Study design and participants

The study protocol was formally approved by the Institutional Review Board of Fuzhou Children’s Hospital (approval number 2014–001) and by the Chinese Clinical Trial Registry (approval number ChiCTR-IPR-14005458). The study was carried out in accordance with the Declaration of Helsinki principles. All participants provided written informed consent. Subjects were included in the study if they were older than 18 years of age, had body mass index (BMI) <26 kg/m 2 , were non-smokers, had scores ≤7 on the Pittsburgh sleep quality index (PSQI), had almost had no daytime sleep and slept only at night, that is, they went to bed between 21:00 and midnight and habitually spent in between 6 and 9 h per night in bed. Exclusion criteria included a history or current diagnosis of other sleep disorders (such as restless leg syndrome, periodic leg movements with arousals, narcolepsy, REM behavior disorder, circadian rhythm sleep disorder, breathing-related sleep disorder, or parasomnia), which was assessed by the clinical manifestation and a diagnostic PSG record (which was also performed to familiarize subjects with the PSG procedures); reduced hearing acuity (>20 dB hearing loss at a single frequency, as tested with an audiometer (Entomed SA 201); blindness (as tested with visual testing and perimetry), and a history of alcohol or medication abuse. Participants with an occupational history that included shift work or recent significant travel across three or more time zones within the prior two weeks were also excluded. In addition, after a screening PSG, participants with an apnea-hypopnea index >15 or a periodic leg-movement arousal index >15, and known allergy to melatonin, were also excluded.

All healthy participants (n = 40) slept in individual private rooms for eight nights (21:00 to 06:00) (Figure 1). The first night served as adaptation, that is, the participants followed the same procedure and data (not to be used in the analyses) were collected just as in the following nights. Then, the study was conducted in two stages. The first stage used a crossover design to investigate the impact of ICU noise and light environment on the sleep quality of healthy subjects. To minimize order effects, half of the healthy subjects (n = 20) were randomly exposed to a simulated ICU noise and light (NL) environment on the second night and to a quiet and dark environment (baseline) on the third night. In the meantime, other participants (n = 20) were exposed in the opposite order. In this stage, all subjects underwent two overnight PSG examinations on the second and third nights. For each subject, study nights were spaced 3 days apart to avoid delay effects. The second stage was to evaluate the effect of melatonin, and earplugs and eye masks, on the sleep quality of healthy subjects exposed to simulated nocturnal ICU noise and light. These 40 participants were assigned randomly to either: (1) simulated ICU noise and light (NL); (2) NL plus placebo (NLP); (3) NL plus melatonin (NLM); or (4) NL plus use of earplugs and eye masks (NLEE) in a 1:1:1:1 ratio.

Randomization was performed using a computer-generated schedule independent of treatment personnel. Subjects in the melatonin and placebo groups did not know they were receiving active therapy, nor did their clinicians. As potential chronophypnotic benefits of melatonin are not immediate and may take at least 3 days to be released, the process of intervention should take 4 days [28-30]. In order to control for possible effects of baseline values on the outcome variable, the baseline data for the simulated ICU environment (Baseline NL ) needed to be collected and analyzed before the 4-day intervention. Therefore, in this stage, all participants slept in the simulated ICU noise and light environment during the fourth night, and were exposed to corresponding intervening factors based on their group assignment for the following four consecutive nights (fifth to eight night) in the laboratory, and underwent two nighttime PSG evaluations on the fourth night for the baseline, and on the eighth night for assessment of the outcome variable, respectively.

Intervention and instruments

Baseline night (quiet and dark environment)

The laboratory is constructed so that sounds or vibrations from the surroundings are completely prevented and the background noise level with full ventilation is less than 15 dB (A). The dB(A) means that the sound level is measured by A weighting sound level meter. Mean nighttime light levels in the sleep laboratory measured 5 lux with the light off and the door to the hallway shut. Therefore, on the baseline night, all healthy subjects slept in a quiet environment with the light off.

Simulated ICU noise and light night (NL night)

Simulated ICU noise exposure

The exposure sounds in our study were recorded digitally during a typical weekday night shift (21:00 to 06:00) in the ICU at Fujian provincial hospital and stored on computer for playback in the sleep laboratory. ICU noise was continuously monitored during the night using a sound meter, model AW5610D (AWAI, Hangzhou, China) in the surgical ICU (SICU) environments. The SICU had noise levels far exceeding the 20 dB (A) at nighttime recommended by the Guidelines of the Chinese Association of Critical Care medicine (2006). The mean (standard deviation) noise value in the SICU was 67.1 ± 10.2 dB (A), the peak noise level recorded was 99.7 dB (A), and the minimal noise level recorded was 47.3 dB (A). The sound recording equipment, model ICD-P320 (Sony Inc., Tokyo, Japan), was positioned at the bed of patients receiving mechanical ventilation. Simultaneous sound meter readings were taken to ensure similar noise levels during playback in the sleep laboratory.

Simulated ICU lighting conditions

Nighttime illumination in the ICU setting and the sleep laboratory were monitored by a light detector model TES1332 (Taiwantes, Shenzhen, China). In both settings, the main light was provided by fluorescent ceiling lights. The light detector was placed by a patient receiving mechanical ventilation, but not so as to interfere with patient care. Light measurements were taken every hour during the night. In the ICU high mean night light levels ranging between 56.0 and 221.3 lux were maintained. The mean nighttime light level in the sleep laboratory measured 100 lux with the light on, and 5 lux with the light off and the door to the hallway shut. Therefore, the study used 100 lux to simulate the ICU lighting conditions.

During Baseline NL , NL, NLM, NLEE and NLP nights, recorded ICU noise was played and the fluorescent lights were turned on in the sleep laboratory. A sound meter was placed at the head of the subject’s bed and the recording time synchronized with the sound meter to ensure playback in a similar range of decibels to that recorded.

Earplugs and eye masks (NLEE night)

Subjects were instructed to wear earplugs with a 29-dB noise reduction rating (3 M Corporation, Beijing, China) and eye masks during the NLEE night. Subjects chose from three sizes of eye mask provided, which were 18 × 6 cm, 21 × 8 cm, and 24 × 10 cm, respectively. All participants chose the most suitable size according to their face size. We offered earplugs and eye masks to the subjects at night (from 21:00 to 06:00).

Melatonin (NLM)

Participants assigned to the melatonin group were given a 1-mg fast-release oral dose of melatonin (Armonia® Retard 1 mg; Nathura, Montecchio Emilia, Italy) administered at approximately 21:00. Dose changes were not permitted. Melatonin is not a licensed drug in China, and it is sold as a food supplement in a variety of preparations. The product used in this study contains a high-purity melatonin preparation (99.9%). This product has been regularly registered in the list of food supplements of the Italian Ministry of Health (cod. 08 29284 Y).

Placebo (NLP)

Participants in this group were treated according to a protocol identical to those receiving active medication. As with melatonin, the placebo was made in the identical formulation, and there were no differences in appearance, smell or flavor between the active and inactive pills.

Sleep measures and laboratory test

Polysomnography

Sleep was assessed by PSG using the Polysmith 2003 sleep acquisition and analysis system (Neurotronics, Gainesville, FL, USA). The standard procedure for sleep measurement described by Rechtschaffen and Kales was followed [32]. Subjects were hooked up for recording of an electroencephalogram (EEG), eye movement, and a submental electromyogram (EMG) in the sleep laboratory. Electrode impedances were within acceptable limits (<10 kQΩ). PSG equipment was located outside the subject’s room. Sleep variables (sleep period time, sleep efficiency index, sleep-onset latency, REM latency, arousal index and percentage of sleep in REM, stage one, two and three and so on) were scored manually and independently by two scorers who were unaware of the experimental conditions, according to standardized criteria. Polysomnographic records were collected from 21:00 to 06:00 on nights 1 to 4 and on night 8.

Serum melatonin concentration

Nocturnal blood was collected at 20:50 (before administration), 22:00, 23:00, 24:00, 01:00, 02:00, 03:00, 04:00, 05:00 and 06:00 h on nights 2, 3 and 8. In order to avoid repeated venipuncture, it is routine to give all subjects an indwelling vein needle. The trained technicians were required to access the participant’s room as quietly as possible and take blood by the light of an electric torch. Blood samples were collected in plastic tubes without anticoagulant agents and stored at −20°C until assayed. Melatonin concentrations were measured using a commercial radioimmunoassay (RIA) kit for human melatonin (BioSource Europe SA, Belgium). In this assay, sensitivity was 2 pg/mL. The intra-assay and inter-assay coefficients of variation (CV) were 5.6% and 8.2%, respectively.

Subjective measurements

Subjective sleep quality was assessed by a visual analog scale developed by the researchers based on previous scales [33]. Subjects evaluated their sleep quality on a scale of 0 to 10 (0 = excellent, 10 = poor) at 7:00 am on the morning after nights 2, 3, 4 and 8, with a higher score indicating poorer habitual sleep quality.

State anxiety level was assessed at 7:00 am on the morning after nights 2, 3, 4 and 8. In our study, the Spielberger state anxiety inventory (SAI) was chosen because it provides evaluation of state anxiety levels, namely a temporary unpleasant emotional arousal in the face of threatening demands or dangers. Subjects rated their feelings of anxiety on a 4-point scale ranging from a score of 1 (almost never anxious) to 4 (almost always anxious), a higher score indicating a higher anxiety level.

Subjects were asked to evaluate the comfort, effectiveness and ease of use of earplugs and eye masks on the morning after the NLEE night, using a 5-point scale ranging from a score of 1 (very uncomfortable, very unhelpful, very awkward) to 5 (very comfortable, very helpful, very easy to use), with low scores indicating a less pleasant experience.

Statistical analysis

Data were analyzed using SPSS version 19.0 (SPSS Inc., Chicago, IL, USA). Data for the adaptation night were excluded from analysis because the first night of sleep in a sleep laboratory room with unfamiliar surroundings differs from sleep on subsequent nights [18]. All data were expressed as mean ± SD. One-way analysis of variance (ANOVA) was used to determine differences in perceived sleep quality and anxiety levels during the four nights of the experiment. The ANOVA for repeated measures can be used to determine differences in sleep variables and melatonin concentrations during the four nights of the experiment. The paired Student t-test or non-parametric Wilcoxon rank sum test were performed to evaluate the effect of melatonin, and earplugs and eye masks on sleep variables and melatonin secretion during exposure to simulated ICU sound and light, where appropriate. The chi-square test was used to compare the gender ratio. P <0.05 was considered significant.