Lingual muscle activity across sleep-wake States in rats with surgically altered upper airway

Abstract

Obstructive sleep apnea (OSA) patients have increased upper airway muscle activity, including such lingual muscles as the genioglossus (GG), geniohyoid (GH), and hyoglossus (HG). This adaptation partially protects their upper airway against obstructions. Rodents are used to study the central neural control of sleep and breathing but they do not naturally exhibit OSA. We investigated whether, in chronically instrumented, behaving rats, disconnecting the GH and HG muscles from the hyoid (H) apparatus would result in a compensatory increase of other upper airway muscle activity (electromyogram, EMG) and/or other signs of upper airway instability. We first determined that, in intact rats, lingual (GG and intrinsic) muscles maintained stable activity levels when quantified based on 2 h-long recordings conducted on days 6 through 22 after instrumentation. We then studied five rats in which the tendons connecting the GH and HG muscles to the H apparatus were experimentally severed. When quantified across all recording days, lingual EMG during slow-wave sleep (SWS) was modestly but significantly increased in rats with surgically altered upper airway [8.6 ± 0.7% (SE) vs. 6.1 ± 0.7% of the mean during wakefulness; p = 0.012]. Respiratory modulation of lingual EMG occurred mainly during SWS and was similarly infrequent in both groups, and the incidence of sighs and central apneas also was similar. Thus, a weakened action of selected lingual muscles did not produce sleep-disordered breathing but resulted in a relatively elevated activity in other lingual muscles during SWS. These results encourage more extensive surgical manipulations with the aim to obtain a rodent model with collapsible upper airway.

Keywords: REM sleep; genioglossus; hyoid bone; obstructive sleep apnea; tongue.

Figure 1

Location of the recording sites in the tongue in the rats of this study. The sites were localized post-mortem and superimposed on a standard sagittal cross-section of the rat tongue. Lingual EMG signals recorded from eight sites in five rats with GH/HG muscles disconnected from H apparatus (filled circles) and from five recording sites in five rats with intact upper airway (open circles) were analyzed. When signals from two sites in the same animal were available, the mean EMG levels recorded from the two sites were averaged to ensure that each animal contributed evenly to the group mean data. The numbers identify different animals.

Figure 2

Examples of polygraphic records collected on days 6 and 22 after instrumentation from a rat with intact upper airway (rat 17 in Figure 1). (A,B) Continuous records of lingual and nuchal EMGs and cortical EEG, and the corresponding hypnograms spanning the entire analyzed recording periods (2 h). Periods with low levels of lingual and nuchal EMG during quiet wakefulness and sleep are interrupted by intense bursts of activity associated with active wakefulness. (C,D) Nine minutes-long segments of the records marked by the vertical gray lines in (A,B) shown using expanded time and amplitude scales. The segments are taken from SWS periods when lingual muscles are atonic or nearly atonic. The records demonstrate a close similarity of activity patterns, sleep–wake patterns, and noise levels recorded from the same animal 16 days apart.

Figure 3

Stable levels of lingual and nuchal EMGs across successive recording days in rats with intact upper airway. (A) Lingual and nuchal EMG levels quantified separately during different sleep–wake states, and the percentage amounts of wakefulness, SWS, and REMS in one rat across all five recording sessions conducted on days 6, 8, 11, 14, and 22 after instrumentation (rat 17 in Figure 1). (B) Average levels of lingual and nuchal EMG, and the mean percentage amounts of wakefulness, SWS, and REMS in five rats with intact upper airway studied on the same 5 days after instrumentation. There was no systematic, time-dependent trend in the levels of lingual or nuchal EMGs, or the amount of sleep–wake states across the 16 days separating the first and last recording session.

Figure 4

Examples of parasagittal cross-sections through the upper airway in an intact rat [(A); rat 11 in Figure 1] and a rat with the GH/HG muscles disconnected from the H apparatus [(B); rat 8 in Figure 1]. The bottom panels show enlarged images of the region where the cut was made and wax barrier inserted to prevent re-connection in the rats with compromised upper airway. In all rats with altered upper airway, the wax barrier remained in place and GH/HG muscles ended with a healed scar [black circle around the lesion in the enlarged image in (B)], with no evidence of re-connection to their original insertion point.

Figure 5

Stable levels of lingual and nuchal EMGs across successive recording days in rats with GH/HG muscles disconnected from the H apparatus. (A) Lingual and nuchal EMG levels quantified separately during different sleep–wake states, and the percentage amounts of wakefulness, SWS, and REMS in one rat across all five recording sessions conducted on days 6, 8, 11, 14, and 22 after instrumentation (rat 15 in Figure 1). (B) Average levels of lingual and nuchal EMG, and the mean percentage amounts of wakefulness, SWS, and REMS in the five rats with compromised upper airway studied on the same 5 days after instrumentation. Within this data set, the level of nuchal EMG during REMS was higher on day 14 when compared to day 6 (p = 0.04); other than that, no significant effects of the recording day were detected.

Figure 6

The mean lingual and nuchal EMG levels during different sleep–wake states in control rats and rats with GH/HG muscles disconnected from H apparatus. When quantified in absolute units, both lingual (A) and nuchal (B) EMGs had similar characteristic patterns of mean levels of activity across wakefulness (W), SWS, and REMS in the control and experimental rats, with only nuchal EMG measured during SWS being significantly higher in the experimental than in the control rats. The rats subjected to surgical intervention tended to have reduced lingual EMG and elevated nuchal EMG during W, suggesting a protective reaction to the injury applied to the ventral neck region. When lingual and nuchal EMG levels during sleep were quantified relative to their levels during wakefulness (C,D), lingual EMG during SWS was significantly higher in the rats with altered upper airway when compared to control rats, whereas there was only an insignificant trend in this direction for nuchal EMG. See text for alternative interpretations of this finding. Bars show average data derived from 24 recording sessions with 5 control rats and 25 recording sessions with 5 experimental rats. ns – not significant difference between the control and experimental rats.

Figure 7

Respiratory modulation of lingual EMG was rare and occurred mainly during SWS. (A) Distribution of inspiratory-modulated segments of lingual EMG in relation to sleep–wake states in a 2 h-long recording sessions obtained on day 22 after instrumentation (rat 17 of Figure 1). Positions of the segments with respiratory modulation (RM) of lingual EMG are marked by vertical lines above the hypnogram. The RM segments occurred mainly during SWS, lasted 2.4–62.2 s, and collectively occupied 2.4% of the entire recording session. (B) Example of a record from the session illustrated in (A) during which inspiratory modulation appeared and then faded away during a period of SWS. The record is 44 s-long and shows the raw and integrated lingual and diaphragmatic (Dia) EMGs. Superposition of the integrated EMGs from both muscles (bottom trace) reveals that lingual EMG is inspiratory-modulated with a stable phase relationship relative to diaphragmatic activity during the 32 s-long period marked by the horizontal bar at the bottom.

Figure 8

Example of two successive central apneic events during REMS. The traces show a 56 s-long record of the raw and integrated diaphragmatic EMG, raw lingual and nuchal EMGs and EEG. Arrows point to two apneas longer than 2 s. The record was collected on day 22 after instrumentation from a rat with altered upper airway (rat 10 in Figure 1).