Leila Kheirandish

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.

Intranasal Steroids and Oral Leukotriene Modifier Therapy in Residual Sleep-Disordered Breathing After Tonsillectomy and Adenoidectomy in Children

OBJECTIVE. Tonsillectomy and adenoidectomy (T&A) is the primary therapeutic approach for sleep-disordered breathing (SDB) in children. However, residual mild SDB will be found in more than one third of these patients after T&A. We hypothesized that combined therapy with the leukotriene receptor antagonist montelukast and intranasal budesonide would result in normalization of residual SDB after T&A. METHODS. During the period of October 2002 to February 2005, children who underwent T&A for SDB underwent a routine postoperative (second) overnight polysomnographic evaluation (PSG) 10 to 14 weeks after T&A surgery. In children with residual apnea hypopnea index (AHI) >1 and <5/hour of total sleep time (TST), treatment with montelukast and intranasal budesonide aqueous solution was administered for a period of 12 weeks (M/B group), at which time a third PSG was performed. Children who had residual SDB and did not receive M/B therapy from their treating physicians were recruited as control subjects. RESULTS. Twenty-two children received M/B, and 14 children served as control subjects. Mean age, gender distribution, ethnicity, and BMI were similar in the 2 treatment groups. The mean AHI at the second PSG was 3.9 ± 1.2/hour of TST and 3.6 ± 1.4/hour of TST in M/B-treated and control patients, respectively. Similar nadir arterial oxygen saturation (87.3 ± 1.2%) and respiratory arousal index (4.6 ± 0.7/hour of TST) were recorded for both groups. However, the M/B group demonstrated significant improvements in AHI (0.3 ± 0.3/hour of TST), in nadir arterial oxygen saturation (92.5 ± 3.0%), and in respiratory arousal index (0.8 ± 0.7/hour of TST) on the third PSG, whereas no significant changes occurred over time in control subjects. CONCLUSIONS. Combined anti-inflammatory therapy that consists of oral montelukast and intranasal budesonide effectively improves and/or normalizes respiratory and sleep disturbances in children with residual SDB after T&A.

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.

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.

Temporal aspects of spatial task performance during intermittent hypoxia in the rat: evidence for neurogenesis

Intermittent hypoxia (IH) during sleep, such as occurs in obstructive sleep apnea, leads to degenerative changes in the hippocampus, and is associated with spatial learning deficits in the adult rat. We report that in Sprague–Dawley rats the initial IH‐induced impairments in spatial learning are followed by a partial functional recovery over time, despite continuing IH exposure. These functional changes coincide with initial decreases in basal neurogenesis as shown by the number of positively colabelled cells for BrdU and neurofilament in the dentate gyrus of the hippocampus, and are followed by increased expression of neuronal progenitors and mature neurons (nestin and BrdU‐neurofilament positively labelled cells, respectively). In contrast, no changes occurred during the course of IH exposures in the expression of the synaptic proteins synaptophysin, SNAP25, and drebrin. Collectively, these findings indicate that the occurrence of IH during the lights on period results in a biphasic pattern of neurogenesis in the hippocampus of adult rats, and may account for the observed partial recovery of spatial function.

Platelet-activating factor receptor-deficient mice are protected from experimental sleep apnea-induced learning deficits

Intermittent hypoxia (IH) during sleep, a hallmark of sleep apnea, is associated with neurobehavioral impairments, regional neurodegeneration and increased oxidative stress and inflammation in rodents. Platelet‐activating factor (PAF) is an important mediator of both normal neural plasticity and brain injury. We report that mice deficient in the cell surface receptor for PAF (PAFR–/–), a bioactive mediator of oxidative stress and inflammation, are protected from the spatial reference learning deficits associated with IH. Furthermore, PAFR–/– exhibit attenuated elevations in inflammatory signaling (cyclo‐oxygenase‐2 and inducible nitric oxide synthase activities), degradation of the ubiquitin–proteasome pathway and apoptosis observed in wild‐type littermates (PAFR+/+) exposed to IH. Collectively, these findings indicate that inflammatory signaling and neurobehavioral impairments induced by IH are mediated through PAF receptors.