involving gross motor control (Rouleau et al. 2002).

Clinical Findings

In more than 50 clinical studies investigators have examined the possible interactions between OSA and cognitive performance. The earliest work, in the 1980s, characterized neurobehavioral performance deficits in individuals with sleep apnea. For example, Anthony Kales and colleagues (1985) described neurobehavioral testing in 50 subjects newly diagnosed with severe OSA and reported that most of them experienced impairments in perception, thought processes, memory, learning capacity, and/or communication. Several subsequent studies identified the severity of nocturnal hypoxemia in sleep apnea as a correlative factor for the severity of impairment in memory and executive function (Berry et al. 1986; Greenberg et al. 1987; Naismith et al. 2004; Telakivi et al. 1993). Sonya Ancoli-Israel and colleagues (1991) examined the association of OSA severity and dementia in over 200 nursing home residents; they found severe dementia in most individuals with severe sleep apnea, but no clear cognitive impairments in individuals with mild to moderate sleep apnea. One of the largest studies, a survey of over 700 individuals with sleep apnea or snoring (Jennum and Sjol 1992), showed that males aged 30 to 60 with polysomnographically confirmed sleep apnea were three times more likely to report difficulty concentrating than individuals whose snoring did not include apneic events. Marc-André Bédard and colleagues (1991) examined adult subjects with moderate and severe sleep apnea and normal controls and found that both sleepiness and nocturnal hypoxemia predicted specific cognitive impairments: hypoxemia predicted executive function impairments, and somnolence predicted attention and memory impairments (Bédard et al. 1991). The same group in a later study found vigilance impairments related to hypoxemia severity (Montplaisir et al. 1992). Thus, the severity of hypoxia in sleep apnea predicts the severity of cognitive impairments.

In addition to neurobehavioral characterization of brain function, electroencephalography (EEG) has revealed abnormalities in the brain activity of humans with obstructive sleep apnea. Florence Morisson and colleagues (1998) examined the spectral analysis of EEG waves in the cerebral cortex during wakefulness and found that subjects with severe OSA with hypoxemia showed diffuse (frontal, parietal, and central) slowing in wakefulness. Brain electrical activity may also change in response to specific stimuli; one research group identified conflict event–related potentials as abnormal even in those with mild to moderate sleep apnea (Zhang et al. 2002).

Newer brain imaging techniques, such as magnetic resonance imaging (MRI) and computed tomography (CT), enable the characterization of regional brain injury and dysfunction in subjects with sleep apnea. For example, clinical studies in subjects with severe sleep apnea have shown gray matter loss in the hippocampus, a region essential for specific spatial and contextual memory tasks (Macey and Harper 2005). More recently researchers have correlated lesions of specific areas (including the hippocampus) with depressive symptoms in persons with OSA (Cross et al. 2008). Richard Thomas has used functional MRI to characterize cognitive dysfunction in sleep apnea; he and coworkers identified inactivation of the dorsolateral prefrontal cortex during memory tasks and noted that this cortical metabolic derangement persisted despite effective continuous positive airway pressure (CPAP) therapy and improved vigilance in subjects (Thomas et al. 2005). It is also possible that hypometabolism in the frontal cortex contributes to mood, attention, and executive function deficits in persons with severe OSA (Macey and Harper 2005).

Determining Causal Factors and Reversibility

A major challenge in human studies of sleep apnea is identification of the causal link between the disease and a slowly developing, potentially irreversible process such as cognitive dysfunction. Bédard and colleagues (1993) examined the reversibility of cognitive dysfunction after successful treatment with nasal CPAP and found that many neurobehavioral impairments resolved; however, vigilance, manual dexterity, and executive function remained impaired despite 6 months of therapy. Reports that CPAP treatment of OSA improves vigilance (Bardwell et al. 2001; Engleman and Joffe 1999; Felver-Gant et al. 2007; Lim et al. 2007), attention, executive planning, memory (Aloia et al. 2003), and brain metabolism (Tonon et al. 2007) support the concept that sleep apnea can induce or contribute to neurobehavioral deficits.

It is possible, however, that the comorbidities of sleep apnea—obesity, unhealthy diet, physical inactivity, diabetes, and cardiovascular disease—are more important modifiers of neurobehavioral function in persons with OSA. Carefully controlled animal models and human studies are necessary to determine whether OSA itself can cause irreversible clinically significant neural injury and neurobehavioral impairments. In addition, further studies are needed to determine whether the other physiological disturbances of sleep apnea—hypercapnia, hemodynamic fluctuations, and sleep disruption—can also contribute to neural injury.

Animal Models of Obstructive Sleep Apnea

The most frequently identified risk factor for obstructive sleep apnea in humans is obesity. Although obese animals (e.g., pigs) exhibit mild sleep-disordered breathing, scientists have not identified a naturally occurring animal model of severe OSA. The English bulldog has snoring and fragmented sleep (Hendricks et al. 1987), but typically has apneas only during rapid eye movement (REM) sleep and demonstrates very mild hypoxemia. Because numerous clinical studies (described above) have suggested that it is the severity of hypoxemia that predicts neural injury, researchers have developed animal models of frequent brief hypoxemic episodes to examine the effects of sleep apnea oxygenation patterns on cognitive function and on other physiological processes described elsewhere in this issue (Dematteis et al. 2009; Jun and Polotsky 2009; Kanagy 2009).

Long-term IH exposure induces neurobehavioral impairment and injury to specific groups of neurons. David Gozal and colleagues (2001) were the first to examine the effects of intermittent hypoxia (IH1) on cognitive function, and his rodent model of long-term IH is still the most widely used for studies of sleep apnea oxygenation patterns. The model changes ambient oxygen by increasing the percentage of nitrogen; the resulting oxygenation pattern in rodents parallels severe OSA patterns in humans with 40 to 60 desaturation events per hour, reducing arterial oxygen levels from 96-99% oxyhemoglobin saturation to 73-87%.

In the first series of experiments Gozal and colleagues (2001) exposed young adult rats (6 to 8 weeks old) to fluctuating oxygen levels during the animals’ sleep-predominant (i.e., light) period for 1 to 14 days. On the first day of exposure, the animals’ sleep was fragmented and reduced, but by the second day they slept right through the IH events and had normal total sleep times for the day and night. (This may therefore be a model of sleep apnea oxygenation changes rather than of oxygenation changes and sleep disruption.) After 14 days, the rats exposed to either sham or true IH were examined for learning abilities in the Morris water maze (a hippocampal-dependent learning/memory assay); those exposed to 14 days of IH had greater difficulty learning the location of the platform. Importantly, after a 14-day recovery, learning impairments improved but were not normalized; studies have not yet shown whether they eventually fully recover. In addition, the investigators identified increased neuronal apoptosis and increased astrocytes in the CA1 but not the CA3 region of the hippocampus and in the frontal cortex. This differential IH susceptibility provides a valuable tool with which to identify mechanisms of injury.

The Gozal model has also been useful in efforts to determine the molecular mechanisms underlying neural injury; for example, studies using this model have shown that long-term IH exposure impairs proteosomal degradation and induces a proinflammatory response in neurons and surrounding glia (Gozal et al. 2003; Li et al. 2003; Row et al. 2004). Evidence that inflammatory mediators contribute to or influence the degree of injury and neurobehavioral impairment comes from observations that inhibition of cyclooxygenase-2 (COX-2) and transgenic ablation of either platelet-activating factor or inducible nitric oxide synthase prevent both neuronal apoptosis and learning impairments in the Morris water maze (Li et al. 2004; Row et al. 2004). Further research might usefully explore how IH activates these proinflammatory pathways in the CA1 region and why the CA3 region is protected.

The long-term (>2 weeks) IH model has also been used to identify important age and environmental factors that may influence the neurobehavioral sequelae of sleep apnea oxygenation exposures. Specifically, IH exposure results in more profound learning decrements in the Morris water maze in old rats (22 to 23 months) than in young rats (3 to 4 months) (Gozal et al. 2003). In addition, a high-fat, simple carbohydrate diet during either IH or sham exposure results in more profound learning impairments, of similar magnitude to those of the old rats (Goldbart et al. 2006). And an absence of apolipoprotein E increases susceptibility to neural injury from intermittent hypoxia (Kheirandish et al. 2005a).

Effects are not limited to the hippocampus but include pyramidal cortical, basal forebrain, cerebellar, and wake-active neurons. The diffuseness of IH-induced injury to areas critical for cognition is consistent with human neurobehavioral sequelae, but hampers the discovery of specific molecular mechanisms underlying such injury as the exact groups of neurons critical for particular neurobehavioral impairments are poorly understood beyond the hippocampus. In contrast to the cortex, the hippocampus is an excellent region to study: it has clear boundaries, allowing for gene therapy studies, and there are behavioral studies that specifically test hippocampal function.

In parallel with changes in the hippocampus, apoptosis occurs in the cortex and basal forebrain after IH exposure (Gozal et al. 2001, 2003; Row 2007). Cortical findings suggest reductions in acetylcholine neurotransmission with increased postsynaptic nicotinic receptor binding affinity. In addition, IH-exposed rats show reduced choline acetyltransferase activity in the basal forebrain (Row 2007) and less dendritic branching (Kheirandish et al. 2005a). Recently, Juliana Perry and colleagues (2008) examined the effects of fewer IH events (15/hr) on norepinephrine content in the striatum and extended previous studies by comparing IH effects to those of sleep restriction. They found that this milder IH paradigm did not cause significant impairments in an avoidance task (memory) but did reduce norepinephrine content in the striatum (Perry et al. 2008; Zhang et al. 2006). IH effects on norepinephrine synthesis or release may be regionally specific; in a separate study, IH increased norepinephrine levels in the prefrontal cortex but not in the hippocampus (Kheirandish et al. 2005b).

Consistent with fine motor deficits and the loss of cerebellar gray matter in some individuals with OSA, Ron Harper and colleagues have identified increased IH susceptibility for Purkinje and fastigial neurons in the cerebellum (Pae et al. 2004). As with the difference in impact on the CA1 and CA3 regions, the differential susceptibility among subtypes of neurons in the cerebellum provides a powerful tool for identifying the mechanisms behind IH-induced neural injury.

In addition, researchers in my laboratory have found that mice exposed to long-term IH develop irreversible wake impairments (Zhu et al. 2007). Wake-active neurons are readily identifiable by brain region and either their neurotransmitters or their synthesizing enzymes, and their function may be ascertained by c-Fos or pCREB (phosphorylation of cAMP-response element binding protein) activity. These neurons are an excellent focus for understanding the mechanisms by which IH injures neurons. Across all groups of wake-active neurons, injury was present only in catecholaminergic neurons where both dopaminergic periaqueductal gray and noradrenergic locus coeruleus neurons were significantly injured and 30-40% of the neurons lost. Thus moderate to severe sleep apnea oxygenation patterns can result in neurodegeneration together with irreversible neuronal dysfunction, in this case, impaired wakefulness (Veasey et al. 2004). This injury also involves a proinflammatory component with increased COX-2 and inducible nitric oxide synthase (Veasey et al. 2004; Zhan et al. 2005). By blocking nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity, whether transgenically or pharmacologically, we prevented both the oxidative and proinflammatory injury and wake impairments. Intriguingly, this injury is far less severe in females, where estrogen may play a neuroprotective role. One of the next steps is to determine why and how IH-related NADPH oxidase contributes to these injuries in male mice but not in female mice.

Summary and Future Directions

Animal models of intermittent hypoxia have greatly increased scientists’ understanding of the clinical significance of chronic IH and its capacity to induce irreversible neural injury as well as neurobehavioral impairments. Having identified specific mediators of neural injury in animal models, scientists can move forward with human studies to determine whether postmortem human brain tissue reveals both injury in the same wake-active neural groups and the same markers of injury (NADPH oxidase, iNOS, and COX-2) as in the rodent IH models. At the same time, researchers can now begin focused translational studies to identify pharmacological targets for the prevention and reversal of neural injury to help humans with sleep apnea.

Acknowledgments

This work was supported in part by grants from the National Institutes of Health (HL079588 and HL080492).

¹Abbreviations used in this article: IH, intermittent hypoxia; OSA, obstructive sleep apnea

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