Diaphragm long-term facilitation following acute intermittent hypoxia during wakefulness and sleep

Abstract

Acute intermittent hypoxia (AIH) elicits a form of respiratory plasticity known as long-term facilitation (LTF). Here, we tested four hypotheses in unanesthetized, spontaneously breathing rats using radiotelemetry for EEG and diaphragm electromyography (Dia EMG) activity: 1) AIH induces LTF in Dia EMG activity; 2) diaphragm LTF (Dia LTF) is more robust during sleep vs. wakefulness; 3) AIH (or repetitive AIH) disrupts natural sleep-wake architecture; and 4) preconditioning with daily AIH (dAIH) for 7 days enhances Dia LTF. Sleep-wake states and Dia EMG were monitored before (60 min), during, and after (60 min) AIH (10, 5-min hypoxic episodes, 5-min normoxic intervals; n = 9), time control (continuous normoxia, n = 8), and AIH following dAIH preconditioning for 7 days (n = 7). Dia EMG activities during quiet wakefulness (QW), rapid eye movement (REM), and non-REM (NREM) sleep were analyzed and normalized to pre-AIH values in the same state. During NREM sleep, diaphragm amplitude (25.1 ± 4.6%), frequency (16.4 ± 4.7%), and minute diaphragm activity (amplitude × frequency; 45.2 ± 6.6%) increased above baseline 0-60 min post-AIH (all P < 0.05). This Dia LTF was less robust during QW and insignificant during REM sleep. dAIH preconditioning had no effect on LTF (P > 0.05). We conclude that 1) AIH induces Dia LTF during NREM sleep and wakefulness; 2) Dia LTF is greater in NREM sleep vs. QW and is abolished during REM sleep; 3) AIH and repetitive AIH disrupt natural sleep patterns; and 4) Dia LTF is unaffected by dAIH. The capacity for plasticity in spinal pump muscles during sleep and wakefulness suggests an important role in the neural control of breathing.

Fig. 1.

Representative traces of vigilance-state change and diaphragm electromyography (Dia EMG) activity in unanesthetized rats depicting each experimental protocol. Sleep-wake state (top traces) and integrated Dia EMG activity (bottom traces) are shown before, during, and after normoxia [top: time control (TC)], acute intermittent hypoxia (AIH; middle), and AIH following preconditioning with daily AIH (dAIH) + AIH (bottom), shown on the last day of AIH exposure. Note: 1) long-lasting increase in diaphragm amplitude above baseline (dotted white line) is diaphragm long-term facilitation (Dia LTF; arrows on right) following AIH and dAIH + AIH. No Dia LTF was observed in TC experiments; 2) sleep-wake states changed fairly rapidly in all groups; and 3) during each hypoxic episode in AIH exposures, non-rapid eye movement (NREM) sleep periods were decreased. AW, active wakefulness; QW, quiet wakefulness.

Fig. 2.

A: representative resting diaphragm activity during normoxia across sleep-wake states. Pretreatment-raw and integrated Dia EMG, neck EMG, and EEG in each sleep-wake state are shown. B and C: absolute values of mean Dia EMG peak activity and respiratory frequency during pretreatment normoxic period (1 h) across vigilance states in all treatment groups (TC, AIH, and dAIH + AIH). Note: baselines during normoxia in all groups show similar values in diaphragm amplitude and frequency. Values are means ± SE. All variables in the dAIH + AIH group were measured on the last day of AIH exposure (day 8). AU, arbitrary units.

Fig. 3.

Percentage of time spent in each vigilance state before, during, and after TC (A), AIH (B), and dAIH + AIH (C) treatments. Time spent in each vigilance state was averaged in every 20-min period (before and after the treatment) or every 5-min period (during treatment). Note (B and C): 1) the time spent in NREM and QW during AIH groups switched (QW > NREM) relative to the pre- and post-treatment periods (NREM > QW); 2) the time spent in NREM sleep during hypoxia (h1–10) gradually increased toward the end of hypoxic episodes; and 3) the amount of time spent in REM sleep was suppressed during AIH. Values are means ± SE. All variables in the dAIH + AIH group were measured on the last day of AIH exposure (day 8). Although AW is not shown, the percentage of sleep-wake state time is always AW + QW + slow-wave sleep + REM = 100.

Fig. 4.

Effect of AIH on NREM sleep. Percentage of time (A), number (B), duration (C), and delta power (D) of NREM sleep pre (1 h)-, during (hypoxia, h1–10, total 50 min; normoxia, n1–10, total 45 min), and post (1 h)-treatments (TC, AIH, and dAIH + AIH) are expressed as averaged values. Note: 1) time spent in NREM sleep was significantly decreased during hypoxia (h1–10) in AIH- and dAIH + AIH-treated rats (A); 2) duration of NREM sleep episode during hypoxia (h1–10) was decreased but was significantly extended post-AIH treatment in both AIH- and dAIH + AIH-treated rats (C). Values are means ± SE. All variables in the dAIH + AIH group were measured on the last day of AIH exposure (day 8). *Significantly different from TC; †significantly different from baseline; P < 0.05.

Fig. 5.

Time course of rat peritoneal temperature before, during, and after treatment (TC, AIH, and dAIH + AIH). Absolute values of average baseline temperature (1 h) were similar in all treatment groups: AIH, 37.4 ± 0.1°C; dAIH + AIH, 37.6 ± 0.1°C; TC, 37.4 ± 0.1°C. Peritoneal temperatures prior to the 6th episode of hypoxia and 40–60 min after treatment were not significantly different among groups. After the 6th hypoxic episode, peritoneal temperature in AIH and dAIH + AIH groups had significantly decreased relative to TC rats, but these values had returned to TC levels by 40–60 min post-AIH. All variables in the dAIH + AIH group were obtained on the last day of AIH exposure (day 8). Values are means ± SE. *Significantly different from TC; †significantly different from baseline; P < 0.05.

Fig. 6.

A: changes in diaphragm activity expressed as percent change from baseline during NREM sleep before, during, and after treatments (TC, AIH, and dAIH + AIH). Significant increases above baseline were observed in diaphragm amplitude (B), frequency (C), and minute activity (amplitude × frequency; D) during NREM sleep, 0–60 min post-treatment in AIH- and dAIH + AIH-treated rats. Similar changes were not observed in TC rats. Thus, AIH induced robust Dia LTF during NREM sleep. There was no significant difference in any variable between AIH and dAIH + AIH rats. All variables in the dAIH + AIH group were obtained on the last day of AIH exposure (day 8). Values are means ± SE. *Significantly different from TC; †significantly different from baseline; P < 0.05.

Fig. 7.

A: changes in diaphragm activity expressed as percent change from baseline during QW before, during, and after treatment (TC, AIH, and dAIH + AIH) in unanesthetized rats. Significant increases above baseline were observed in diaphragm amplitude (B) and minute activity (amplitude × frequency) (D) during QW, 0–60 min post-treatment in AIH- and dAIH + AIH-treated rats. Similar effects were not observed in TC rats. Thus AIH elicits Dia LTF during QW, although frequency LTF was not observed during QW (C). There was no significant difference in any variable between the AIH and dAIH + AIH groups. All variables in the dAIH + AIH group were measured on the last day of AIH exposure (day 8). Values are means ± SE. *Significantly different from TC; †significantly different from baseline; P < 0.05.