Patients with Obstructive Sleep Apnea (OSA) commonly manifest cognitive impairment and neurodegeneration that may be due to hypoxic damage during sleep. This study investigated the effects of intermittent hypoxia on spatial learning in a rat model using a Barnes maze paradigm. We hypothesized that spatial learning would be increasingly impaired as a function of hypoxia severity. Twenty-one male Sprague-Dawley rats were split into three hypoxia conditions: severe (6% oxygen; n=7), moderate (11% oxygen; n=5), and sham (21% oxygen; n=9). Rats were exposed to hypoxic conditions (180 second periods of cyclical oxygen between 21% and condition-specific nadir O2) for 12 hours during the day in enclosed housing chambers designed to output appropriate air compositions. Barnes maze testing was performed monthly. Rats were placed on a platform containing 19 shallow holes with a single “target box” to escape the maze. At each month, rats had 3 minutes to find the “target box” during four daily trials over four consecutive days. Maze completion time and number of errors were recorded. Rats from the three hypoxia groups did not differ in metrics of spatial learning at baseline. By month 3, the sham group showed a faster improvement in maze completion time across four days compared to both moderate (p=0.0071) and severe (p=0.0281) groups. Within the severe hypoxia group, maze completion time was significantly longer at month 3 compared to baseline on day 2 (p=0.0057), day 3 (p=0.0023) and day 4 (p=0.0336). Similarly, the sham group showed a quicker improvement in number of errors made across four days compared to the severe group at month 3 (p=0.0087). Our findings indicate that spatial learning, measured by shorter completion times and fewer errors, is impaired in rats undergoing 3 months of cyclical intermittent hypoxia when compared to sham animals. Results add to the growing body of evidence on the relationship between obstructive sleep apnea and cognitive impairment. Funded by NIH P01 HL094307.
Posture is a determinant of UA caliber and apnea frequency and severity. However, the effect of neck-bending on UA caliber and soft-tissues has not been well studied. We hypothesized that neck extension and flexion would increase and decrease UA caliber in controls and apneics. UA MRIs were obtained in 24 controls (AHI<5; 1.5 ± 1.5 events/hour) and 33 apneics (AHI≥5; 33.2 ± 28.7) during neck flexion, extension, and neutral position and analyzed for airway measures in the retropalatal (RP) and retroglossal (RG) regions and soft-tissue movement. Apneics were older (48.9 ± 11.9 vs. 40.8 ± 14.6, p=0.0256) and more obese (33.9 ± 4.4 vs. 28.4 ± 5.6 kg/m2, p=0.0001) than controls; subjects were 49.1% male. During extension (23.9 ± 14.9°) controls showed increased minimum cross-sectional area (CSA), anteroposterior (AP) and lateral dimension in the RP (CSA: 38.2mm2, p=0.0068; AP: 2.6mm, p=0.0145; lateral: 3.8mm, p=0.0046) and RG (CSA: 71.5mm2, p=0.0176; AP: 2.9mm, p=0.0029; lateral: 4.9mm, p=0.0120) airway relative to neutral. Apneics (19.7 ± 9.3°) showed increased RP (CSA: 16.8mm2, p=0.0021; lateral: 2.2mm, p=0.0009) and RG (CSA: 70.6mm2, p<0.0001; AP: 2.8mm, p=0.0001; lateral: 4.7mm, p<0.0001) measures. During flexion, both controls (-16.5 ± 8.2°) and apneics (-11.9 ± 5.7°) showed reduced RP lateral dimension (-2.2mm [p=0.0120] and -1.1mm [p=0.0245]) and RG CSA (-28.2mm2 [p=0.0072] and -12.3mm2 [p=0.0595]). Compared to extension, controls in flexion showed reduced RP (CSA: p=0.0011; AP: p=0.0030; lateral: p=0.0016) and RG (CSA: p=0.0033; AP: p=0.0010; lateral: p=0.0022) measures, RP lateral wall narrowing (p=0.0305), and greater posteroinferior movement of the soft palate (p=0.0019) and all tongue quadrants (p≤0.0042). Compared to extension, apneics in flexion showed reduced RP (CSA: p=0.0006; AP: p=0.0502; lateral: p<0.0001) and RG (CSA: p<0.0001; AP: p=0.0001; lateral: p<0.0001) measures, significant total lateral wall narrowing (p=0.0010), and greater posteroinferior movement of the soft palate (p=0.0005) and all tongue quadrants (p<0.0008). Controls and apneics showed reductions in both RP and RG airway caliber during neck flexion and increases during extension, primarily due to movement of surrounding soft-tissues. These data provide important insights into the role of head and neck position on UA caliber. Funded by NIH P01 HL094307.
Although the mechanisms through which obesity contributes to obstructive sleep apnea (OSA) are unknown, OSA patients have increased tongue fat compared to controls. Animal models can be used to study the effect of weight gain on tongue fat. We studied obese (OBZ) and lean (NBZ) Zucker rats longitudinally (3 months), and hypothesized: (1) the amount of tongue fat would increase in both OBZ and NBZ rats following weight gain; and (2) mechanical properties of the tongue would be adversely affected because of fat deposition. A subset of 12 NBZ and 12 OBZ males were euthanized monthly to identify longitudinal changes in tongue force and composition. Spin-echo and Dixon MRI were used to obtain the total and fat volumes of the tongue and masseter at baseline and monthly. Tongue force values were obtained by stimulating the hypoglossal nerves of anesthetized animals. Tongue volume, fat volume and fat percentage increased longitudinally in both groups (p<0001). There was no change in masseter fat percentage despite the volume of this muscle increasing (p<0.001). Changes in tongue volume and fat measures were similar between groups. Among all rats, there were significant positive associations between weight and tongue volume, tongue fat volume, and tongue fat percentage (p<0.0001). There were no significant associations between weight gain and tongue force outcome variables. Tongue volume, tongue fat volume, and tongue fat percentage were positively associated with weight gain in OBZ and NBZ rats. Fat preferentially deposited in the tongue compared to the masseter. Increased tongue fat may contribute to the relationship between obesity and OSA. There were no significant associations between weight and tongue force measures. Rats had baseline tongue fat percentages averaging 2.6% compared to 27–33% seen in humans. It is possible the absolute amount of tongue fat in these animals was insufficient to produce significant changes in tongue force output. Future studies should consider examining weight gain in other animal models (pig/cow) with tongue fat percentages similar to humans. Supported by NIH P01 HL094307.
Previous studies indicated the upper airway narrows in the retropalatal (RP) region during sleep in normal subjects due to thickening of the lateral pharyngeal walls. We hypothesized that apneics would exhibit state-dependent upper airway narrowing in the RP and retroglossal (RG) regions and narrowing in apneics would be greater than controls. Upper airway MRIs were obtained in 15 sleep-deprived controls (AHI<5; 0.3 ± 0.5 events/hour) and 12 apneics (AHI≥5; 36.2 ± 19.9) during wake and sleep and analyzed for airway measures and soft-tissue movement. We evaluated whether there were significant changes during sleep and if changes differed between groups. Apneics were older (39.2 ± 12.2 vs. 26.4 ± 6.9 years, p=0.005) and more obese (33.0 ± 6.2 vs. 24.2 ± 3.5 kg/m2, p=0.0003) than controls; 96.3% of subjects were male. In the RP region, controls showed reduced average cross-sectional airway area (CSA; -18.7%, p=0.003) and minimum CSA (-40.5%, p=0.0001), anteroposterior (AP; -25.0%, p=0.0004), and lateral (-17.7%, p=0.037) dimensions. Apneics showed reduced average CSA (-31.3%, p=0.0001) and minimum CSA (-65.4%, p<0.0001), AP (-29.2%, p=0.0016), and lateral (-44.2%, p=0.0004) measures. Apneics had greater reductions in RP minimal CSA (p=0.0204) and lateral dimensions (p=0.0326) than controls. In the RG region, controls showed no significant changes with sleep. Apneics had reductions in minimal CSA (-45.2%, p=0.0011), lateral dimensions (-26.3%, p=0.0137), and trending changes in AP dimension (-18.9%, p=0.070). Reductions in RG minimal CSA (p=0.0096) and lateral dimension (p=0.0291) were greater in apneics than controls. The soft palate (p=0.0451), anterior-superior (p<0.0001), and posterior-inferior (p=0.0113) tongue quadrants exhibited greater posterior movement in apneics. Similar increases in RP lateral wall thickness (p=0.3332) were seen in controls (10.8%, p=0.0214) and apneics (21.8%, p=0.0549). Controls and apneics showed significant reductions in RP airway caliber during sleep, while only apneics showed RG airway changes. Apneics had greater narrowing in both regions compared to controls. State-dependent reductions in AP and lateral airway dimensions are primarily due to posterior movement of the soft palate and tongue and thickening of lateral walls. These data provide important insights into OSA pathogenesis. Funded by NIH P01 HL094307.
Changes in airway caliber during periods of negative intraluminal pressure are fundamental in understanding the pathogenesis of obstructive sleep apnea (OSA). We evaluated the change in airway caliber in normal subjects and apneics during a normalized Muller maneuver (a forced inspiration). We hypothesized that apneics would exhibit greater airway collapse during negative intraluminal pressures compared to controls. Nasopharyngoscopy with the Muller maneuver was performed on 18 normal subjects (AHI<5; 0.7 ± 1.1 events/hour) and 30 apneics (AHI>15; 32.6 ± 28.5). Maximal effort maneuvers were performed in the supine and seated positions and changes in retropalatal (RP) and retroglossal (RG) regions quantified. Measures in the RP region were also quantified during graded pressures (-10, -20, -30, -40mmH2O) while sitting. Apneics were older (44.7 ± 12.1 vs. 30.2 ± 10.5 years, p=0.0001) and more obese (33.1 ± 7.6 vs. 25.2 ± 3.7 kg/m2, p<0.0001) than controls; 80% of subjects were male. During maximal effort, controls showed reduced minimum cross-sectional area (CSA) in only the RP region in the supine (-63%, p<0.0001) and seated (-59%, p<0.0001) positions. 34–57% reductions were seen in the lateral dimension in both positions and regions (all p<0.0001). Apneics showed significantly reduced airway caliber during maximal pressures for all measures, regions and positions, except RG seated AP dimension. Reductions ranged from 22% in the RG supine AP dimension (p=0.0004) to 88% in seated RP minimal CSA (p<0.0001). Reductions in apneics were greater (all p<0.023) than those in controls for nearly all measures, regions and positions, except RG lateral and RP supine AP dimension. Seated graded efforts in the RP region showed reductions in all measures in both groups and greater reductions in apneics for pressures ≤ -20mmH2O. Both groups showed significant reductions in RP airway caliber during maximal and graded efforts in both positions, while only apneics showed consistent reductions in the RG airway. Reductions in apneics were greater than those seen in controls, despite similar negative pressures. Examining a normalized Muller maneuver may provide important information about the site of airway narrowing in apneics. Funded by NIH P01 HL094307.
Abstract Introduction Cognitive and spatial dysfunction is common among patients with obstructive sleep apnea (OSA). The cause of these abnormalities may be related to the effects of hypoxic damage in the brain during sleep. Here we report a rodent model for chronic intermittent hypoxia (CIH) that examines spatial performance tasks via a Barnes Maze paradigm. We hypothesized that increased severity of CIH yields decreased cognitive and spatial performance. Methods Three groups of rats were subject to varying levels of hypoxia conditions: sham (21% oxygen; n = 19), moderate (11% oxygen; n = 14), and severe (6% oxygen; n = 21). To deliver hypoxia, rats were exposed to three-minute cycles of oxygen between 21% and condition-specific nadir oxygen for 12 hours daily (during sleep) in specialized chambers. Barnes maze testing was performed at 0, 1, 2, and 3 months. Rats were placed on a circular platform with 19 shallow holes and one deeper target hole to escape the noxious sound. Each month, rats had 3 minutes to find the target hole in four daily trials over four consecutive days. Average maze completion time on day 4 was recorded. Results Rats from the three hypoxia groups did not differ significantly in mean maze completion time at baseline (0 months). Throughout the three months of exposure to hypoxic conditions, maze completion time on day 4 did not differ significantly from baseline for sham rats. However, by month 3, rats exposed to severe hypoxic conditions had a significantly larger percent increase from baseline compared to sham rats (p = 0.0358). Conclusion Our findings indicate that rats undergoing intermittent hypoxia perform worse than normoxic rats in spatial performance tasks. These data suggest there is a relationship between CIH and cognitive/spatial impairment. Support Funded by NIH P01 HL094307
Abstract Introduction Patients with obstructive sleep apnea (OSA) commonly exhibit grey and white matter loss, which may be related to hypoxic damage in the brain during sleep. Our preliminary data demonstrated lower values of cerebral metabolic rate of oxygen (CMRO2) consumption in apneics versus controls. As such, reduced CMRO2 may be an important contributor to the neurologic consequences of OSA. Here we report a rodent model for chronic intermittent hypoxia (CIH) to quantify effects on CMRO2 consumption. We hypothesized that increased severity of CIH results in decreased CMRO2 levels. Methods Three groups of rats were subject to varying levels of hypoxia: sham (21% oxygen; n = 19), moderate (11% oxygen; n = 14), and severe (6% oxygen; n = 21). To deliver hypoxia, rats were exposed to three-minute cycles of oxygen between 21% and condition-specific nadir O2 for 12 hours daily during their sleep cycle. CMRO2 values were measured with MRI techniques, performed on anesthetized rats before and after 3 months exposure to CIH. Results Rats from the three hypoxia groups did not differ significantly in CMRO2 values at baseline (0 months). After 3 months of exposure to hypoxic conditions, there was a trending difference (p=0.0726) in percent change from baseline between severely hypoxic (-35.3%) and sham (+12.3%) rats. Moderately hypoxic rats demonstrated an intermediate decrease from baseline after 3 months (-19.0%). Conclusion Our findings suggest that increased severity of intermittent hypoxia yields a dose-response decrease in brain oxygen consumption. Our data add to the growing body of evidence on the relationship between obstructive sleep apnea and hypoxic damage in the brain, suggesting that CMRO2 levels may be an indicator of the neurologic consequences of OSA. Support Funded by NIH P01 HL094307
Increased volume of upper airway soft-tissues, tongue fat deposition, and restricted craniofacial dimensions are anatomic risk factors for Obstructive Sleep Apnea (OSA). A new strategy for identifying associated genes is to leverage outbred mouse models and examine associations with intermediate phenotypes. We quantified phenotypes in Diversity Outbred (DO) mice and the 8 inbred and wild-derived Founder strains. We hypothesized that these phenotypes would significantly differ among Founders, suggesting associations with underlying genetic architecture. Magnetic Resonance Imaging (MRI)-base phenotypes were quantified within 6–7 month-old male DO mice (n=47) and male mice from the 5 inbred [A/J (n=3), C57BL/6J (n=3), 129S1/SvlmJ (n=3), NOD/LtJ (n=3), NZO/HILtJ (n=3)] and 3 wild-derived [CAST/EiJ (n=2), PWK/PhJ (n=3), WSB/EiJ (n=3)] Founder strains. All mice were group housed and fed the same diet. MRI was performed to quantify tongue volume, tongue fat volume, tongue fat percentage, mandibular divergence and length, and the intramandibular volume (IMV) using Amira 5.4.1 software. Phenotypes were compared among Founders and the variability within DO mice was evaluated. There were significant differences among the Founder strains for tongue volume (p<0.0001), tongue fat volume (p=0.0002), and tongue fat percentage (p=0.0007), as well as mandibular width at the gonion (p=0.0011) and 2nd molar (p=0.0047) and the IMV (p=0.0010). No differences among Founders were seen for other mandibular measures (divergence [p=0.4211], length [p=0.0896], or width at 1st molar [p=0.5040], and condyle [p=0.5961]). There was high variability for MRI-based phenotypes in the DO mice; values spanned or extended beyond the ranges observed across Founders, reflecting increased genetic diversity. We quantified tongue size and craniofacial dimensions using MRI-based imaging in DO and Founder mice. Differences in tongue size, tongue fat and fat percentage, mandibular width and IMV among Founders suggest genetic associations with these phenotypes. High variability in DO mice indicates these phenotypes are suitable for genetic association studies in outbred mice populations. Genetic analyses in these mice may identify specific genes related to craniofacial size or tongue fat involved in OSA pathogenesis. Funded by NIH P01 HL094307.
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