J Appl Physiol 119: 1088–1096, 2015. First published August 27, 2015; doi:10.1152/japplphysiol.00030.2015.

Effect of age on long-term facilitation and chemosensitivity during NREM sleep Susmita Chowdhuri,1,2 Sukanya Pranathiageswaran,2 Rene Franco-Elizondo,1,2 Arunima Jayakar,1,2 Arwa Hosni,1,2 Ajin Nair,1 and M. Safwan Badr1,2 1

Medical Service, John D. Dingell Veterans Affairs Medical Center, Detroit, Michigan; and 2Division of Pulmonary/Critical Care and Sleep Medicine, Department of Medicine, Wayne State University School of Medicine, Detroit, Michigan

Submitted 9 January 2015; accepted in final form 4 August 2015

aging; intermittent hypoxia; episodic hypoxia; ventilation; hypoxic ventilatory response; peripheral chemoreceptor activity

is increased with age, with a predilection for central sleep apnea in older adults (1). Increased prevalence of central apnea in older adults remains unexplained and is likely multifactorial, including unfavorable upper-airway anatomy (24, 25), higher upper-airway collapsibility (14), or increased ventilatory instability (36, 37). In addition, increased propensity to apneas may be due to increased chemoreflex sensitivity (45, 46). However, studies investigating age differences in chemoresponsiveness during wakefulness and sleep reached conflicting conclusions. A number of studies noted a similar response to hypoxia and hypercapnia in old vs. young adults (7, 39, 43). Conversely, a study THE PREVALENCE OF SLEEP APNEA

noted an increase in the ventilatory response to isocapnic hypoxia during wakefulness (9), and a longitudinal study found an increase in the hypoxic ventilatory response (HVR) with aging, which was more prominent in men (21). However, there are no studies addressing the age effect on the HVR during sleep. The occurrence of recurrent central apneas depends on the balance between excitatory and inhibitory influences (28). For example, episodic hypoxia (EH), as seen in recurrent sleep apnea, is associated with a prolonged increase in ventilatory motor output, referred to as long-term facilitation (LTF), which may stabilize respiration and mitigate the recurrence of obstructive apnea (22, 28). Conversely, the propensity to central apnea is increased in the aftermath of EH (12), suggesting that LTF may promote breathing instability. There is evidence, in animal studies, that aging is associated with diminished LTF following EH (30, 48). We have previously shown that EH elicits LTF of the genioglossus (GG) muscle activity during sleep (11). The magnitude of the GG LTF during the recovery period correlated with the magnitude of GG activation during hypoxia and inversely with age, suggesting a potential interaction between age and hypoxic response. Therefore, we sought to determine the effect of age on ventilatory LTF during sleep. We reasoned that the magnitude of HVR may influence the development of ventilatory LTF. We hypothesized that aging would be associated with diminished ventilatory LTF during sleep and decreased HVR when compared with young adults. METHODS

Participants The Human Investigation Committees of Wayne State University School of Medicine and Detroit Veterans Affairs Medical Center approved the experimental protocols. Informed, written consent was obtained from 14 healthy, older adult participants, free of symptoms of sleep apnea or other medical disorders, except for one individual with hypertension. The participants had normal pulmonary function and ECG. They also underwent an overnight polysomnography (PSG) study (Table 1). Nine young participants without sleep-disordered breathing were recruited as controls for the brief hyperoxia protocol. Ten young adults without sleep-disordered breathing also served as controls for the HVR protocol. Effects of EH in the young are not presented here, as these findings were published previously [Chowdhuri et al. (12); also see DISCUSSION and Table 4]. Breathing Circuit

Address for reprint requests and other correspondence: S. Chowdhuri, John D. Dingell VA Medical Center (11M), 4646 John R, C3427, Detroit, MI 48201 (e-mail: [email protected]). 1088

Each participant was connected to the breathing circuit via a nasal mask. This has been described previously (1, 2, 11, 12, 38, 40). An appropriate-sized, airtight silicone nasal mask (Respironics, Murryshttp://www.jappl.org

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Chowdhuri S, Pranathiageswaran S, Franco-Elizondo R, Jayakar A, Hosni A, Nair A, Badr MS. Effect of age on longterm facilitation and chemosensitivity during NREM sleep. J Appl Physiol 119: 1088 –1096, 2015. First published August 27, 2015; doi:10.1152/japplphysiol.00030.2015.—The reason for increased sleep-disordered breathing with a predominance of central apneas in the elderly is unknown. We speculate that ventilatory control instability may provide a link between aging and the onset of unstable breathing during sleep. We sought to investigate potential underlying mechanisms in healthy, elderly adults during sleep. We hypothesized that there is 1) a decline in respiratory plasticity or long-term facilitation (LTF) of ventilation and/or 2) increased ventilatory chemosensitivity in older adults during non-, this should be hyphenated, nonrapid rapid eye movement (NREM) sleep. Fourteen elderly adults underwent 15, 1-min episodes of isocapnic hypoxia (EH), nadir O2 saturation: 87.0 ⫾ 0.8%. Measurements were obtained during control, hypoxia, and up to 20 min of recovery following the EH protocol, respectively, for minute ventilation (VI), timing, and inspiratory upper-airway resistances (RUA). The results showed the following. 1) Compared with baseline, there was a significant increase in VI (158 ⫾ 11%, P ⬍ 0.05) during EH, but this was not accompanied by augmentation of VI during the successive hypoxia trials nor in VI during the recovery period (94.4 ⫾ 3.5%, P ⫽ not significant), indicating an absence of LTF. There was no change in inspiratory RUA during the trials. This is in contrast to our previous findings of respiratory plasticity in young adults during sleep. Sham studies did not show a change in any of the measured parameters. 2) We observed increased chemosensitivity with increased isocapnic hypoxic ventilatory response and hyperoxic suppression of VI in older vs. young adults during NREM sleep. Thus increased chemosensitivity, unconstrained by respiratory plasticity, may explain increased periodic breathing and central apneas in elderly adults during NREM sleep.

Aging and Long-Term Facilitation

Table 1. Characteristics of older adults (n ⫽ 14) Age, yr Gender BMI, kg/m2 Neck circumference, cm AHI, per hour Sex hormone, women* Sex hormone, men

62 ⫾ 8 8 women/6 men 26.3 ⫾ 2.4 35.5 ⫾ 3.2 4.7 ⫾ 4.1 LH: 36.3 ⫾ 19.1 mIU/ml FSH: 83.1 ⫾ 51.3 mIU/ml Testosterone: 299.4 ⫾ 144.3 ng/dl

Values are reported as means ⫾ SD. BMI, body mass index; AHI, apnea hypopnea index; LH, luteinizing hormone; FSH, follicle-stimulating hormone; *Postmenopausal range for LH: 11.13– 65.07 mIU/ml and FSH: 20 –138 mIU/ml, respectively. Women were postmenopausal. Normal male testosterone levels, ⱖ300 ng/dl (10, 33).

Measurements For the PSG study, the apnea hypopnea index was measured using 4% desaturation with a concomitant 30% decline in flow to define hypopneas in keeping with U.S. Medicare guidelines for diagnosing sleep apnea that would be relevant to the elderly population. EEG, electrooculograms (EOG), and chin electromyograms were recorded using the International 10-20 system of electrode placement (EEG: C3-A2 and C4-A1; EOG: O-A2). Inspiratory airflow was measured by a heated pneumotachometer (Model 3700A; Hans Rudolph) that was attached to a pressure transducer (Validyne, Northridge, CA). The tidal volume (VT) was obtained from the electronic integration of the flow signal (Model FV156 integrator; Validyne). To confirm the central etiology of apnea and to ascertain upper-airway mechanics, supraglottic pressure (PSG) was measured using a pressure transducertipped catheter (Model TC-500XG; Millar Instruments, Houston, TX) with the tip positioned in the hypopharynx. The hypopharyngeal position was obtained by advancing the catheter tip for 2 cm after it disappeared behind the tongue. PETCO2 readings were obtained continuously by an infrared analyzer (Model CD-3A; AEI Technologies, Pittsburgh, PA) from tubing placed in the nares via a port in the nasal mask. Arterial O2 saturation (SaO2) was measured by a pulse oximeter (Ohmeda Biox 3700). The signals were displayed on a polygraph recorder (Grass Model 15; Astro-Med, West Warwick, RI) and recorded using PowerLab data acquisition software (ADInstruments, Colorado Springs, CO) for detailed analyses. Upper-airway resistance (RUA) was measured at a constant flow rate on the linear portion of the flow/pressure loop of each breath. This methodology has been used by our laboratory, as noted in prior publications (11, 12, 38, 40). In six individuals, in whom hypopneas were noted with EEG arousals and fragmented sleep, expiratory positive airway pressure (EPAP) of 4 –5 cm H2O was added at the start of the study to allow reduction of hypopneas and stable sleep (no central apneas emerged on this PAP

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setting). EPAP was held at a constant level throughout the study protocol. The same EPAP setting was also added during the corresponding sham study. Protocol 1: EH during NREM Sleep The study was conducted during the individual’s usual nocturnal sleep period. The EH protocol also has been described previously (12, 38). In brief, the participants underwent EH trials on night 1 and sham study on a night 2 for a comparable duration on room air. During the study run-in period, some of the participants underwent an experimental night of EH without zolpidem. However, it was noted that the elderly participants were consistently unable to maintain sleep through the 15 EH trials; subsequently, the protocol was changed to allow the patients to receive zolpidem before the study, and this allowed successful completion of the EH protocol. The participants received zolpidem 10 mg during both experimental and sham study nights to allow stable stage N2 sleep. All trials were conducted in stable stage N2 or N3 sleep. During stable NREM sleep, the participants breathed room air for 15–20 min of the baseline control period, followed by 15, 1-min isocapnic EH, each episode separated by room-air breathing for ⬃1 min. Hypoxia was induced rapidly by adding nitrogen, for four to six breaths, to the breathing circuit to produce a hypoxic gas mixture to attain a fraction of inspired O2 (FIO2) goal of 7– 8%. Supplemental FICO2 21% was added to the circuit to maintain isocapnia by aiming to raise the inspired CO2 by ⬃0.5% and with the goal of maintaining the average PETCO2 during the hypoxia trials within 1 mmHg of the eupneic CO2 (see Fig. 2). The gases were delivered through a gas blender that allowed “fine tuning” of gas flow as needed. The administration of nitrogen was terminated when the oxyhemoglobin saturation decreased to 89%; thereafter, O2 saturation continued to decline spontaneously an additional 3–5% in the milieu of the hypoxic gas mixture in the circuit. Hypoxia was terminated abruptly at the end of 1 min, with two breaths of 100% O2 (Fig. 1). Once the O2 saturation returned to control levels, a short recovery period for 1 min was allowed on room air before the next EH. Minute ventilation (VI), timing, and PSG were measured during baseline control, hypoxia trial, immediately after each hypoxia trial, and up to 45 min after the final hypoxia trial. To ensure that changes during the recovery period were not due to time-dependent phenomena, independent of EH, participants underwent a sham study on a different night for a similar time duration as the experimental night, with identical measurements but without the hypoxia intervention. The sham protocol involved flow of room air into the inspiratory line that was similar to the flow of gases during the hypoxia runs. Protocol 2: Chemosensitivity during NREM Sleep Measurements. We measured VI, timing, and PSG, as described above in Protocol 1. BRIEF HYPEROXIA PROTOCOL. The brief hyperoxia protocol was performed on a separate night from the EH protocol. The breathing circuit is described above. We studied 10 healthy, older [age 62.7 ⫾ 8.5 yr, body mass index (BMI) 26.4 ⫾ 2.4 kg/m2; 6 women] and 9 healthy, young (age 22.6 ⫾ 2.7 yr, BMI 25.6 ⫾ 3.4 kg/m2; three women) individuals. The same older individuals from Protocol 1 also underwent Protocol 2. The participants breathed room air or the contents of a gas tank with 100% O2 through a one-way valve. During stable NREM sleep, a trial of brief hyperoxia, to mimic Dejours’ test (13, 18), was introduced with few breaths of FIO2 100% to reach a desired inspired O2 concentration of ⬃70% or partial pressure of inspired O2 ⬎ 350 mmHg, which is sufficient to reduce the sensitivity of the peripheral chemoreceptor in humans (13, 18). This was followed by a quick termination of the trial by turning off the valve and allowing room air to flow into the circuit. The trials were repeated three times at intervals of 3 min to ensure reproducibility of results and to avoid random effects from background variation in ventilation.

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ville, PA) was glued to the face to prevent mask leaks. The mask was connected to a Plateau Exhalation Valve (Respironics), via a heated pneumotachometer (Model 3700A; Hans Rudolph, Shawnee, KS). The valve, which provides a continuous leak path in the breathing circuit and serves as an exhaust vent, was connected to the inspiratory line. Mask leak was corrected during the protocols. Two cylinders containing 100% N2 or 100% O2 were connected to the inspiratory line. To maintain isocapnia, supplemental partial pressure of CO2 [PCO2; fraction of inspired CO2 (FICO2): 21%, balanced with N2] was added to the inspiratory line from the gas cylinder via a gas blender to maintain end-tidal CO2 (PETCO2) at or near baseline control levels. Room air was also added to the inspiratory line, as per the protocol. We performed multiple protocols, as described below, which included the following: 1) Protocol 1, which examines the effect of EH on LTF during nonrapid eye movement (NREM) sleep in elderly adults, and 2) Protocol 2, which examines peripheral chemosensitivity in elderly vs. young adults during NREM sleep.

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Fig. 1. A representative polygraph segment from a participant, age 60 yr, during an isocapnic hypoxia trial, demonstrating increased tidal volume (VT) and supraglottic pressure (PSG) during the hypoxic episode (EH) compared with the control period. The EH was terminated by 2 breaths of 100% O2. EOG, electrooculogram; PETCO2, end-tidal CO2; Pmask, mask pressure; SaO2, arterial O2 saturation.

Thus when FIO2 returned to normoxic baseline values, a period of breathing for 3 min on room air was allowed for all ventilatory parameters to return to baseline. The FIO2 achieved during hyperoxia was 82 ⫾ 13%. In case stable sleep was not maintained, the individual was allowed to return to stable-stage sleep before resuming the trial. HYPOXIC VENTILATORY RESPONSE. HVR was determined from the hypoxia trials in Protocol 1. The participants underwent exposure to multiple 1-min isocapnic EH, each trial interspersed with room air (12, 19). Isocapnia was maintained, because a low PCO2 in arterial blood (PaCO2) is known to attenuate the hypoxic response of carotid chemoreceptors. Thirteen healthy, older (age 62.5 ⫾ 8.5 yr, BMI 26.4 ⫾ 2.3 kg/m2; 7 women) and 10 healthy, young (age 26.4 ⫾ 4.3 yr, BMI 22.3 ⫾ 2.2 kg/m2; 6 women) individuals without significant sleep apnea were exposed to multiple trials of isocapnic hypoxia during NREM sleep. Through a one-way valve, the subjects breathed room air or the contents of one of three tanks of gas attached by tubing to a five-way stopcock. The gas mixtures used were 8% O2, 21% CO2, and 100% O2. The number of breaths of 8% O2 was titrated to maintain O2 saturation between 80 and 85% by maintaining the partial pressure of inspired O2 (PIO2) at 8 –10% (55 mmHg). Simultaneously, during the EH, 21% CO2 was introduced via a side port attached to the inspiratory tubing to maintain PETCO2 at control values (isocapnia). The nadir SaO2 attained was 87.2 ⫾ 2% in the young adults in 11 ⫾ 3 trials and 87.6 ⫾ 3% in the older adults in 12 ⫾ 2 trials. At the completion of each episode, hypoxia was terminated abruptly with two breaths of 100% O2 to bring the PIO2 rapidly back to the normoxic range. Data Analysis Episodic hypoxia. The methodology for analyses has also been described previously (12, 19, 38, 40). Data recorded during exposure to EH were used for analysis only if there was stable NREM sleep. Sleep was scored using the American Academy of Sleep Medicine scoring criteria (6). Inspired VT, inspiration time, total breath time,

breathing frequency (fR), VI, PETCO2, and SaO2 were calculated breath by breath. Inspiratory RUA was measured at a constant flow rate on the linear portion of the pressure-flow loop during inspiration of each breath (11, 12, 38, 40). For each variable, an average value was computed during the control period, during each EH, and during recovery periods that followed each EH. Average values during the control period were determined by averaging the data obtained from 20 breaths, recorded immediately before the onset of the first EH. Average values for all variables were also obtained using the last five breaths recorded during each normoxic period that preceded each EH. The average values of five to six breaths during the nadir hypoxia period were obtained. Lastly, following the final EH, the recovery period was analyzed. Average value for each variable was obtained from consecutive breaths after 15–20 min of recovery during stage N2 sleep following the final EH. The presence of periodic breathing cycles was examined visually as oscillatory changes in the breathing pattern. Periodic breathing cycles were identified as the number of peaks in the crescendo-decrescendo oscillatory breathing pattern. The number of cycles of periodic breathing cycles was assessed for 5 min during the control period and for the first 15 min of the recovery period. Sham protocol. During the sham protocol, measurements were obtained before (control) and after breathing room air for 50 min. The 50th min corresponded to an equivalent time period during the experimental night. Brief hyperoxia. Eupneic breaths before each trial were analyzed for VI and timing. Similarly, the nadir breath immediately following hyperoxic breaths was analyzed. The nadir VI, achieved immediately upon exposure to hyperoxia as a percent of eupneic VI, was analyzed (45) (see Fig. 3). Brief hyperoxia protocol was repeated three times at intervals of 3 min during stable NREM sleep, and the average results are reported. Hypoxic ventilatory response. Ten eupneic breaths were analyzed, as well as five breaths, during nadir hypoxia. HVR was calculated as

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the change in VI for a change in SaO2 during a given EH (12, 19). HVR was calculated for 11.5 ⫾ 2.3 (means ⫾ SD) hypoxia trials. Statistical Analysis

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data. When the normality test failed (e.g., for HVR) nonparametric tests were performed. RESULTS

The participant characteristics for older adults are reported in Table 1. A polygraph segment obtained from a participant in stage N2 sleep, before, during, and immediately following exposure to isocapnic hypoxia, is shown in Fig. 1. A compressed polygraph segment obtained from the same participant at baseline, during hypoxia trials, and 20 min into the recovery period, demonstrating maintenance of isocapnia during the trials, is also shown in Fig. 2. The brief hyperoxia protocol is represented in a polygraph segment in Fig. 3. Protocol 1: EH Hypoxia trials. Group data for ventilation, timing, and upper-airway mechanics during control, nadir hypoxia, and recovery periods are shown in Table 2. The nadir SaO2 attained was 87% in 11 ⫾ 3 (means ⫾ SD) trials. Induction of hypoxia was associated with a significant increased VI at 158 ⫾ 11% of control values, due to increased VT (Fig. 1) and fR (Table 2). Isocapnia was maintained during the majority of the EH trials. However, there was no evidence of a change in inspiratory RUA during the trials, and there was no progressive augmentation of ventilation during either the room-air or hypoxia periods as the trials ensued from hypoxia trials (Hx)1 to Hx15 [P ⫽ not significant (ns); see Fig. 6].

Fig. 2. A compressed polygraph segment from an older participant during room-air control, hypoxia trials (Hx1–Hx5 and Hx12–Hx15), and recovery period. Note that isocapnia was maintained during the hypoxic exposure.

O2

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A commercially available computer statistical package was used to analyze the data (SigmaStat 3.11.0; SPSS, IBM, Armonk, NY). The results are presented as means ⫾ SE unless specified otherwise. The level of statistical significance was set at P ⱕ 0.05. Protocol 1. EXPERIMENTAL PROTOCOL. For normally distributed data, comparisons among time points for control, hypoxia, and recovery were made using one-way ANOVA with repeated measures, followed by post hoc analysis for all pair-wise comparisons using the Holm-Sidak method. When the normality test failed (RUA), Friedman’s ANOVA on rank test was performed. In addition, a two-way ANOVA with repeated measures was performed to compare control and recovery parameters with EH vs. sham intervention, followed by multiple pair-wise comparisons using the Student-Newman-Keuls method. To ascertain the time course of increased VI, we plotted the VI during successive room air and hypoxia periods, respectively, for each individual subject. We then performed linear regression analysis to determine if there was a significant increase in the slope of VI. A significantly increased slope would reflect augmentation of VI (11, 38, 41, 47). Similarly, we also used regression analysis to determine the slope of HVR in the older individuals to determine the presence of augmentation of HVR. Relationship between male testosterone levels and recovery VI was evaluated using Spearman’s correlation. SHAM PROTOCOL. Paired t-tests were performed to compare the variables between control and sham periods. If normality test failed (expiratory time), then the signed-rank test was used. Protocol 2. Comparisons of ventilatory parameters between young and old individuals were made using t-tests for normally distributed

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10 sec

Fig. 3. A representative polygraph segment showing immediate suppression of VT, “Dejours’ effect” (see arrow), with brief hyperoxia in an elderly participant without sleep apnea.

Table 2. Results of episodic hypoxia protocol presented as grouped data for timing, ventilation, and resistance during the 3 periods: room-air control, hypoxia, and recovery (n ⫽ 14) Variables

Baseline Control

Hypoxia Trials

Recovery

TI, s TE, s TTOT, s fR, breath/min PETCO2, mmHg SaO2, % VT, liter VI, l/min Inspiratory RUA, cm H2O·l⫺1·s⫺1

1.8 ⫾ 0.1 2.0 ⫾ 0.1 3.8 ⫾ 0.2 16.1 ⫾ 0.7 42.1 ⫾ 0.9 96 ⫾ 0.3 0.41 ⫾ 0.02 6.6 ⫾ 0.5

1.6 ⫾ 0.1*† 1.9 ⫾ 0.1 3.6 ⫾ 0.2*† 17.6 ⫾ 0.8*† 41.0 ⫾ 1.5 87 ⫾ 0.7†§ 0.56 ⫾ 0.05†§ 9.2 ⫾ 0.7†§

1.9 ⫾ 0.1‡ 2.1 ⫾ 0.1 4.0 ⫾ 0.1 15.4 ⫾ 0.6 41.9 ⫾ 1.0 96 ⫾ 0.4 0.40 ⫾ 0.03 6.2 ⫾ 0.5

5.5 ⫾ 2.1

3.4 ⫾ 1.2

7.9 ⫾ 3.5

Values are means ⫾ SE. *P ⬍ 0.05 control vs. hypoxia; †P ⬍ 0.001 hypoxia vs. recovery; ‡P ⬍ 0.05 control vs. recovery; §P ⬍ 0.001 control vs. hypoxia. TI, inspiratory time; TE, expiratory time; TTOT, total time; fR, respiratory rate; PETCO2, end-tidal CO2 level; SaO2, arterial O2 saturation; VT, tidal volume; VI, minute ventilation; RUA, upper-airway resistance.

There was no gender difference in ventilatory LTF, i.e., no difference in recovery VI as a percent of control VI, men vs. women: 95.9 ⫾ 4.1% vs. 92.5 ⫾ 6.5% (P ⫽ ns). In the subgroup of male participants, there was a negative correlation between serum testosterone levels and VI as a percent of control (ventilatory LTF; r ⫽ ⫺0.8, P ⫽ 0.1, Spearman’s correlation); however, it was not statistically significant, likely due to a small sample size. There was no correlation with HVR (r ⫽ 0.2, P ⫽ ns). In women, there was no correlation among the magnitude of ventilatory LTF to luteinizing hormone levels, estrogen levels, or the ratio of progesterone to estrogen (P ⫽ ns). There was also no correlation of respective hormone 12

Inspiratory RUA (cmH2O/L/s)

Recovery period. The measured ventilatory parameters, including VT, timing, and VI, were unchanged (94.4 ⫾ 3.5%, P ⫽ ns) during the post-EH recovery period (at 15–20 min after the final EH) compared with the control period (Table 2), indicating absence of ventilatory LTF. Furthermore, note that there was no significant change in the inspiratory RUA during the recovery period (P ⫽ ns; Fig. 4). There were no significant changes in ventilation, timing, or resistance data during the sham recovery period compared with baseline control (Table 3).

10

Control Hypoxia Recovery

8

6

4

2

0

Fig. 4. Group data for inspiratory upper-airway resistance (RUA) in older adults. There was no significant difference in resistance among control, hypoxia, and recovery periods (P ⫽ not significant).

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Variables

Control

Sham Recovery

TI, s TE, s TTOT, s fR, breath/min PETCO2, mmHg SaO2, % VT, liter VI, l/min Inspiratory RUA, cm H2O · l⫺1 · s⫺1

1.6 ⫾ 0.1 1.9 ⫾ 0.1 3.6 ⫾ 0.1 17.1 ⫾ 0.6 38.7 ⫾ 1.1 96 ⫾ 0.4 0.40 ⫾ 0.02 6.9 ⫾ 0.5 2.3 ⫾ 0.9

1.7 ⫾ 0.1 2.1 ⫾ 0.1 3.7 ⫾ 0.1 16.5 ⫾ 0.6 38.9 ⫾ 1.0 96 ⫾ 0.4 0.39 ⫾ 0.03 6.4 ⫾ 0.6 2.4 ⫾ 1.1

Values are reported as mean ⫾ SE; no significant change in measured variables during sham recovery compared with control period. See Table 2 for abbreviations.

Protocol 2: Chemosensitivity—Brief Hyperoxia and HVR Exposure to brief hyperoxia was associated with decreased VT and VI in the older group but not in the young adults (Fig. 5). Similarly, older adults demonstrated higher HVR compared with young individuals (Fig. 6A). The nadir O2 saturations obtained in young and older adults were similar at 87.0%. In addition, there was no significant increase in the slope of HVR across Hx1–Hx15 (P ⫽ ns), thus confirming an absence of augmentation of HVR across the hypoxia trials (Fig. 6B). DISCUSSION

Summary of Findings The aim of this study was to determine whether LTF was present after repetitive hypoxic exposure in older adults. Our study demonstrated several novel findings regarding the control of breathing in older adults during NREM sleep. 1) Ventilatory LTF was absent following acute isocapnic EH. 2) Peripheral chemoreceptor activity was elevated in older men and women, as evidenced by increased HVR and greater suppression of VI following brief hyperoxia. 3) There was no augmentation of ventilation or HVR with repetitive hypoxia. 4) There were no significant changes observed in ventilation, timing, or resistance parameters following sham exposure in lieu of EH. 5) There was evidence of increased periodic breathing in older adults following EH in the absence of ventilatory LTF. Aging Is Associated with Diminished Ventilatory LTF The ventilatory control system demonstrates considerable plasticity in response to changing conditions. For example, exposure to intermittent hypoxia is associated with several changes that influence subsequent respiration; the net effect of EH depends on the balance between stabilizing and destabilizing factors. EH leads to sustained elevation of the ventilatory motor output, referred to as LTF, an excitatory mechanism

characterized by a sustained elevation in ventilatory motor output following intermittent hypoxia (1–3, 8, 16, 17, 22, 26, 32, 34). We noted that exposure to EH during sleep in older adults was not associated with ventilatory LTF. This is in contrast to the robust LTF that we have observed previously in young, sleeping humans (2, 11, 12, 38, 40). LTF in young adults, manifested by increased GG muscle activity, decreased RUA and increased VI (38, 40); thus EH may promote upperairway patency during sleep. Interestingly, we have previously demonstrated an inverse correlation between age and GG LTF in sleeping humans (11). We hypothesize that the occurrence of GG LTF and the ensuing improvement in upper-airway mechanics may be a protective mechanism mitigating recurrent upper-airway obstruction in the aftermath of a series of apneaand hypopnea-obstructive events. LTF following EH is believed to be a central neural mechanism resulting from episodic chemoafferent stimulation of the Raphe medullary neurons, with a subsequent increase in serotonin release in the vicinity of respiratory motoneurons (4). We speculate that the lack of LTF following exposure to EH may be due to diminished peripheral chemoafferent activity, Raphe activity, serotonin availability, or responsiveness to chemoafferent stimulation. We found that HVR and hyperoxic responsiveness were elevated in older adults compared with young adults. The presence of increased peripheral chemoafferent activity, despite the absence of LTF in older adults, negates diminished chemoafferent activity as a likely explanation for the absence of LTF in older adults. Thus we speculate that decreased Raphe neuronal activity or responsiveness is the likely culprit underlying diminished LTF in older adults. Studies in animal models revealed that the development of LTF is age related. Zabka et al. (48) demonstrated decreased phrenic and hypoglossal nerve (XII) LTF in old vs. young rats, suggesting that serotonergic modulation of respiratory motor output is reduced with aging male rats. Interestingly, the effect of age varied among different studies. For example, age (49) and gonadectomy attenuated XII but not phrenic LTF in male rats in one study (50). Other studies in middle-aged and gonadectomized male Sprague-Dawley rats demonstrated a similar attenuation of phrenic as well as XII LTF (48). Overall, it appears that aging diminishes the magnitude of LTF through decreased sex-hormone levels (49, 50). We noted that there

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*

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0

Fig. 5. Hyperoxic effect on ventilation. Note lower nadir minute ventilation with brief hyperoxia in older vs. young adults, indicating higher peripheral chemoresponsiveness in older adults (*P ⫽ 0.03).

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levels with HVR in women or men (r ⫽ 0.2, P ⫽ ns). Of note, there were significantly increased number cycles of periodic breathing per 5-min segments during the recovery period compared with a 5-min control period: 2.6 ⫾ 0.53 vs. 1.6 ⫾ 0.48 (P ⫽ 0.046), potentially indicating increased breathing instability during the recovery period.

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Nadir Minute Ventilation, % control

Table 3. Results of sham protocol presented as grouped data for timing, ventilation, and resistance during control and sham recovery periods (n ⫽ 12)

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B

0.8

HVR, L/min/%

* Fig. 6. A: hypoxic ventilatory response (HVR) was higher in older vs. young adults (*P ⫽ 0.04). B: lack of augmentation of HVR (progressive increase in HVR) during the course of the hypoxia trials in both groups.

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0.6

0.4

0.2

1.0

Old Young

HVR (L/min/SaO 2)

A

•

0.8

Old Young

0.6

0.4

0.2

0.0

0.0

Hx1 Hx2 Hx3 Hx4 Hx5 Hx6 Hx7 Hx8 Hx9 Hx10 Hx11Hx12 Hx13 Hx14 Hx15

be an immutable age-related phenomenon that could promote breathing instability during sleep (see below). Pathophysiological Implications We have previously shown that LTF can be evoked in humans during NREM sleep under an isocapnic condition (12, 38, 40). Evidence in the literature suggests that repetitive hypoxia elicits LTF of the XII, phrenic, diaphragmatic, or intercostal inspiratory activity (16, 17, 26, 49). Therefore, exposure to EH may promote upper-airway patency during sleep via increased upper-airway muscle activity and decreased RUA (38, 40). Similarly, increased VI and the ensuring reduction of PaCO2 promote central breathing stability by decreasing plant gain, whereby a greater change in ventilation is required to elicit a reduction in PaCO2 (12). Conversely, EH may promote breathing instability by increasing the hypocapnic ventilatory response, hence increasing the propensity to central apnea and recurrent breathing instability (12). Notably, the increase in periodic breathing cycles following EH in older adults may be indicative of increased breathing instability in the absence of ventilatory LTF. LTF: friend or foe? Our findings have significant implications to the pathogenesis of sleep apnea in older adults. Studies from our laboratory have demonstrated that LTF following EH is associated with salutatory effects on upper-airway patency, as evidenced by increased GG muscle activity (11) and decreased RUA and increased VI (38, 40). Whereas hypoxia per se may trigger upper-airway collapsibility (35, 44), in fact, LTF following EH may mitigate recurrent upper-airway obstruction following a series of apneas or hypopneas. We speculate that the lack of LTF in older adults following EH eliminates a protective mechanism and promotes recurrent upper-airway obstruction during sleep. Table 4. Studies demonstrating ventilatory LTF in young individuals References

Control VI, l/min

Recovery VI, l/min

Shkoukani et al. (40) Babcock et al. (3) Pierchala et al. (38) Chowdhuri et al. (12)

7.1 ⫾ 1.8 6.7 ⫾ 1.9 6.0 ⫾ 0.3 6.2 ⫾ 0.4

8.3 ⫾ 1.8 8.2 ⫾ 2.7 6.5 ⫾ 0.3 6.7 ⫾ 0.4

LTF, long-term facilitation.

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Recovery VI, % Control

117%, 122%, 108%, 108%,

P P P P

⬍ ⬍ ⬍ ⬍

0.05 0.05 0.05 0.05

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was a trend for a negative correlation between male testosterone levels and recovery VI. The average total testosterone level in the older males was lower than measured levels in healthy, young adults from our lab (10). Lower average sex-hormone levels in our male study participants are consistent with the hypothesis that sex-hormone levels may influence the decline of LTF with aging. However, our study cannot ascertain the relative contribution of aging vs. decreased sex hormones to the magnitude of LTF in older adults. The lack of LTF may be due to decreased sex-hormone levels or to an age-related decline of serotonergic modulation of ventilatory motor output. These possibilities are speculations that require experimental proof. In our study, there were no observed gender differences in ventilatory LTF. However, the study was not designed nor powered to detect gender differences. The trend for a potential negative correlation between recovery VI and testosterone levels requires further exploration, as the study was not powered to detect a significant difference. It is possible that the elderly could have a different threshold for CO2 level for developing LTF. There were several reasons for maintaining isocapnia and not hypercapnia during the protocol. The current protocol mimicked our prior published protocols in young adults, allowing that direct comparisons with this group have been shown to develop LTF, despite the absence of hypercapnia. In other words, isocapnia elicited LTF in the young but not in the elderly. This methodology also emulates a real-life scenario in most individuals with obstructive sleep apnea or central sleep apnea (without morbid obesity or neuromuscular disease) who would not develop hypoventilation or hypercapnia during sleep. Hypercapnia has been used during wakefulness (20) to elicit LTF. This is likely because LTF is less robust during wake than during sleep (42). However, hypercapnia elicits sleep fragmentation (15) and could potentially promote more arousals in these elderly individuals, fragment sleep, and dampen ventilatory LTF. The underlying mechanism of increased peripheral chemoreceptor activity could not be determined from our data. One possible explanation is the presence of sensory LTF secondary to chronic intermittent hypoxia. This is an unlikely possibility in this group of healthy, older adults in the absence of ventilatory LTF following acute intermittent hypoxia. Therefore, we believe that increased peripheral chemoreceptor activity and responsiveness were due to age-related changes rather than adaptation to chronic intermittent hypoxia. Accordingly, increased peripheral chemoreceptor activity in older adults may

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hypocapnia (28) and enhanced during wake with hypercapnia (20, 27). It is possible that upper-airway and diaphragmatic responses may be different with hypercapnia. It is known from animal studies that LTF is less robust during wakefulness than during sleep (42). However, given that hypercapnia can elicit sleep fragmentation (15), a relevant caveat is that hypercapnia could potentially dampen LTF by fragmenting sleep in the elderly participants who are already easily arousable. This needs to be studied further. Third, we used moderate sleep curtailment on the night preceding the sleep study to facilitate natural, unaided sleep. Our previous experience demonstrated no difference in the findings between subjects who obtained normal vs. curtailed nocturnal sleep, and the available literature supports our observation. In addition, the participants underwent similar sleep curtailment for both the EH and sham nights; so any potential alteration in airway resistance or ventilation would be present in both nights and thus would not alter our conclusions. Finally, our study addressed acute EH and did not address the effects of chronic intermittent hypoxia. In summary, the results confirm our hypothesis that LTF declines with aging in humans during sleep. The absence of LTF may be related to alterations in sex-hormone levels that act via serotonergic pathways. We speculate that manipulations with serotonergic drugs that enhance LTF may provide a potential pathway for the pharmacologic treatment of sleep apnea in the elderly. ACKNOWLEDGMENTS The authors thank Ms. Simranjit Narula and Ms. Nicole Nickert for their technical assistance with the study.

Methodological Considerations Our protocol for EH has been successful in eliciting ventilatory LTF in the past (12, 38); however, several methodological considerations need to be addressed. First, arousals from sleep may have prevented ventilatory LTF, as LTF is most easily induced during deep NREM sleep and is harder to elicit during light sleep or wakefulness in male rats (31, 42); however, we allowed sleep restriction before the study and a low dose of zolpidem to ensure a stable sleep state during the study. A similar protocol was followed for the sham studies. Therefore, any change in ventilation and resistance cannot be attributed to sleep restriction or zolpidem per se. Moreover, prior published studies did not find that zolpidem influences ventilatory parameters during sleep (5, 23, 29, 46). In our study, despite increased VI during the hypoxia trials, the effect did not persist into the recovery period. If zolpidem suppressed ventilation, leading to the absence of ventilatory LTF, then one would expect VI to be reduced even during the hypoxia trials. In fact, we would also expect VI to be reduced, not elevated, compared with young adults (the latter did not receive zolpidem). Finally, animal studies show that ventilatory LTF is “more robust” during sleep than during wakefulness (42); thus improved sleep due to zolpidem could potentially help elicit LTF. There are no studies to suggest that zolpidem preferentially enhances chemosensitivity. In fact, apneic threshold studies by Dempsey’s group (46) used zolpidem to maintain sleep in patients with heart failure and noted the absence of effect of zolpidem on occlusion pressure, ventilatory response, saturation, and PaCO2. Second, CO2 was added to the breathing circuit to maintain isocapnia; studies have shown that the full expression of ventilatory LTF is prevented if accompanied by

GRANTS Support for this work was provided by a Career Development Award-2 (#CDA-2-019-07F), the Department of Veterans Affairs (to S. Chowdhuri), and a Merit Review Award of the Department of Veterans Affairs (#1I01CX000194; to M. S. Badr). DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the authors. AUTHOR CONTRIBUTIONS Author contributions: S.C. and M.S.B. conception and design of research; S.C., S.P., R.F-E., A.J., and A.H. performed experiments; S.C., S.P., R.F-E., A.J., A.H., A.N., and M.S.B. analyzed data; S.C., S.P., R.F-E., A.J., A.H., and M.S.B. interpreted results of experiments; S.C. and S.P. prepared figures; S.C., R.F-E., and M.S.B. drafted manuscript; S.C. and M.S.B. edited and revised manuscript; S.C., S.P., R.F-E., A.J., A.H., A.N., and M.S.B. approved final version of manuscript. REFERENCES 1. Aboubakr SE, Taylor A, Ford R, Siddiqi S, Badr MS. Long-term facilitation in obstructive sleep apnea patients during NREM sleep. J Appl Physiol (1985) 91: 2751–2757, 2001. 2. Babcock M, Shkoukani M, Aboubakr SE, Badr MS. Determinants of long-term facilitation in humans during NREM sleep. J Appl Physiol (1985) 94: 53–59, 2003. 3. Babcock MA, Badr MS. Long-term facilitation of ventilation in humans during NREM sleep. Sleep 21: 709 –716, 1998. 4. Bach KB, Mitchell GS. Hypoxia-induced long-term facilitation of respiratory activity is serotonin dependent. Respir Physiol 104: 251–260, 1996. 5. Beaumont M, Goldenberg F, Lejeune D, Marotte H, Harf A, Lofaso F. Effect of zolpidem on sleep and ventilatory patterns at simulated altitude of 4,000 meters. Am J Respir Crit Care Med 153: 1864 –1869, 1996. 6. Berry RB, Budhiraja R, Gottlieb DJ, Gozal D, Iber C, Kapur VK, Marcus CL, Mehra R, Parthasarathy S, Quan SF, Redline S, Strohl KP, Davidson Ward SL, Tangredi MM; American Academy of Sleep

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LTF may also have deleterious effects of central breathing stability. We have previously demonstrated that exposure to EH is associated with increased propensity to hypocapnic central apnea (12). The lack of LTF in older adults suggests that propensity to central apnea does not change in response to abnormal respiratory events. Additionally, we noted increased periodic breathing cycles in the aftermath of EH in the elderly that may be indicative of breathing instability. We did not include results from young subjects to avoid duplicating already published data. Instead, we refer the reader to Table 4, which presents a summary of a spectrum of ventilatory LTF data in young adults, with and without flow limitation from prior publications (2, 12, 38, 40), to allow comparison with current results obtained in older adults. The recovery VI in the elderly adults was only 94% control. Based on our findings, we speculate that the lack of LTF may contribute to increased periodic breathing or breathing instability during sleep in the elderly. Finally, we have noted increased HVR and suppression of VI following brief hyperoxia, indicating increased chemoresponsiveness during sleep in older adults. Studies in patients with heart failure have shown that increased chemoresponsiveness mediates the propensity of breathing instability and central sleep apnea in these individuals (46). Thus increased chemoresponsiveness may contribute to the development of breathing instability and central apnea in older adults. However, the underlying mechanism for increased chemoresponsiveness cannot be determined from our study.

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