RATS DEMONSTRATE CIRCADIAN VARIATION IN VENTILATION AND SLEEP APNEA http://dx.doi.org/10.5665/sleep.3576

Brown Norway and Zucker Lean Rats Demonstrate Circadian Variation in Ventilation and Sleep Apnea Anne M. Fink, PhD, RN1,2; Irina Topchiy, PhD1,2,3; Michael Ragozzino, PhD4; Dionisio A. Amodeo, MA4; Jonathan A. Waxman, BS1,5; Miodrag G. Radulovacki, MD, PhD1,6; David W. Carley, PhD1,2,3

Center for Narcolepsy, Sleep and Health Research, College of Nursing, University of Illinois at Chicago, Chicago, IL; 2Department of Biobehavioral Health Science, College of Nursing, University of Illinois at Chicago, Chicago, IL; 3Department of Medicine, University of Illinois at Chicago, Chicago, IL; 4Department of Psychology, University of Illinois at Chicago, Chicago, IL; 5Department of Bioengineering, University of Illinois at Chicago, Chicago, IL; 6Department of Pharmacology, University of Illinois at Chicago, Chicago, IL 1

Study Objectives: Circadian rhythms influence many biological systems, but there is limited information about circadian and diurnal variation in sleep related breathing disorder. We examined circadian and diurnal patterns in sleep apnea and ventilatory patterns in two rat strains, one with high sleep apnea propensity (Brown Norway [BN]) and the other with low sleep apnea propensity (Zucker Lean [ZL]). Design/Setting: Chronically instrumented rats were randomized to breathe room air (control) or 100% oxygen (hyperoxia), and we performed 20-h polysomnography beginning at Zeitgeber time 4 (ZT 4; ZT 0 = lights on, ZT12 = lights off). We examined the effect of strain and inspired gas (twoway analysis of variance) and analyzed circadian and diurnal variability. Measurements and Results: Strain and inspired gas-dependent differences in apnea index (AI; apneas/h) were particularly prominent during the light phase. AI in BN rats (control, 16.9 ± 0.9; hyperoxia, 34.0 ± 5.8) was greater than in ZL rats (control, 8.5 ± 1.0; hyperoxia, 15.4 ± 1.1, [strain effect, P < 0.001; gas effect, P = 0.001]). Hyperoxia reduced respiratory frequency in both strains, and all respiratory pattern variables demonstrated circadian variability. BN rats exposed to hyperoxia demonstrated the largest circadian fluctuation in AI (amplitude = 17.9 ± 3.7 apneas/h [strain effect, P = 0.01; gas effect, P < 0.001; interaction, P = 0.02]; acrophase = 13.9 ± 0.7 h; r 2 = 0.8 ± 1.4). Conclusions: Inherited, environmental, and circadian factors all are important elements of underlying sleep related breathing disorder. Our method to examine sleep related breathing disorder phenotypes in rats may have implications for understanding vulnerability for sleep related breathing disorder in humans. Keywords: Brown Norway rat, sleep apnea, sleep related breathing disorder, Zucker Lean rat Citation: Fink AM; Topchiy I; Ragozzino M; Amodeo DA; Waxman JA; Radulovacki MG; Carley DW. Brown Norway and Zucker Lean rats demonstrate circadian variation in ventilation and sleep apnea. SLEEP 2014;37(4):715-721.

INTRODUCTION Sleep related breathing disorder (SRBD) reflects a complex array of phenotypes influenced by factors including anatomical characteristics, genetic susceptibilities, sleep stage-specific activities, and circadian variability.1-10 Animal models will become increasingly important for elucidating mechanisms by which these factors mediate or modulate SRBD. We showed that laboratory rats express spontaneous central sleep apneas, and apnea frequency varies among rat strains.11-15 We also demonstrated that hyperoxia increased the frequency of spontaneous apneas in outbred Sprague-Dawley rats.14 It is unknown how SRBD may vary across the circadian day. In the current study, we examined inbred rat strains that exhibit low (Zucker Lean [ZL]) or high (Brown Norway [BN]) sleep apnea frequency and hypothesized that sleep related breathing behaviors, and responses to hyperoxia, would exhibit diurnal and circadian patterns in both strains.

Vertebrate Animals and were approved by the University of Illinois at Chicago institutional animal care and utilization committee. Adult male BN and ZL rats (n = 40, weighing 345.9 g ± 7.0, Harlan, Indianapolis, IN) were housed individually under optimal conditions (22°C, 60% humidity, and 12:12 h light:dark cycle with lights on at 08:00). With the rats under anesthesia (ketamine:xylazine 80:5 mg/kg), electroencephalography (EEG) electrodes (stainless steel screws placed bilaterally into the frontal and parietal skull bones) and bilateral electromyography (EMG) electrodes were implanted and placed in the nuchal muscles. The EEG/EMG leads were soldered to a connector affixed to the skull with acrylic dental cement. The skin was sutured around the connector, and rats were permitted 7 days to recover. After recovery, rats underwent 3 days of adaptation to the recording apparatus, a cylindrical bias-flowventilated single-chamber plethysmograph (Buxco Electronics, Sharon CT, [6 inches in diameter with a 10-inch vertical chimney]) in which animals had unlimited access to food and water. EEG and EMG signals were obtained from the animal’s headset using a flexible recording apparatus tethered to the top of the plethysmograph chimney, allowing for free movement of the EEG/EMG recording cable. Respiration was transduced by measuring pressure changes within the plethysmograph with respect to an integrated reference chamber. For animals randomized to the control condition (BN rats, n = 10; ZL rats, n = 10), chambers were ventilated with room air; for animals randomized to the hyperoxia condition (BN rats, n = 11; ZL rats, n = 9), chambers were ventilated with 100% oxygen. Plethysmographs were continuously ventilated

MATERIALS AND METHODS All procedures conformed to the American Physiological Society’s Guiding Principles for the Care and Use of

Submitted for publication August, 2013 Submitted in final revised form October, 2013 Accepted for publication November, 2013 Address correspondence to: Anne M. Fink, PhD, RN, Center for Narcolepsy, Sleep and Health Research, University of Illinois at Chicago, 845 South Damen Ave, Office 750 (MC 802), Chicago, IL 60612; Tel: (312) 355-0689; Fax: (312) 996-4979; E-mail: [email protected] SLEEP, Vol. 37, No. 4, 2014

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Circadian Variation in Ventilation and Sleep Apnea—Fink et al

paired t tests (data not shown). Analysis of ƒ (breaths per min) recorded during wakefulness in the first hour of recording demonstrated that BN rats (control) took fewer breaths on average (196.1 ± 11.4) than ZL rats (control; 229.5 ± 11.1, respectively; P = 0.05). Neither BN nor ZL rats demonstrated significant changes in N-ƒ values between light and dark phases across sleep-wake states (Table 1). In both strains, hyperoxia was associated with a reduction in N-ƒ during NREM and REM sleep, but this association only reached statistical significance in the light phase (Table 1). BN rats demonstrated higher AI than ZL rats throughout sleep, but this difference achieved statistical significance during the light phase (P < 0.001) and not during the dark phase (P = 0.06; Table 1). In the control condition (Figure 1A), BN rats demonstrated a significantly greater AI than ZL rats only during NREM sleep. Hyperoxia exacerbated sleep apnea in both BN rats (Figure 1B) and ZL rats (Figure 1C), and the effects of hyperoxia were more prominent during the light phase (Figure 1B and 1C). When exposed to hyperoxia, BN rats demonstrated elevated AIs during both the light (NREM and REM sleep) and dark phases (REM sleep; Figure 1B). We observed similar patterns for ZL rats, but the exacerbation of AI was statistically significant only during the light phase (Figure 1C). During REM sleep in the light phase, the increase in AI induced by hyperoxia was significantly greater in BN rats than ZL rats (P = 0.04 for strain by gas interaction; Table 1). Compared with ZL rats, BN rats demonstrated an AI approximately twofold greater during NREM sleep and threefold greater during REM sleep when exposed to hyperoxia during the light phase (Table 1; Figure 1D). Also, hyperoxia increased apnea duration in both strains in the light phase (Table 1). There were few statistically significant differences between light and dark phases among stratified AI variables (as assessed by paired t tests; Figure 1); however, NREM sleep AI was significantly elevated during the dark phase compared with the light phase in both control BN rats (P = 0.01) and control ZL rats (P = 0.03; Figure 1A-1C). Animals exposed to hyperoxia did not exhibit any statistically significant differences between AI values for light and dark phases. Circadian rhythms were evident in all variables. Amplitudes, acrophases, and r2 statistics are shown in Table 2. In general, the fraction of explained variance (r2) was greatest for animals exposed to hyperoxia. Sample data illustrating the best fit cosine waves are shown in Figure 2 for individual animals. Hyperoxia was associated with increased circadian amplitudes in all measures of apnea (apnea duration, AI in total, NREM, and REM sleep) but not for any breath-by-breath measures (ƒ, N-ƒ, N-VT, N- E). When exposed to hyperoxia, BN rats demonstrated the largest circadian amplitudes for the sleep apnea variables. None of the acrophase values for any sleep apnea variables differed significantly according to strain or inspired gas; all animals demonstrated their peak amplitudes for these variables approximately 11-13 h into the recording (i.e., beginning of the dark phase; Table 2).

at 2.5 L/min, which was sufficient to prevent rebreathing.12,16 We performed 20-h polysomnographic recordings starting at Zeitgeber time 4 [ZT4; ZT0 represented lights on and ZT12 lights off). All polysomnography signals were amplified, filtered (low-pass filter set at 100 Hz), and digitized at 250/sec (Bio-Logic Systems Sleepscan Premier, Natus, Mundelein, IL). Polysomnographic recordings were visually scored in 30-sec epochs as wakefulness, nonrapid eye movement (NREM) sleep, and rapid eye movement (REM) sleep. Sleep state distributions (min) were determined for every 2-h increment. Using software developed in our laboratory, we determined the hourly values for respiratory frequency (ƒ), tidal volume (VT), and minute ventilation ( E) for each sleep-wake stage (i.e., wake, NREM sleep, REM sleep). Because the plethysmograph was not sealed, pressure fluctuations were not calibrated on a volume scale. We normalized VT and E values relative to each rat’s mean value obtained for wakefulness in the first hour of recording. For comparison, we also calculated normalized respiratory frequency (N-ƒ). These normalizations permitted assessment of the effects of sleep-wake state, inspired gas, nocturnal-(dark)/diurnal-(light) period, and circadian time on each animal’s respiratory pattern. Sleep apneas, defined as cessation of respiration for ≥ 2.0 sec, were detected and associated with either NREM or REM sleep. The ≥ 2.0 sec criterion was selected because this duration represents at least two missed breaths, which corresponds to a 10-sec apnea in humans.13 We did not differentiate between spontaneous and postsigh apneas. Sleep apnea indices (AI, apneas/h of sleep) were calculated for total, NREM, and REM sleep. Statistical Analysis Data were analyzed using the Statistical Package for the Social Sciences (SPSS; Version 20.0, Chicago, IL) and STATA (Version 12.0, College Station, TX). To assess diurnal patterns, we aggregated the sleep-wake and respiratory data to reflect mean values during light and dark phases of recording. We conducted one-way and two-way analysis of variance (ANOVA) with least significant difference post hoc tests to examine the effect of strain and inspired gas (strain × gas) on sleep-wake states, AI, respiratory patterns, and circadian variability. We also conducted paired t tests to determine whether any sleep and breathing variables differed significantly between light and dark phases. Circadian variation was analyzed by least squares regression to fit a cosine wave with a period of 24 h. For each variable, we calculated an amplitude, acrophase, and r2 value for each animal. All data are reported as mean ± standard error of the mean; statistical significance was set at P ≤ 0.05. RESULTS Table 1 summarizes the effects of rodent strain and inspired gas on sleep architecture, respiratory pattern, and apnea expression in light and dark phases of the recording. Hyperoxia influenced sleep architecture in the light phase, but the effects depended on rat strain. For example, hyperoxia provoked a significant reduction in NREM sleep in BN rats but augmented NREM sleep in ZL rats (Table 1). There were no significant differences in the light or dark phases between BN and ZL rats for N-ƒ, N-VT, and N- E (Table 1), nor within each strain for ventilatory patterns, as assessed by SLEEP, Vol. 37, No. 4, 2014

DISCUSSION Our findings demonstrate that two inbred rat strains exhibit different vulnerabilities for SRBD despite demonstrating similar diurnal and circadian rhythms in respiratory pattern and 716

Circadian Variation in Ventilation and Sleep Apnea—Fink et al

Table 1—Comparisons of sleep, ventilation, and apnea expression in the light and dark phases by strain and inspired gas Brown Norway rats Sleep architecture (light phase) Wake (%) NREM sleep (%) REM sleep (%) Sleep architecture (dark phase) Wake (%) NREM sleep (%) REM sleep (%) Ventilatory patterns (light phase) N-f Wake N-f NREM N-f REM N-VT Wake N-VT NREM N-VT REM N- V E Wake N- VE NREM N- VE REM Ventilatory patterns (dark phase) N-f Wake N-f NREM N-f REM N-VT Wake N-VT NREM N-VT REM N- VE Wake N- VE NREM N- VE REM Sleep apnea (light phase) AI (apnea/h) total sleep Apnea duration (sec) total sleep AI NREM (apnea/h) AI REM (apnea/h) Sleep apnea (dark phase) AI (apnea/h) total sleep Apnea duration (sec) total sleep AI NREM (apnea/h) AI REM (apnea/h)

Zucker Lean rats

Strain Gas Interaction effect Strain effect Gas effect (P) differences (P) differences (P)

Control

Hyperoxia

Control

Hyperoxia

42.2 ± 3.1 50.0 ± 2.3 7.8 ± 1.0

47.0 ± 1.8 45.8 ± 1.1 7.0 ± 0.8

46.6 ± 2.4 46.9 ± 1.8 6.5 ± 1.2

41.4 ± 1.7 50.4 ± 1.5 8.1 ± 0.6

0.82 0.58 0.71

NS NS NS

0.99 0.79 0.78

NS NS NS

0.05 0.05 0.13

38.9 ± 2.2 45.2 ± 2.4 8.3 ± 0.7

39.5 ± 3.2 42.5 ± 3.4 7.7 ± 1.0

40.8 ± 1.9 44.6 ± 2.2 6.5 ± 1.0

46.1 ± 3.6 40.0 ± 3.5 6.9 ± 1.1

0.21 0.70 0.24

NS NS NS

0.27 0.28 0.81

NS NS NS

0.25 0.63 0.87

1.0 ± 0.2 0.7 ± 0.1 0.7 ± 0.1 1.0 ± 0.1 0.8 ± 0.1 0.8 ± 0.1 1.0 ± 0.1 0.7 ± 0.1 0.7 ± 0.1

0.9 ± 0.1 0.4 ± 0.1 0.5 ± 0.04 1.0 ± 0.03 0.7 ± 0.03 0.7 ± 0.1 1.0 ± 0.1 0.5 ± 0.1 0.6 ± 0.1

0.9 ± 0.04 0.6 ± 0.1 0.6 ± 0.1 1.0 ± 0.1 0.7 ± 0.1 0.7 ± 0.1 0.9 ± 0.1 0.7 ± 0.1 0.7 ± 0.1

0.9 ± 0.03 0.4 ± 0.1 0.5 ± 0.03 0.9 ± 0.03 0.8 ± 0.03 0.7 ± 0.1 0.9 ± 0.03 0.5 ± 0.1 0.5 ± 0.1

0.51 0.64 0.48 0.21 0.33 0.18 0.21 0.58 0.27

NS NS NS NS NS NS NS NS NS

0.82 0.02 0.01 0.29 0.97 0.68 0.61 0.08 0.27

NS C>H C>H NS NS NS NS NS NS

0.60 0.67 0.82 0.63 0.70 0.60 0.83 0.70 0.80

1.0 ± 0.1 0.6 ± 0.1 0.6 ± 0.1 1.0 ± 0.1 0.7 ± 0.1 0.7 ± 0.1 1.0 ± 0.1 0.7 ± 0.1 0.7 ± 0.1

0.9 ± 0.1 0.5 ± 0.1 0.5 ± 0.1 0.9 ± 0.03 0.8 ± 0.05 0.8 ± 0.05 0.9 ± 0.1 0.6 ± 0.1 0.6 ± 0.1

0.8 ± 0.1 0.6 ± 0.1 0.6 ± 0.1 1.0 ± 0.07 0.8 ± 0.07 0.8 ± 0.07 0.8 ± 0.1 0.7 ± 0.1 0.7 ± 0.1

0.9 ± 0.1 0.5 ± 0.04 0.5 ± 0.3 0.9 ± 0.03 0.9 ± 0.1 0.8 ± 0.1 0.9 ± 0.04 0.6 ± 0.1 0.6 ± 0.1

0.68 0.73 0.49 0.39 0.95 0.74 0.84 0.58 0.40

NS NS NS NS NS NS NS NS NS

0.54 0.12 0.04 0.46 0.21 0.42 0.89 0.55 0.32

NS NS C>H NS NS NS NS NS NS

0.96 0.68 0.94 0.47 0.69 0.62 0.25 0.89 0.77

16.9 ± 0.9 2.9 ± 0.1 14.7 ± 1.0 32.4 ± 4.6

34.0 ± 5.8 3.2 ± 0.1 26.0 ± 4.6 90.5 ± 15.9

8.5 ± 1.0 2.9 ± 0.2 6.6 ± 0.8 31.8 ± 7.3

15.4 ± 1.1 3.6 ± 0.1 11.1 ± 1.0 42.2 ± 8.6

< 0.001 0.20 < 0.001 0.02

BN > ZL NS BN > ZL BN > ZL

0.001 < 0.001 0.01 0.002

C

Brown Norway and Zucker Lean rats demonstrate circadian variation in ventilation and sleep apnea.

Circadian rhythms influence many biological systems, but there is limited information about circadian and diurnal variation in sleep related breathing...
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