Inducible nitric oxide synthase in long-term intermittent hypoxia: hypersomnolence and brain injury

Abstract

Rationale: Long-term intermittent hypoxia (LTIH) exposure in adult mice, modeling oxygenation patterns of moderate-severe obstructive sleep apnea, results in lasting hypersomnolence and is associated with nitration and oxidation injuries in many brain regions, including wake-active regions.

Objectives: We sought to determine if LTIH activates inducible nitric oxide synthase (iNOS) in sleep/wake regions, and if this source of NO contributes to the LTIH-induced proinflammatory gene response, oxidative injury, and wake impairments.

Methods: Mice with genetic absence of iNOS activity and wild-type control animals were exposed to 6 weeks of long-term hypoxia/reoxygenation before behavioral state recordings, molecular and biochemical assays, and a pharmacologic intervention.

Measurements and main results: Two weeks after recovery from hypoxia/reoxygenation exposures, wild-type mice showed increased iNOS activity in representative wake-active regions, increased sleep times, and shortened sleep latencies. Mutant mice, with higher baseline sleep times, showed no effect of long-term hypoxia/reoxygenation on sleep time latencies and were resistant to hypoxia/reoxygenation increases in lipid peroxidation and proinflammatory gene responses (tumor necrosis factor alpha and cyclooxygenase 2). Inhibition of iNOS after long-term hypoxia/reoxygenation in wild-type mice was effective in reversing the proinflammatory gene response.

Conclusions: These data support a critical role for iNOS activity in the development of LTIH wake impairments, lipid peroxidation, and proinflammatory responses in wake-active brain regions, and suggest a potential role for inducible NO inhibition in protection from proinflammatory responses, oxidative injury, and residual hypersomnolence in obstructive sleep apnea.

Figure 1.

Effects of long-term intermittent hypoxia (LTIH) on total nitric oxide synthase (NOS) and inducible NOS (iNOS) activity. ¹⁴C-arginine to citrulline conversion was measured in homogenates from the laterobasal forebrain of adult wild-type mice exposed to intermittent hypoxia for 6 weeks (n = 5) or sham intermittent hypoxia (n = 5) for total NOS activity (left columns) and iNOS activity (right columns). *p < 0.05.

Figure 2.

Effect of LTIH on 24-hour sleep times and average sleep latencies in iNOS null and wild-type mice. (A) The 24-hour mean ± SE sleep times in minutes, with sample sizes of 15–24 mice/strain and condition. NREM = non–REM; SIH = sham intermittent hypoxia; TST = total sleep time. (B) Average multiple sleep latency test values (minutes) for the same groups of mice as above (use same color legend as A to identify strain/condition). Left bars represent baseline average sleep latencies from multiple sleep latency testing, and right bars show sleep latency responses after 6 hours forced wakefulness. *p < 0.05 for Bonferroni-corrected comparisons.

Figure 3.

Proinflammatory gene responses to LTIH vary with presence of iNOS activity. Tumor necrosis factor α (TNF-α; top panel), cyclooxygenase-2 (COX-2; middle panel), and mRNA copy numbers (lower panel) were measured in 20 μg RNA from micropunches from the following brain regions: frontal cortex (Cortex); magnocellular preoptic/substantia inominata/horizontal diagonal band or laterobasal forebrain (MCPO/SI/HDB); hippocampus CA1; and posterior and lateral hypothalamus (Lat Hypothalamus). sh = sham. *Bonferroni-corrected p < 0.05.

Figure 4.

Effects of LTIH on lipid peroxidation (top) and carbonylation (bottom) in the laterobasal forebrains of iNOS null and wild-type mice. Macrodissections, as performed for gene response assays, of the laterobasal forebrain were used to measure isoprostane (8,12-iso-iPF_2α) levels. Average isoprostane values ± SE for sham LTIH wild-type (^+/+sh LTIH, n = 10), sham LTIH iNOS null (^−/−sh LTIH, n = 11), LTIH wild-type (^+/+LTIH, n = 10), LTIH iNOS null (^−/−sh LTIH, n = 11). *p < 0.05 Bonferroni-corrected IH and strain comparisons.

Figure 5.

Effects of iNOS inhibition on LTIH proinflammatory gene responses. After LTIH, a series of mice received varying doses of a selective iNOS inhibitor (1400W 0.01–2 mg/kg) or saline vehicle systemically. iNOS activity was measured and analyzed in two brain regions: laterobasal forebrain (Lat Basal Forebrain; solid triangles) and the lateral and posterior regions of the hypothalamus (Lat/Post Hypothalam; open circles) as percentage reduction from vehicle iNOS activity to perform linear regression with percentage of iNOS inhibition after LTIH and copy numbers of TNF-α (top) and COX-2 (bottom).