ON-LINE REPOSITORY MATERIAL DIFFERENCES IN SLEEP-INDUCED HYPOXIA BETWEEN A/J AND DBA/2J MOUSE STRAINS

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1 37 ON-LINE REPOSITORY MATERIAL DIFFERENCES IN SLEEP-INDUCED HYPOXIA BETWEEN A/J AND DBA/2J MOUSE STRAINS Arnon E. Rubin, Vsevolod Y. Polotsky, Alex Balbir, Jerry A. Krishnan, Alan R. Schwartz, Philip L. Smith, Robert S. Fitzgerald, Clark G. Tankersley, Machiko Shirahata, and Christopher P. O'Donnell,

2 38 Adult, male A/J (24.3 ± 0.5 g; 109 ± 7 days of age) and DBA/2J (25.6 ± 0.9 g; 96 ± 6 days of age) mice from Jackson Laboratory (Bar Harbor, ME) were used in the study. The study was approved by the Johns Hopkins University Animal Use and Care Committee and complied with the American Physiological Society Guidelines. For all surgical procedures, anesthesia was induced and maintained using 1-2% isoflurane administered through a facemask. At the completion of experiments, animals were euthanized with pentobarbital (60 mg i.p.) Procedures Polysomnography: Three electroencephalographic (EEG; E363/1, Plastics One Inc., VA) electrodes were fastened to the skull, two nuchal electromyographic (EMG; E363/76, Plastics One Inc., VA) electrodes were stitched flat onto the surface of the muscle immediately posterior to the dorsal area of the mouse skull as previously described 1. The three EEG and two EMG electrodes were inserted into a pedestal (MS363, Plastics One Inc., VA) and cemented to the skull with dental acrylic. Each animal was allowed 5-7 days to recover from surgery and then attached via a connector cable (363-SL/6 80CM 6TCS, Plastics One Inc., VA) and electrical swivel to preamplifiers. Animal Maintenance The connector cable from the animal was fixed above to a low friction mercury swivel (Dragonfly R&D Inc., Ridgeley, WV) allowing 360 degree unrestricted movement of the tethered mouse. The mouse was housed in a pyramid-shaped customized chamber

3 39 (volume = 1.4 l), which had four inlet ports on each side of the base and an open hole at the apex for gas exhaust and the connector cable to exit. The upper portion of the base was covered with a flat mesh screen and regular bedding material to ensure an even distribution of gases throughout the chamber. A water bottle was attached to the outside of the chamber with the spout inserted through a hole into interior. Approximately 4 cm above the top of the base three ports were inserted to continuously monitor the ambient FiO 2 in the chamber. The polysomongraphic leads and the sampling line for inspired O 2 exited out of a larger sound proof holding container (BRS/LVE 9381-D, Laurel Maryland) to connect to amplifiers. A timer operating a 4-watt light bulb controlled the 12-hour light/dark cycle inside the holding container. Sleep/Wake Detection System The techniques and procedures related to our on-line sleep/wake detection system have been previously described 1. In brief, the program used a combination of the frequency distribution of the EEG and amplitude of the nuchal EMG to determine sleep/wake state over five second epochs. Each mouse underwent an initialization period in which threshold values for the B2/D1 EEG frequency ratio, two EMG thresholds, and the EEG threshold were optimized visually from the polysomnography over several hours by one or more investigators. Once initial values for the thresholds were determined, we recorded polysomnography and computer sleep/wake assessment for a subsequent hours and made minor adjustments in the thresholds where appropriate. In the

4 40 current study using DBA/2J and A/J strains the computer algorithm detected wakefulness and NREM sleep with accuracy consistent with our previously published values in C57BL/6J mice (wake, 97.2 ± 1.1%; NREM sleep, 96.0 ± 0.9%) 1. The accuracy of the computer algorithm at detecting REM sleep in DBA/2J and A/J mice fell below our previously published levels of 85.6 ± 5.0% 1. However, misclassified REM periods were always scored as NREM sleep and did not influence our experimental interventions (see below) that were based on the presence of sleep versus wake and not specific sleep stages. Overall sleep architecture data are reported as wakefulness and sleep (NREM and REM sleep combined). Hypoxia and Sleep Fragmentation Sleep-Induced Hypoxia: During continuous periods of wakefulness, room air was delivered through the cage at a rate of 4 l/min. When either NREM or REM sleep was detected for a continuous 15 sec period, the output signal from the computer caused the airflow to cease and 100% N 2 to be delivered at 4 l/min. The N 2 gas delivery continued until arousal occurred, at which time room air was once again delivered at a rate of 4 l/min. In addition, for the first 10 sec following arousal, a separate solenoid was used to deliver a second source of room air at 5 l/min. The profile of change in FiO 2 was designed to effectively replicate the timing, pattern and magnitude of oxyhemoglobin changes that characterize human OSA 1. Sleep Fragmentation Without Hypoxia: A system was developed to cause arousal using a tactile and auditory stimulus after a comparable period of time from sleep onset as that

5 41 produced with hypoxia. The sampling ports for measurement of FiO 2 during sleepinduced hypoxia (see above) were adapted and used to deliver a sequentially increasing air flow stimulus until arousal occurred (10 l/min from 0-5sec, 20 l/min from 5-10sec, 50 l/min from 10-15sec). Recording Apparatus Physiologic signals were amplified and filtered (Grass Instruments; Quincy, MA) as follows: EEG (3-35 Hz) and EMG (10-75 Hz) activity and a Beckman Analyzer (Anaheim, CA) sampled oxygen levels from the mouse housing chamber. Both instruments were connected to the pen recorder and data from the recorder were sampled at 300 Hz and converted to digital format (DI-200 data acquisition board; Dataq Instruments; Akron, OH) and acquired to optical disk for storage with Windaq/200 acquisition software (Dataq Instruments; Akron, OH). Protocol Animals were given 5-7 days to recover after surgical implantation of the polysomongraphic electrodes and pedestal assembly before beginning a three to five day acclimation period in the housing chamber. At the completion of the acclimation period, sleep/wake data were collected for a 24-hour baseline period. After the baseline period, animals were exposed to either (a) five consecutive days of sleep-induced hypoxia, or (b) five consecutive days of sleep fragmentation without hypoxia, followed by a 24 hour recovery period in which sleep/wake state was monitored in the absence of

6 42 either sleep-induced hypoxia or sleep fragmentation in an equivalent manner to the baseline period. Analyses Sleep Stage Assignment: Sleep architecture data and air/nitrogen solenoid status data were imported into a database. We determined the time and duration of each event and time and duration of periods of wakefulness and sleep (NREM and REM sleep combined). Furthermore, the sleep/wake state data were subjected to combining rules to produce contiguous epochs of sleep/wake as previously described 1. Analysis of the Nadir FiO 2 from the Event Duration: A least squares correlation analysis was used to assess the relationship between event duration and nadir FiO 2. Statistical Analyses: All results are presented as mean ± SEM. Statistical significance between factors of sleep-induced hypoxia or sleep fragmentation (baseline, Day 1-5, recovery) and Light Cycle (light, dark), was determined by ANOVA. If the ANOVA was significant for a factor, a Dunnett test was used to determine which means were significantly different. All the results are presented as mean ± s.e. (standard error of mean). Survival Analysis: The relative propensity of strains (DBA/2J vs. A/J) to fall asleep was evaluated using survival analyses. Because each mouse had multiple events (e.g., falling asleep after each hypoxia-induced awakening), the Andersen-Gill model of multiple

7 43 failure-time survival analysis was used 2. A two-tailed p-value less than 0.05 was used to denote statistical significance for all analyses. Computations for survival analyses were performed using STATA version 7.0 (College Station, TX). REFERENCES E 1. Tagaito, Y., V. Y. Polotsky, M. J. Campen, J. A. Wilson, A. Balbir, P. L. Smith, A. R. Schwartz, and C. P. O'Donnell A model of sleep-disordered breathing in the C57BL/6J mouse. J.Appl.Physiol 91: E 2. Andersen, P. K and Gill, R. D. Cox's regression model for counting process: A large sample study. Annals of Statistics 10,