Barry W. Row

Increased oxidative stress is associated with chronic intermittent hypoxia-mediated brain cortical neuronal cell apoptosis in a mouse model of sleep apnea

Chronic intermittent hypoxia (CIH), as occurs in obstructive sleep apnea (SA), is associated with substantial cortico-hippocampal damage leading to impairments of neurocognitive, respiratory and cardiovascular functions. Previous studies in a rat model have shown that CIH increases brain cortical neuronal cell death. However, the molecular events leading to CIH-mediated neuronal cell death remain largely undefined. The oscillation of O2 concentrations during CIH remarkably mimics the processes of ischemia/re-oxygenation and could therefore increase cellular production of reactive oxygen species (ROS). We extended the CIH paradigm to a mouse model of SA to identify the molecular mechanisms underlying cortical neuronal cell death. A significant increase of ROS production in mouse brain cortex and cortical neuronal cells was detected by fluorescent oxidation assays upon exposure of mice to CIH, followed by increased expression of oxidative stress response markers, c-Fos, c-Jun and NF-kappaB in mouse brain cortex, as revealed by immunohistochemical and LacZ reporter assays respectively. Long-term exposure of mice to CIH increased the levels of protein oxidation, lipid peroxidation and nucleic acid oxidation in mouse brain cortex. Furthermore, exposure of mice to CIH induced caspase-3 activation and increased some cortical neuronal cell apoptosis. On the other hand, transgenic mice overexpressing Cu,Zn-superoxide dismutase exposed to CIH conditions had a lower level of steady-state ROS production and reduced neuronal apoptosis in brain cortex compared with that of normal control mice. Taken together, these findings suggest that the increased ROS production and oxidative stress propagation contribute, at least partially, to CIH-mediated cortical neuronal apoptosis and neurocognitive dysfunction.

Impaired Spatial Learning and Hyperactivity in Developing Rats Exposed to Intermittent Hypoxia

Obstructive sleep apnea (OSA) is a frequent medical condition and is associated with cognitive impairments in adults and with hyperactivity and decreased school performance in children. In an adult rodent model, intermittent hypoxia (IH), such as occurs in OSA, is associated with neurodegenerative changes in the hippocampus and cortex and with spatial learning deficits. Because a unique developmental window of neural vulnerability to IH is present, we hypothesized that exposure to IH throughout the vulnerable ages would result in increased behavioral impairments in the juvenile rat. Rat pups were therefore exposed to either room air or IH beginning at postnatal (PN) d 10 until PN d 30. Learning and memory were assessed via a standard place-training version of the Morris water maze beginning at PN d 25. Locomotor activity was assessed on PN d 29 and 30. Pups exposed to IH displayed significant spatial learning impairments, and exposed male rats but not female rats displayed increased locomotor activity in the open field. Collectively, these findings indicate that exposure to IH at an age that corresponds to the peak incidence of OSA in children induces substantial learning impairment and gender-dependent behavioral hyperactivity in the juvenile rat. We postulate that this novel experimental model may allow for future exploration of mechanisms underlying the neurobehavioral deficits of children with OSA.

Developmental differences in cortical and hippocampal vulnerability to intermittent hypoxia in the rat

Obstructive sleep apnea is characterized by intermittent hypoxic events during sleep, and is associated with substantial neurocognitive morbidity, particularly in children. Intermittent hypoxia (IH) leads to increases in apoptosis in the cortex and hippocampus of the adult rat, peaking at 48 h of exposure. To examine whether the susceptibility to IH exhibits developmental differences, rats were exposed to 48 h of IH at ages 2, 5, 10, 15, 20, 25, 30, 60, and 120-day postnatally, and apoptosis was determined by terminal deoxy-nucleotidyl transferase-mediated in situ end labeling and immunohistochemical staining for single-stranded DNA. Although IH induced apoptosis at all postnatal ages, smaller increases were apparent in 2 and 5-day old (P < 0.01 vs. any other age) while peak apoptosis occurred at 10-25 days (P < 0.001 vs. 30, 60, and 120 days). We conclude that a unique window of vulnerability to IH is present in the cortex and hippocampus during post-natal maturation, and may underlie the high frequency of neurobehavioral deficits associated with obstructive sleep apnea in children.

Nitric oxide synthase and intermittent hypoxia-induced spatial learning deficits in the rat.

Intermittent hypoxia (IH) during sleep induces significant neurobehavioral deficits in the rat. Since nitric oxide (NO) has been implicated in ischemia-reperfusion-related pathophysiological consequences, the temporal effects of IH (alternating 21% and 10% O(2) every 90 s) and sustained hypoxia (SH; 10% O(2)) during sleep for up to 14 days on the induction of nitric oxide synthase (NOS) isoforms in the brain were examined in the cortex of Sprague-Dawley rats. No significant changes of endothelial NOS (eNOS) and neuronal NOS (nNOS) occurred over time with either IH or SH. Similarly, inducible NOS (iNOS) was not affected by SH. However, increased expression and activity of iNOS were observed on days 1 and 3 of IH (P < 0.01 vs. control; n = 12/group) and were followed by a return to basal levels on days 7 and 14. Furthermore, IH-mediated neurobehavioral deficits in the water maze were significantly attenuated in iNOS knockout mice. We conclude that IH is associated with a time-dependent induction of iNOS and that the increased expression of iNOS may play a critical role in the early pathophysiological events leading to IH-mediated neurobehavioral deficits.

Intermittent hypoxia during development induces long-term alterations in spatial working memory, monoamines, and dendritic branching in rat frontal cortex.

Exposure to intermittent hypoxia (IH), such as occurs in sleep-disordered breathing, is associated with increased apoptosis in vulnerable brain regions as well as with spatial reference memory deficits in adult and developing rats. The latter are more susceptible to IH, suggesting that early exposure to IH may have long-term consequences. Rats were exposed to 14 d of room air (RA) or IH starting at postnatal d 10. Working memory was then assessed in the water maze at 4 mo of age using a delayed matching to place task in which the rats were required to locate a submerged platform hidden in a novel location on the first trial (T1 or acquisition trial), and then remember that position after a delay (T2 or test trial). Mean escape latencies and swim distances were derived and the savings (T1–T2) were used as a measure of working memory. Male but not female rats exposed to IH showed working memory deficits at both a 10- and 120-min delay (for both latency and pathlength). Additionally, Sholl analysis of Golgi-stained neurons revealed decreased dendritic branching in the frontal cortex, but not the hippocampus, of male rats exposed to IH. Norepinephrine concentrations, dopamine turnover, and tyrosine hydroxylase activity were increased similarly in males and females. However, increased dopamine concentrations were present only in the frontal cortex of female rats. In conclusion, exposure to IH during a critical developmental period is associated with long-term alterations in frontal cortical dopaminergic pathways that may underlie gender differences in neurobehavioral deficits.

Increased susceptibility to intermittent hypoxia in aging rats: changes in proteasomal activity, neuronal apoptosis and spatial function

Obstructive sleep apnea (OSA) is a frequent medical condition characterized by intermittent hypoxia (IH) during sleep, and is associated with neurodegenerative changes in several brain regions along with learning deficits. We hypothesized that aging rats exposed to IH during sleep would be particularly susceptible. Young (3–4 months) and aging (20–22 months) Sprague–Dawley rats were therefore exposed to either room air or IH for 14 days. Learning and memory was assessed with a standard place‐training version of the Morris water maze. Aging rats exposed to room air (RA) or IH displayed significant spatial learning impairments compared with similarly exposed young rats; furthermore, the decrements in performance between RA and IH were markedly greater in aging compared with young rats (p < 0.01), and coincided with the magnitude of IH‐induced decreases in cyclic AMP response element binding (CREB) phosphorylation. Furthermore, decreases in proteasomal activity occurred in both young and aging rats exposed to IH, but were substantially greater in the latter (p < 0.001). Neuronal apoptosis, as shown by cleaved caspase 3 expression, was particularly increased in aging rats exposed to IH (p < 0.01 versus young rats exposed to IH). Collectively, these findings indicate unique vulnerability of the aging rodent brain to IH, which is reflected at least in part, by the more prominent decreases in CREB phosphorylation and a marked inability of the ubiquitin‐proteasomal pathway to adequately clear degraded proteins.

Intermittent hypoxic exposure during light phase induces changes in cAMP response element binding protein activity in the rat CA1 hippocampal region: water maze performance correlates.

Intermittent hypoxia (IH) during sleep, a characteristic feature of sleep-disordered breathing (SDB) is associated with time-dependent apoptosis and spatial learning deficits in the adult rat. The mechanisms underlying such neurocognitive deficits remain unclear. Activation of the cAMP-response element binding protein (CREB) transcription factor mediates critical components of neuronal survival and memory consolidation in mammals. CREB phosphorylation and DNA binding, as well as the presence of apoptosis in the CA1 region of the hippocampus were examined in Sprague-Dawley male rats exposed to IH. Spatial reference task learning was assessed with the Morris water maze. IH induced significant decreases in Ser-133 phosphorylated CREB (pCREB) without changes in total CREB, starting as early as 1 h IH, peaking at 6 h-3 days, and returning toward normoxic levels by 14-30 days. Double-labeling immunohistochemistry for pCREB and Neu-N (a neuronal marker) confirmed these findings. The expression of cleaved caspase 3 (cC3) in the CA1, a marker of apoptosis, peaked at 3 days and returned to normoxic values at 14 days. Initial IH-induced impairments in spatial learning were followed by partial functional recovery starting at 14 days of IH exposure. We postulate that IH elicits time-dependent changes in CREB phosphorylation and nuclear binding that may account for decreased neuronal survival and spatial learning deficits in the adult rat. We suggest that CREB changes play an important role in the neurocognitive morbidity of SDB patients.

High fat/refined carbohydrate diet enhances the susceptibility to spatial learning deficits in rats exposed to intermittent hypoxia

BACKGROUND Intermittent hypoxia during sleep (IH), as occurs in sleep disordered breathing (SDB), induces spatial learning deficits associated with regulation of transcription factors associated with learning and memory in the hippocampal CA1 region in rats. high fat refined carbohydrate diet (HF/RC) can induce similar deficits and associated changes in signaling pathways under normoxic conditions. METHODS Sprague-Dawley adult male rats were fed either with (HF/RC) or low fat/complex carbohydrate diet (LF/CC) starting at post-natal day 30 for 90 days, and were then exposed for 14 days during light phase (12 h/day) to either normoxia (RA) or IH (21% and 10% O2 alternations every 90 s). Place-training reference memory task deficits were assessed in the Morris water maze. Total and ser-133 phosphorylated CREB were assessed in different brain regions by Western blotting and immunostaining in rats exposed to normoxia or IH and to LF/CC or HF/RC. RESULTS Substantial decreases in CREB phosphorylation occurred in CA1 but not in motor cortex following either IH, HF/RC, and HF/RC + IH. Place-training reference memory task deficits were observed in rats exposed to IH and to HF/RC, and to a much greater extent in rats exposed to HF/RC + IH. CONCLUSIONS Nutritional factors alter recruitment of transcription factors, possibly via oxidative-related pathways, and modulate the vulnerability of the CA1 region of the hippocampus to the episodic hypoxia that characterizes SDB, thereby enhancing neurocognitive susceptibility in SDB patients.

Intermittent Hypoxia and Cognitive Function: Implications from Chronic Animal Models

Obstructive sleep apnea syndrome (OSAS) is a frequent sleep disorder in which the upper airway collapses repeatedly during sleep, resulting in intermittent hypoxia (IH) and asphyxia, and leading also to sleep fragmentation due to the recurrent nocturnal arousals necessary to relieve the upper airway obstruction. In addition to cardiovascular and metabolic morbidities, OSAS also causes serious neurocognitive daytime dysfunction and is associated with regional alterations in brain morphology in humans. These findings suggest that the anatomical brain lesions may underlie the behavioral deficits associated with the disease. In rodents, chronic exposure to intermittent hypoxia (IH) during sleep, which model the hypoxia/re-oxygenation patterns observed in moderate to severe OSAS patients, replicates many of the neurocognitive features of OSAS in humans, such as learning and memory deficits and impaired vigilance. Exposure to experimentally-induced IH in the rodent is also associated with age- and time-related neurodegenerative changes in addition to alterations in brain regions and neurotransmitter systems involved in learning and memory, attention, and locomotor activity. Multiple pathophysiological processes appear to be involved in the mechanistic aspects of the behavioral and neuronal susceptibility to IH during sleep, and include pathways leading to increased oxidative stress and inflammation, altered gene regulation, and decreases in the cellular and molecular substrates of synaptic plasticity. In addition, both environmental and genetic factors modulate the end-organ susceptibility to IH-induced cognitive dysfunction in rodents. Collectively, the available data indicate that exposure to IH during sleep is associated with adverse behavioral and neuronal consequences in the rodent. Improved understanding of the determinants of IH-related susceptibility may help explain the phenotypic variance in OSAS-associated morbidities, and enable improved therapeutic approaches in the future.

Impaired spatial working memory and altered choline acetyltransferase (CHAT) immunoreactivity and nicotinic receptor binding in rats exposed to intermittent hypoxia during sleep

Exposure to intermittent hypoxia (IH), such as occurs in sleep-disordered breathing (SDB), is associated with cognitive impairment, neurodegeneration, oxidative stress, and inflammatory responses within rodent brain regions such as the basal forebrain. In this region, damage to cholinergic neurons correlates with working memory deficits in a number of neurodegenerative disorders, suggesting that degeneration of cholinergic systems may also contribute to the working memory impairments observed after IH exposures. We therefore examined basal forebrain choline acetyltransferase (CHAT) immunohistochemistry, nicotinic receptor binding in the prefrontal cortex (PFC), and working memory, in male rats tested on a delayed matching to place (DMP) task in the water maze following exposure to either room air (RA) or intermittent hypoxia (IH; alternating 90s epochs of 21% and 10% O(2) during sleep). IH-treated animals displayed impaired working memory with respect to controls, along with significant reductions in CHAT-stained neurons in the medial septal nucleus, in both the vertical and horizontal limbs of the diagonal band, and the substantia inominata after 14 days of IH exposure. In addition, increases in nicotinic binding and receptor affinity in the PFC were observed after 14 days of IH exposure. Thus, a loss of cholinergic neuronal phenotype in the basal forebrain may contribute to the cognitive impairments associated with CIH exposure. However, compensatory mechanisms may also be activated in other brain regions, and may provide potential therapeutic targets for the cognitive impairments associated with SDB.