Ethanol abolishes ventilatory long-term facilitation and blunts the ventilatory response to hypoxia in female rats

Abstract

Obstructive sleep apnea (OSA) is a breathing disorder in which airway obstruction during sleep leads to periodic bouts of inadequate (hypopneic) or absent (apneic) ventilation despite neurorespiratory effort. Repetitive apneic and hypopneic exposures can induce intermittent hypoxemia and lead to a host of maladaptive behavioral and physiological outcomes. Intermittent hypoxia treatment (IH), which consists of alternating exposure to hypoxic and normal air, can induce a long-lasting increase in breathing motor outputs called long term facilitation (LTF). IH models key aspects of the hypoxemia experienced during OSA and LTF might serve to prevent OSA or ameliorate its severity by stimulating ventilatory output during or after apnea/hypopnea. Ethanol consumption prior to sleep exacerbates existing OSA, but it is unknown how ethanol affects LTF expression. Thus, we hypothesized that ethanol treatment would attenuate LTF expression and the magnitude of the ventilatory response during acute hypoxic exposure. We administered either low-dose (0.8 g/kg) or high-dose (3 g/kg) ethanol or saline to adult female Sprague-Dawley rats through intraperitoneal injection and then measured subjects' ventilatory output by whole-body plethysmography during baseline, a 5 by 3-minute moderate IH protocol (hypoxia: F_iO₂ = 0.11, Normoxia: room air), and for one hour following the end of IH. Results indicate that low-dose ethanol abolishes LTF of respiratory rate and minute ventilation and trends suggest that low-dose ethanol might attenuate respiratory rate and minute ventilation during acute hypoxic exposure. While high-dose ethanol significantly diminished subjects' respiratory rate and minute ventilation during hypoxia, LTF expression was not significantly different between high-dose ethanol and saline-treated subjects. Overall, data indicate that ethanol exposure dramatically attenuates LTF expression following IH treatment and impairs ventilatory responses to hypoxia in a dose-dependent manner. Such findings inspire further consideration of ethanol's negative effects upon endogenous compensatory mechanisms for repeated hypoxic exposure, both in the context of OSA and beyond.

Keywords: Ethanol; Hypoxia; Intermittent hypoxia; Long-term facilitation; Plasticity; Respiratory function.

Fig. 1:. Depiction of the crossover study design for the low-dose (A) and high-dose cohorts (B) as well as the experimental timeline (C).

On the experimental day indicated by panels A and B, subjects received an intraperitoneal injection of low (A) or high dose (B) ethanol or saline immediately prior to placement in a whole-body plethysmography chamber (C) for ventilatory measurement at baseline, during intermittent hypoxia (IH), and for 1 hour after the end of IH to measure LTF.

Fig. 2:. Low dose (0.8 g/kg) ethanol abolishes LTF of minute ventilation (V_E) following intermittent hypoxia treatment and trends towards attenuating V_E and RR during hypoxic exposure.

Panel A depicts sample ventilatory waveforms (recorded by WBP) during the final 10 minutes of baseline, the fourth hypoxic exposure, and the final 10 minutes of the post-IH recording for one EtOH and one saline-treated rat. Quantified data is shown as mean ± SEM. In columns B and C open data symbols indicate subjects whose raw waveform data is displayed in panel A. Rows from i-iii contain V_T, RR, and V_E data, respectively. Panel B demonstrates no significant differences between low dose EtOH and saline-treated groups at baseline as evaluated by Mann-Whitney Rank Sum Test (Bii) or Student’s t-test (Bi & Biii). Columns C and D contain ventilatory data expressed as a percent of baseline. C shows that EtOH exposure trends towards depressing RR and V_E during an average of the five hypoxic bouts (Student’s t-test). Panel D contains minute-by-minute averages of ventilatory output during the first hour post-IH which demonstrate that ethanol abolishes LTF of V_E, indicated by RM 2-way ANOVA with a significant main effect of time for all three ventilatory metrics, and a significant interaction of time and treatment for V_E. The datapoint depicting pre-IH baseline is represented as a black star symbol for column D. P values < 0.05 are bolded. * indicates a significant pairwise comparison of the time main effect vs. pre-IH. # indicates a significant pairwise comparison of timepoint vs. pre-IH for the saline-treated group. ^ indicates a significant pairwise comparison between ethanol and saline-treated groups at 58, 59, and 60 minutes post-IH.

Fig. 3:. High dose (3.0 g/kg) ethanol (EtOH) diminishes baseline tidal volume (V_T) and minute ventilation (V_E), blunts respiratory rate (RR) and V_E during hypoxic exposure and trends towards attenuating LTF of V_E and RR following IH treatment.

Panel A depicts sample ventilatory waveforms (recorded by WBP) during the final 10 minutes of baseline, the fourth hypoxic exposure, and between 35 and 45 minutes post-IH for one EtOH and one saline-treated rat. Quantified data is shown as mean ± SEM. In columns B and C open data symbols indicate subjects whose raw waveform data is displayed in panel A. Rows from i-iii contain V_T, RR, and V_E data, respectively. Column B demonstrates that high dose EtOH significantly depressed ventilatory output of V_T and RR at baseline as evaluated by Student’s t-test. Columns C and D contain ventilatory data expressed as a percent of baseline. C shows that EtOH exposure significantly attenuates RR and V_E during an average of the five hypoxic bouts (Student’s t-test). Panel D contains minute-by-minute averages of ventilatory output during the first hour post-IH which demonstrate trends indicating that ethanol might diminish LTF of RR and V_E, as evaluated by RM 2-way ANOVA. The datapoint depicting pre-IH baseline is represented as a black star symbol for column D. P values < 0.05 are bolded. * indicates a significant pairwise comparison of the time main effect vs. pre-IH.