To address the aims we studied volunteer healthy participants throughout two 11-day ‘inpatient’ protocols that combined circadian disruption with extended sleep opportunity (i.e., sufficient sleep) or short sleep (i.e., insufficient sleep). We sought healthy, non-obese, habitually active, male and female volunteers, aged 35-55 years, with no history of chronic medical disorders, no medications, and no substance abuse. This profile was designed to emulate the typical astronaut crew profile. Planned enrollment was 16 participants (8 male, 8 female). Based on flyers, plus newspaper, transportation and web based advertising, we received 2148 inquires. Overall 14 healthy, habitually active participants completed all of the screening and both of the in-laboratory phases of the study (8 male; 6 female).
Each participant was studied in both the Short Sleep and Long Sleep conditions (randomized). These two protocols were called ‘forced desynchrony’ protocols because the day length was shorter than 24 hours (h) such that the behavioral rest/activity cycle became desynchronized from the internal circadian cycle. This was achieved by scheduling participants to live on recurring 20 h ‘days’ in dim light (<3 lux), allowing the endogenous circadian pacemaker to oscillate at its inherent period rather than being reset by daily exposure to the light-dark cycle . One protocol (Short Sleep) permitted sleep for 5 h per 20 h ‘day’, which is equivalent to 6 h sleep opportunity per 24 h for an entire week. The other protocol (Long Sleep) allowed 8 h 20 min sleep per 20 h-‘day’, which is equivalent to 10 h sleep opportunity per 24 h for an entire week. This approach allows us to uniformly distribute the sleep/wake cycle across the circadian cycle to quantify the independent influences of the circadian system and behaviors and also their interacting effects. Under these carefully controlled conditions we tested the hypothesis that circadian disruption combined with sleep restriction would result in unfavorable changes in cardiovascular (CV) function during behavioral challenges commonly faced by astronauts. A behavioral test battery was performed at the same time relative to time since scheduled waking during each laboratory protocol (starting at 2 h after scheduled wake time). Before each test battery, participants had been supine for at least 5 h during the sleep periods in both the Long Sleep and Short Sleep conditions. Subsequently, when lights were switched from darkness to the dim light condition (3 lux), participants remained in a semi-recumbent posture (upper body at 45º) for 2 h during which time they were provided with a urinal or bedpan as needed. During this time participants consumed breakfast 1 h after scheduled wake time. An identical behavioral test battery was repeated on each 20 h ‘day’ and included three standardized stressors performed in the following order: (i) a mental challenge; (ii) a postural challenge; and (iii) an exercise stressor.
In brief, the following dependent variables were assessed at the beginning and at the end of each protocol (i.e., without and with sleep loss, and before and after circadian disruption):
• Maximal oxygen consumption (incremental treadmill exercise to maximal tolerable level
• Hemodynamic response to a strong postural challenge (passive 80° head-up tilt for 30 minutes)
• Cardiac structure and function (echocardiography)
• Endothelial function (reactive hyperemia index and endothelial-independent vasodilation)
• ECG arrhythmias (24 hour Holter recording)
For practical reasons or due to concerns that invasive or intensive tests would affect subsequent measurements (e.g., maximal exercise challenge can induce training effects), for the middle parts of the study (i.e., during the forced desynchrony) the following less invasive/ intensive identical test battery sessions were performed throughout each 20 h forced desynchrony:
• Core body temperature (CBT) for assessment of internal circadian phase
• Wrist actigraphy (and polysomnography is 4 participants) for estimation of sleep duration
• Hemodynamic, autonomic and endocrine responses to:
• Mental Stress (serial addition test for 10 minutes)
• Mild autonomic [postural) challenge (passive 60° head-up tilt for 15 minutes)
• Aerobic exercise challenge (bicycle exercise at 60% maximal heart rate for 15 minutes)
• Circadian Phase Assessment from core body temperature.
• Blood pressure and heart rate.
• Blood Sampling for autonomic, fibrinolytic and metabolic assays.
• Immune function: Astronauts experience various stressors that may result in inhibition of cell mediated immunity and increased reactivation of latent viruses [15,16]. Thus, comprehensive immune assessment was performed from whole blood samples collected with heparin at the beginning of study before either protocol, and twice during each of the short sleep and the long sleep protocols. Immune function was assessed by Dr. Brian E. Crucian and colleagues at NASA Johnson Space Center using standard techniques including peripheral leukocyte distribution by flow cytometry, T cell function, intracellular cytokine profiles, and secreted cytokine production profiles following T cell or monocyte stimulation . In addition, innate reactivation of latent EBV (Epstein Barr Virus), HSV1 (herpes simplex virus 1), and VZV (Varicella Zoster Virus) was assessed from the DNA in liquid saliva samples taken at the beginning of study before either protocol, and every alternate day across both the short and long sleep protocols. These assays were performed by Dr. Satish K. Mehta and colleagues at NASA Johnson Space Center using real time polymerase chain reaction techniques .
• Rest/Activity cycles and Sleep (Actigraphy and Polysomnography): Verification of the rest/activity cycles imposed by the protocols and estimation of sleep was made throughout the entire study (baseline and in-laboratory phases) by wrist actigraphy worn on the non-dominant arm. In a subset of 4 participants, sleep was assessed on each study sleep opportunity during the laboratory phases by using polysomnography. Sleep data were visually scored according to standard criteria .
Statistical Comparisons: The main comparisons were between CV outcome measurements collected at baseline (Wake Periods 3 or 4 from each protocol) and after circadian disruption [with or without sleep loss] (Wake Periods 10 or 11 from each protocol). Differences across the long sleep protocol are attributed to a week of circadian disruption. Differences across the short sleep protocol relative to differences across the long sleep protocol are attributable to sleep loss. This sleep loss effect is seen as a significant statistical interaction between Condition [long sleep vs. short sleep] and Wake Period [beginning vs. end of protocol].
At time of this final report, no results have been published in peer-reviewed scientific journals, although updates were presented as posters at the NASA Human Research Program (HRP) Investigators’ Workshops in 2013 and 2014, and the main results were presented in an oral presentation at the HRP Investigators’ Workshop in 2015. It is anticipated that publications will appear in 2016 or 2017.
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