Benefits of napping and an extended duration of recovery sleep on alertness and immune cells after acute sleep restriction

Brain, Behavior, and Immunity 25 (2011) 16–24

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Benefits of napping and an extended duration of recovery sleep on alertness and immune cells after acute sleep restriction q Brice Faraut a,b, Karim Zouaoui Boudjeltia b, Michal Dyzma a,b, Alexandre Rousseau b, Elodie David a, Patricia Stenuit a, Thierry Franck c, Pierre Van Antwerpen d, Michel Vanhaeverbeek b, Myriam Kerkhofs a,b,* a

Sleep Laboratory, (ULB 222 Unit), CHU de Charleroi, A. Vésale Hospital, Université Libre de Bruxelles, Montigny-le-Tilleul, Belgium Laboratory of Experimental Medicine, (ULB 222 Unit), CHU de Charleroi, A. Vésale Hospital, Université Libre de Bruxelles, Montigny-le-Tilleul, Belgium Anesthésiologie et Pathologie Chirurgicale des Grands Animaux, Université de Liège, Liège, Belgium d Laboratory of Pharmaceutical Chemistry, Université Libre de Bruxelles, Brussels, Belgium b c

a r t i c l e

i n f o

Article history: Received 27 April 2010 Received in revised form 16 July 2010 Accepted 3 August 2010 Available online 8 August 2010 Keywords: Sleep restriction Extended sleep recovery Nap Immune cells Inflammation Alertness Myeloperoxidase

a b s t r a c t Understanding the interactions between sleep and the immune system may offer insight into why short sleep duration has been linked to negative health outcomes. We, therefore, investigated the effects of napping and extended recovery sleep after sleep restriction on the immune and inflammatory systems and sleepiness. After a baseline night, healthy young men slept for a 2-h night followed by either a standard 8-h recovery night (n = 12), a 30-min nap (at 1 p.m.) in addition to an 8-h recovery night (n = 10), or a 10-h extended recovery night (n = 9). A control group slept 3 consecutive 8-h nights (n = 9). Subjects underwent continuous electroencephalogram polysomnography and blood was sampled every day at 7 a.m. Leukocytes, inflammatory and atherogenesis biomarkers (high-sensitivity C-reactive protein, interleukin-8, myeloperoxidase, fibrinogen and apolipoproteins ApoB/ApoA), sleep patterns and sleepiness were investigated. All parameters remained unchanged in the control group. After sleep restriction, leukocyte and – among leukocyte subsets – neutrophil counts were increased, an effect that persisted after the 8-h recovery sleep, but, in subjects who had a nap or a 10-h recovery sleep, these values returned nearly to baseline. Inflammatory and atherogenesis biomarkers were unchanged except for higher myeloperoxidase levels after sleep restriction. The increased sleepiness after sleep restriction was reversed better in the nap and extended sleep recovery conditions. Saliva cortisol decreased immediately after the nap. Our results indicate that additional recovery sleep after sleep restriction provided by a midday nap prior to recovery sleep or a sleep extended night can improve alertness and return leukocyte counts to baseline values. Ó 2010 Elsevier Inc. All rights reserved.

1. Introduction Sleep deprivation is widely described as being associated with sleepiness and impaired neurobehavioral performance. Moreover, accumulating evidence from experimental studies links sleep loss to alterations in the immune and inflammatory systems that may have clinical relevance. It is not clearly established how these post-sleep-deprivation changes in immune and inflammatory functions – mediated via the neuroendocrine system – can affect health. However, epidemiological surveys implicate poor sleep as a predictor of cardiovascular risk, and meta-analyses have reported that shorter sleep duration, an emerging condition in the western population, is associated with a higher incidence of cardiovascular

q

Please see Brief Commentary by Tanja Lange and Born in this issue. * Corresponding author at: Sleep Laboratory, CHU A. Vésale, Université Libre de Bruxelles, Unit 222, Rue de Gozée 706, 6110 Montigny-le-Tilleul, Belgium. Fax: +32 71 921469. E-mail address: [email protected] (M. Kerkhofs). 0889-1591/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbi.2010.08.001

events (Heslop et al., 2002; King et al., 2008; Ferrie et al., 2007; National Sleep Foundation, 2009). Increased peripheral circulation of leukocytes (Dinges et al., 1994; Born et al., 1997; Kerkhofs et al., 2007) and elevated levels of C-reactive protein (CRP) and inflammatory cytokines (Meier-Ewert et al., 2004; Vgontzas et al., 2004; Irwin et al., 2006) have been reported in healthy humans after experimental sleep deprivation. CRP, leukocyte count, fibrinogen, and emerging biomarkers, such as myeloperoxidase (MPO), interleukin-8 (IL-8), and apolipoproteins (Apo), are all associated with the inflammatory and atherogenesis pathways that lead to cardiovascular disease (Danesh et al., 1998; Inoue et al., 2008; Ruggiero et al., 2007; Zhang et al., 2008; Sniderman et al., 2003). Interestingly, several sleep deprivation studies have indicated that one 8-h night of recovery sleep is not sufficient to normalize alertness and performance or cardiovascular risk markers, such as leukocytes and CRP (Lamond et al., 2007; Belenky et al., 2003; Dinges et al., 1994; Meier-Ewert et al., 2004). To our knowledge, the effects of a short nap in addition to recovery sleep or an extended duration of recovery sleep as

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countermeasures to acute sleep restriction have never been investigated in an integrated design on sleepiness and immune and cardiovascular risk markers. We hypothesized that following a sleep-restricted night of 2 h, a 30-min midday nap prior to a normal 8-h recovery sleep or an extended 10-h recovery sleep would improve restoration of these parameters. We, therefore, examined peripheral blood immune cells, inflammatory and atherogenesis biomarkers (CRP, IL-8, MPO, ApoB/ApoA, and fibrinogen) together with sleep patterns, sleepiness and saliva cortisol in young healthy adults. 2. Methods 2.1. Subjects All volunteers were non-smokers, not regular nappers, had a body mass index between 19 and 25, and were aged between 18 and 27 years. They were free of any disease and/or sleep complaints and slept regular nights of 7–9 h as indicated by a rigorous medical evaluation that included psychiatric and medical histories, screening laboratory tests, sleep questionnaires and one adaptation night of polysomnography monitoring. 2.2. Experimental design and procedure The protocol was approved by the Ethic’s Committee of the hospital. All volunteers gave their written informed consent and received financial compensation. During the week preceding their admission to the Sleep Laboratory, the participants followed 1 week of regular sleep-wake behavior of 8 h in bed (11:00 p.m.– 7:00 a.m.) documented by actigraphic recordings and sleep diaries. Before admission, ongoing infection was excluded based on the CRP concentration and the leukocyte count. The protocol included a control group and three groups with distinct recovery conditions. In the control group, nine healthy young men underwent three consecutive nights of 8 h sleep (in bed from 11:00 p.m. to 7:00 a.m.). In the first recovery condition group, 12 healthy young men underwent one baseline night of 8 h (in bed from 11:00 p.m. to 7:00 a.m.), one restricted night of 2 h (in bed from 2:00 a.m. to 4:00 a.m.), and one recovery night of 8 h (in bed from 11:00 p.m. to 7:00 a.m.). In the second recovery condition group, 10 healthy young men underwent one baseline night of 8 h (in bed from 11:00 p.m. to 7:00 a.m.), one restricted night of 2 h (in bed from 2:00 a.m. to 4:00 a.m.), a nap from 1:00 p.m. to 1:30 p.m. after the restricted night, and one recovery night of 8 h (in bed from 11:00 p.m. to 7:00 a.m.). In the third recovery condition group, 9 healthy young men underwent one baseline night of 8 h (in bed from 11:00 p.m. to 7:00 a.m.), one restricted night of 2 h (in bed from 2:00 a.m. to 4:00 a.m.), and one extended recovery night of 10 h (in bed from 9:00 p.m. to 7:00 a.m.). To control the state of alertness of the subjects and to avoid sleep episodes outside the permitted hours, continuous electroencephalogram (EEG), electrooculogram and electromyogram recordings were taken using an ambulatory device (DreamÒ, Medatec, Brussels, Belgium) with the following EEG derivations (C4/A1, C3/ A2, O2/A1, O1/A2 F4/A1, F3/A2), which were also used for sleep recordings. During the study, volunteers were free to move within the unit carrying this ambulatory device. Subjects received standard hospital meals of a maximum of 2500 cal/day with a balanced proportion of nutrients (protein, fat, carbohydrates). Intake of any medication, alcohol, or xanthine derivatives (coffee, tea, chocolate and cola) was prohibited throughout the study period. Controlled drinks and snacks were available during the sleep-restricted nights until 11 p.m. Sleep recordings were scored visually in all subjects according to the 2007 American Academy of Sleep Medicine criteria (Silber et al., 2007). Fasting blood samples were obtained from

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an antecubital vein at 7:00 a.m. each day of the experiment. To assess the effects of the nap on cortisol, saliva samples were taken for cortisol measurement every 2 h from 9:30 a.m. to 5:30 p.m. on the three study days in the 8 h-sleep recovery and 30-min nap + 8 hsleep recovery groups. 2.3. Sleepiness evaluation Objective and subjective sleepiness were assessed at 1:00 and 3:00 p.m. on the three study days using the Maintenance of Wakefulness Test (MWT; Mitler et al., 1982) and the Stanford Sleepiness Scale (Hoddes et al., 1985), respectively, as previously described (Stenuit and Kerkhofs, 2005). Briefly, the MWT assessed the ability to sustain wakefulness and was performed during 20 min. During the test, the subjects were comfortably installed in a chair in a dimly light room and instructed to remain awake while polysomnographic recordings were performed. The test was interrupted at the onset of sleep (stage 1) and the subject was immediately awakened. In the absence of sleep, the test ended after 20 min. 2.4. Assays and measurements Fasting blood samples were obtained from an antecubital vein by a needle stick at 7:00 a.m. each morning. Serum samples were collected in vacuum tubes without anticoagulant (VenojectÒ). Plasma samples were harvested in citrated vacuum tubes (Buffer Sodium Citrate, 0.109 mol/L:3.2 W/V%, VenojectÒ), immediately processed and put on melting ice. Whole blood was collected in EDTA-treated tubes (K3EDTA, VenosafeÒ). High sensitivity CRP (Hs-CRP), Apolipoproteins A and B were evaluated by antibodybinding and turbidity measurement on SYNCHRON LXÒ. Leukocyte counts and subsets were determined on a CELL-DYN4000Ò hemocytometer (Abbott, Abbott Park, IL). Fibrinogen was determined by thrombin time on a STAÒ automate (Stago, Parsippany, NJ). Plasma MPO was determined by ELISA (ZentechÒ, Angleur, Belgium). MPO activity was determined by the SIEFED method as previously described (Franck et al., 2009). Serum IL-8 concentrations were quantified using an ELISA test (Becton DickinsonÒ, Franklin Lakes, NJ). Saliva samples for cortisol analysis were stored at 20 °C until assayed. After thawing, samples were mixed well prior to assay and cortisol concentrations measured by radioimmunoassay (Stockgrand Ltd., Guildford, UK). 2.5. Statistics Data were analyzed using the SigmaStatÒ 3.5 software (SystatÒ, San Jose, CA). Values are expressed as mean values (SEM). We performed two types of comparisons with the objectives of testing the effects of sleep restriction and of the distinct recovery modalities. The first comparisons were between experimental points within each experimental group using a within-subjects one-way repeated measure ANOVA completed by a pairwise comparisons post hoc test (Student–Newman–Keuls’ test). The second comparisons involved the 3 sleep-restricted groups with distinct recovery conditions. We first tested whether the effects of sleep restriction were similar in each sleep-restricted group by comparing the normalized differences observed between sleep restriction and baseline values for each sleep-restricted group (normalized d = (Restriction–Baseline)/Baseline). There were no significant differences in the normalized d scores, indicating that the physiological effects of sleep restriction were similar for the 3 sleep-restricted groups. To increase the sample size, we therefore pooled the results for all subjects from the 3 sleep-restricted groups together and compared sleep restriction to baseline values. Comparisons between the different sleep recovery conditions were made using normalized d scores for each recovery condition, i.e., (recovery 8 h –

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restriction)/restriction; (nap + recovery 8 h – restriction)/restriction); and (recovery 10 h – restriction)/restriction). For sleep analyses, comparisons among groups were performed for each sleep stage for baseline, sleep restriction, and recovery nights. Additional analyses to compare cortisol values between no-nap and nap conditions were performed for each time point. All these comparisons were performed using a one way ANOVA completed by a pairwise comparisons post hoc test (Student–Newman–Keuls’ test). A probability level of p < 0.05 was considered as statistically significant. 3. Results 3.1. Within-group comparisons 3.1.1. Peripheral blood leukocytes There were no significant changes in leukocyte counts in the control group during the three consecutive baseline nights (F (2, 8) = 0.07, p = 0.93; Fig. 1A). Changes in leukocyte count were significant over time in the 8-h recovery group (F (2, 11) = 6.03, p = 0.009), the nap + 8-h recovery group (F (2, 9) = 21.09, p < 0.001), and the 10-h recovery group (F (2, 8) = 7.85, p = 0.005). Pairwise comparisons showed that after the sleep-restricted night, leukocyte counts increased significantly compared to baseline in the three study groups. After recovery sleep, this increase in leukocyte count persisted in the 8 h-recovery condition but not in the nap + 8 h-recovery or the 10 h-recovery conditions. Among the leukocyte subtypes, changes in neutrophil count were significant over the three study days in the 8-h recovery group (F (2, 11) = 6.27, p = 0.008), the nap + 8-h recovery group (F (2, 9) = 16.26, p < 0.001), and the 10-h recovery group (F (2, 8) = 15.25, p < 0.001) (Fig. 1B). Pairwise comparisons indicated that after sleep restriction, neutrophil counts increased significantly in each of the three study groups compared to baseline. After the recovery night, the neutrophil increase persisted in the 8 h-recovery group but there was a significant reduction in neutrophil count in the nap + 8-h recovery and 10-h recovery groups. Sleep restriction did not significantly affect lymphocyte levels in any of the groups. In the 8-h recovery group (F (2, 11) = 4.27, p = 0.027) and the nap + 8-h recovery group (F (2, 9) = 6.56, p = 0.007), pairwise comparisons showed that the lymphocyte level was significantly reduced after 8 h of recovery sleep and nap + 8 h of recovery sleep but not after extended recovery sleep (Fig. 1C). Monocyte counts were not significantly altered in any of the groups (Fig. 1D). 3.1.2. Peripheral blood inflammatory and atherogenesis biomarkers MPO levels did not change significantly over the three days in the control group. The MPO level tended to increase after the sleep-restricted night and the increase was significant compared to baseline in the 8-h recovery group (F (2, 9) = 4.60, p = 0.02; Fig. 2A). The concentrations of Hs-CRP, IL-8, fibrinogen and the ApoB/ApoA ratio did not change significantly in any of the groups (Fig. 2B–E). 3.1.3. Subjective and objective sleepiness Stanford Sleepiness Scale scores did not change significantly in the control group with no onsets of sleep detected with the MWT. As expected, Stanford Sleepiness Scale scores indicated that after sleep restriction volunteers felt sleepier at 1 p.m. in the 8-h recovery group (F (2, 11) = 13.90, p < 0.001), the nap + 8-h recovery group (F (2, 9) = 5.28, p = 0.016) and the 10-h recovery group (F (2, 8) = 9.64, p = 0.002 (Fig. 3A). Pairwise comparisons indicated significantly higher sleepiness values in the three study groups compared to respective baseline. At 3 p.m., Stanford Sleepiness Scale scores indicated that volunteers also felt sleepier after sleep restriction in the 8-h recovery group (F (2, 11) = 26.90, p < 0.001)

Fig. 1. Mean (SEM) counts of peripheral blood leukocytes (A), neutrophils (B), lymphocytes (C) and monocytes (D) at baseline (Bas), after sleep restriction (Res) and after 8-h recovery (Rec 8 h), 30 min-nap + 8-h recovery (Nap + Rec 8 h), or 10-h recovery (Rec 10 h).  indicates p < 0.05 between conditions.

and the 10-h recovery group (F (2, 8) = 11.64, p < 0.001) but not in the nap + 8-h recovery group (Fig. 3C). Interestingly, while sleep-

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iness returned to baseline values in the groups with extended recovery sleep or with a nap before recovery sleep, the group with just 8 h of recovery sleep had sleepiness scores that were still significantly higher than baseline. 3.1.4. Sleep architecture Pairwise comparisons with the corresponding baseline values indicated that the rebound in slow-wave sleep (SWS) observed during the 8-h recovery night (F (1, 11) = 9.16, p = 0.01) was no longer significant in the group who took a nap (Table 1). The 30min midday nap was mainly composed of SWS. Significant increases in stage 2 (F (1, 8) = 23.56, p = 0.005), slow-wave (F (1, 8) = 55.42, p < 0.001) and rapid eye movement (REM) (F (1, 11) = 14.93, p = 0.01) sleep were noted during the extended recovery night. There were no significant variations in the distribution of sleep stages during the three nights in the control group. Analysis of the continuous EEG recordings showed only two short sleep episodes (7 and 8 min of stage 2 sleep, respectively) outside the permitted hours, demonstrating the success of the sleep restriction procedure. 3.2. Between-group comparisons 3.2.1. Peripheral blood leukocytes The increases in leukocyte count after sleep restriction were comparable in the 3 sleep-restricted groups (F (2, 28) = 0.12, p = 0.886), so we pooled the values for the three groups to increase the sample size and confirmed a significant increase in leukocytes after sleep restriction (F (1, 60) = 11.35, p = 0.001). The effects of sleep recovery on leukocyte counts were different in the three recovery conditions (F (2, 28) = 4.53, p = 0.021). Pairwise between-group comparisons showed a significant difference between the ‘‘8-h recovery group” and both the ‘‘nap + 8-h recovery group” and the ‘‘10-h recovery group” indicating a higher reduction in leukocyte count in these two groups (Fig. 4A). The neutrophil increases after sleep restriction were comparable in the 3 sleeprestricted groups (F (2, 28) = 0.05, p = 0.947), so we pooled the values and noted a significantly higher neutrophil count after sleep restriction compared to controls (F (1, 60) = 11.35, p = 0.001). The effects of sleep recovery on neutrophil counts varied according to the recovery condition (F (2, 28) = 4.20, p = 0.028). Pairwise between-group comparisons showed a significant difference between the ‘‘8-h recovery group” and both the ‘‘nap + 8-h recovery group” and the ‘‘10-h recovery group” indicating a greater reduction in neutrophil counts in the subjects who had a nap and in those who had extended recovery sleep (Fig. 4B). There were no significant differences in the recovery of lymphocyte (F (2, 28) = 0.31, p = 0.733); Fig. 4C) or monocyte (F (2, 28) = 0.75, p = 0.48; Fig. 4D) counts. 3.2.2. Peripheral blood inflammatory and atherogenesis biomarkers The increases in MPO level after sleep restriction were similar in the 3 sleep-restricted groups (F (2, 28) = 0.73, p = 0.491), so we pooled the values and noted a significantly higher level of MPO after sleep restriction (F (1, 60) = 5.63, p = 0.02). Between-group comparisons did not show significant differences in MPO levels after the recovery night (F (2, 28) = 0.01, p = 0.98; Fig. 5A). The effects of sleep restriction on Hs-CRP, IL-8, fibrinogen and ApoB/ ApoA ratio were not significant when the values for all subjects were pooled and between-group comparisons did not show significant differences after the recovery night for the three recovery conditions (Fig. 5B–E). Fig. 2. Myeloperoxidase (A), C-reactive protein (B), interleukin-8 (C), fibrinogen (D) and ApoB/ApoA ratio (E) at baseline (Bas), after sleep restriction (Res) and after 8-h recovery (Rec 8 h), 30 min-nap + 8-h recovery (Nap + Rec 8 h), or 10-h recovery (Rec 10 h). Values are expressed as mean (SEM).  indicates p < 0.05 between conditions.

3.2.3. Subjective and objective sleepiness The increases in sleepiness at 1 p.m. after sleep restriction were comparable in the 3 sleep-restricted groups (F (2, 28) = 1.05,

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p = 0.362) so we pooled the values for the three groups and confirmed a significantly higher level of sleepiness after sleep restriction (F (1, 60) = 40.83, p < 0.001). Between-group comparisons did not show significant differences in sleepiness at 1 p.m. after the recovery night (F (2, 28) = 0.40, p = 0.67, Fig. 6A). The increases in sleepiness at 3 p.m. after sleep restriction were similar in the 8-h recovery group and the 10-h recovery group but different in subjects who had had a nap (F (2, 28) = 5.01, p = 0.01). When values for the subjects in the 8-h recovery group and the 10-h recovery group were pooled, sleepiness was higher than baseline values (F (1, 39) = 40.94, p < 0.001). Between-group comparisons showed significant differences in sleepiness at 3 p.m., which was greater with 8 h of recovery sleep than with 10 h of recovery sleep (F (1, 19) = 7.41, p = 0.014, Fig. 6B). These subjective evaluations were confirmed by the objective findings with the MWT. Indeed, only one sleep onset (sleep latency 18 min) was recorded at 3 p.m. in the subjects who had a nap while in subjects who had no nap, 4 sleep onsets (sleep latency 13.57 ± 2.07 min) were recorded in the 8-h recovery group and 6 sleep onsets (sleep latency 9.25 ± 3.00 min) in the 10-h recovery group (Fig. 4D). During the recovery day, 2 sleep onsets (one at 1 p.m., sleep latency 19 min and one at 3 p.m., sleep latency 9 min.) were observed in the 8-h recovery group while 1 sleep onset (sleep latency 12.5 min) was recorded in the extended recovery group, similar to baseline, and none in the nap + 8-h recovery group (Fig. 6B and D). 3.2.4. Sleep architecture The durations of each sleep stage during baseline and sleep-restricted nights were not significantly different between groups. SWS (F (2, 28) = 1.88, p = 0.17), and the duration of stage 1 sleep (F (2, 28) = 2.94, p = 0.07) during the recovery night was not significantly different in the 3 recovery conditions. Pairwise betweengroup comparisons indicated a higher amount of Stage 2 sleep during the 10-h recovery night compared to the 8-h recovery night (F (2, 28) = 4.85, p = 0.01) and a greater amount of REM sleep compared to the 8-h recovery night and the nap + 8-h recovery night (F (2, 28) = 4.92, p = 0.01). 3.2.5. Saliva cortisol To assess the effects of the nap on cortisol levels, we quantified cortisol values in saliva as shown in Fig. 7. There was no significant effect of sleep restriction or recovery on cortisol levels. However, there was a significant decrease in saliva cortisol at 1.30 p.m. in subjects who had a nap compared to the same time point in subjects who did not (F (1, 20) = 18.93, p < 0.001).

4. Discussion

Fig. 3. Stanford Sleepiness Scale scores and number of sleep onsets as assessed by the Maintenance of Wakefulness Test at baseline (Bas), after sleep restriction (Res) and after 8-h recovery (Rec 8 h), 30 min-nap + 8-h recovery (Nap + Rec 8 h), or 10-h recovery (Rec 10 h). Values are shown at 1 p.m. (A and B) and 3 p.m. (C and D) and are expressed as mean (SEM).  indicates a p < 0.05 between conditions.

Our results provide confirmation of the interactions between sleep and the immune system. An increase in peripheral blood leukocytes was observed after a night restricted to 2 h of sleep, an increase which persisted after the recovery night. Interestingly, both a short midday nap prior to the recovery night and an extended night of recovery sleep normalized this increase. Analysis of the leukocyte subsets indicated that neutrophils were the most sensitive subtype to our sleep restriction and recovery protocols. There were no significant correlations between the changes in neutrophil count after recovery sleep and the time spent in a particular stage of sleep during the recovery night. Polysomnography analysis indicated that the 30-min nap was sufficient to reduce the rebound in SWS observed after sleep restriction, suggesting that even a short episode of SWS during a nap can reduce the homeostatic pressure of sleep.

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Table 1 Sleep stages of baseline, sleep restriction, and 8-h recovery, 30 min-nap + 8-h recovery, or 10-h recovery nights. Values are expressed as mean (SEM).  indicates p < 0.05 versus within-group corresponding baseline nights. a and b indicate p < 0.05 versus 8-h recovery sleep and nap + 8-h recovery sleep conditions, respectively.

Baseline 1 Baseline 2 Baseline 3 Baseline Restriction Recovery 8 h Baseline Restriction Nap Recovery 8 h Baseline Restriction Recovery 10 h

Stage 1 (min)

Stage 2 (min)

SWS (min)

REM (min)

9.12 ± 4.65 9.0 ± 2.0 14.0 ± 9.33 9.55 ± 0.94 1.09 ± 0.39 5.5* ± 1.38 14.14 ± 10.33 4.00 ± 1.73 4.22 ± 0.79 10.33 ± 1.52 10.33 ± 1.76 3.62 ± 0.82 7.37 ± 1.14

205.4 ± 17.92 199.25 ± 26.62 196.14 ± 20.04 216.77 ± 12.61 30.27 ± 4.86 190.60 ± 12.27 213.14 ± 10.59 33.66 ± 5.45 10.55 ± 1.44 211.33 ± 11.68 199.50 ± 7.96 30.62 ± 4.7 241.0*a ± 7.49

107.4 ± 21.68 100.5 ± 18.0 107.83 ± 18.55 97.00 ± 10.95 62.54 ± 5.42 130.10* ± 10.81 91.62 ± 8.59 61.11 ± 3.40 14.77 ± 1.73 113.00 ± 11.68 94.16 ± 8.34 65.25 ± 4.14 144.0* ± 9.10

87.62 ± 13.62 90.57 ± 20.04 106.88 ± 15.45 92.88 ± 8.66 16.45 ± 3.67 116.55 ± 8.16 104.71 ± 11.02 12.00 ± 3.18 3.1 ± 2.50 113.83 ± 8.38 87 ± 6.07 10.75 ± 2.34 151.5*ab ± 11.1

As expected, there was an increase in objective and subjective sleepiness after sleep restriction. These increases were reversed during the post-nap period and, interestingly, returned to baseline during the recovery day only in the nap and extended sleep conditions. The same trend was observed at 9 a.m. and 5 p.m. of the recovery day with better alertness in subjects who took a nap than in those who just had 8 h of recovery sleep (data not shown). A short nap, especially during the post-noon nap zone, has been shown to restore alertness and promote performance without the inconvenience of sleep inertia that is associated with longer naps (Takahashi and Arito, 2000; Mednick et al., 2003; Brooks and Lack, 2006; Milner and Cote, 2009). Similar to our findings, less overall fatigue and reduced subjective sleepiness were observed when resident interns sleeping 2–3 h during extended work shifts were assigned to a short nap schedule (Arora et al., 2006). Lamond et al. (2007) also reported that subjective sleepiness and psychomotor vigilance performance recovered better with an extended 9-h compared to a 6-h recovery sleep following sleep deprivation. However, although the effects of nap and extended sleep recovery on neurobehavioral performance and alertness have previously been examined after sleep deprivation, these previous studies did not simultaneously evaluate the effects on immune parameters. Our multi-parameter integrated study has some limitations but also certain strengths. For the nap group, we cannot determine at which time the effect took place after the nap, i.e., before or after the recovery night. We chose not to perform multiple blood samplings to avoid local inflammation from the blood drawing procedure as has previously been reported (Haack et al., 2002). The effectiveness of our sleep restriction procedure was confirmed by the continuous EEG recordings. All subjects had leukocytosis and neutrophilia after sleep restriction. After the recovery night, a decrease in leukocyte and neutrophil counts was observed for all participants who had taken a nap and all subjects who had the extended recovery sleep, but in the group with a standard recovery night, the effect was heterogeneous. Total sleep deprivation has been shown to affect hematocrit (Goldman et al., 1990) which could also affect leukocyte subset counts. However, we found no significant hematocrit changes after partial sleep deprivation (data not shown). Several studies have indicated that sleep deprivation can lead to an inflammatory response that may trigger the development of cardiovascular disease processes (Meier-Ewert et al., 2004; Vgontzas et al., 2004; Irwin et al., 2006, 2010; Frey et al., 2007; van Leeuwen et al., 2009; Mullington et al., 2009). We did not note any significant changes in the stable inflammatory marker CRP, the pro-inflammatory IL-8, or fibrinogen after sleep restriction. Because CRP has been shown to induce IL-8 gene expression in human peripheral blood monocytes, the lack of change in IL-8 levels could in part be the result of the unaltered CRP level (Xie

et al., 2005). Elevated concentrations of CRP and of pro-inflammatory cytokines, such as IL-6 or tumor necrosis factor (TNF)-a, have been reported following chronic partial or total sleep deprivation. (Meier-Ewert et al., 2004; Vgontzas et al., 2004; van Leeuwen et al., 2009). However, decreased or unchanged concentrations of CRP and reduced levels of IL-6 have also been reported after one night of total sleep loss (Frey et al., 2007; Dimitrov et al., 2006). Decreases in habitual sleep duration were associated with reduced CRP and IL-6 concentrations but elevated TNF-a levels in a large sample of 614 subjects (Patel et al., 2009). In a cohort of 210 healthy men and women, poor sleep quality was associated with higher fibrinogen and CRP concentrations but only for women, and a laboratory study reported that increases in IL-6 and TNF-a were still present in the evening following partial sleep deprivation in women but not in men (Suarez, 2008; Irwin et al., 2010). The increased leukocyte and neutrophil counts observed in our study are consistent with previous studies showing similar effects in healthy young adults after sleep restriction or deprivation (Zouaoui-Boudjeltia et al., 2008; Dinges et al., 1994). These increases have not yet been linked with any clinically significant effect on cardiovascular pathology. However, leukocyte count, which is mainly determined by neutrophil count in healthy humans, is also important in the absence of acute medical events. Increased leukocyte counts have long been associated with increased allcause mortality and are considered a biomarker of inflammatory processes that contribute to vascular injury and atherosclerosis (Loimaala et al., 2006). Increased leukocyte and neutrophil counts have been shown to be an independent risk factor for cardiovascular mortality in numerous studies and meta-analyses (Brown et al., 2004, Wheeler et al., 2004, Horne et al., 2005; Danesh et al., 1998). In a prospective study that was conducted over 44 years, higher leukocyte counts, even within the normal range (mainly neutrophils), were associated with higher mortality (Ruggiero et al., 2007). Interestingly, this study found that leukocyte counts (mostly neutrophils) increased progressively, starting several years before death, in participants who died during follow-up, although leukocyte counts remained stable over time in those who survived. Hence, we suggest that repetitive acute sleep restriction, as commonly observed in extended workshifts or in individuals with sleep disorders, could result in progressively elevated leukocyte and neutrophil levels over time, with potentially negative longterm effects on mortality. Neutrophils can provoke oxidative and proteolytic damage to coronary arteries and may influence the development of cardiovascular diseases. We noted an increase in MPO – mainly released by neutrophils – after sleep restriction, which has potential negative consequences because of its role in catalyzing the formation of oxidizing agents that can convert LDL into an atherogenic form (Podrez et al., 1999; Zouaoui Boudjeltia et al., 2004). The same profile was also observed for MPO activity

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Fig. 4. Changes (normalized d) in leukocyte (A), neutrophil (B), lymphocyte (C) and monocyte (D) counts after 8-h recovery sleep, 30 min-nap + 8-h recovery sleep, or 10-h recovery sleep. Values are expressed as mean (SEM).  indicates p < 0.05 between conditions.

Fig. 5. Changes (normalized d) in myeloperoxidase (A), C-reactive protein (B), interleukin-8 (C), fibrinogen (D) and ApoB/ApoA ratio (E) after 8-h recovery sleep, 30 min-nap + 8-h recovery sleep, or 10-h recovery sleep. Values are expressed as mean (SEM).  indicates p < 0.05 between conditions.

B. Faraut et al. / Brain, Behavior, and Immunity 25 (2011) 16–24

Fig. 6. Changes (normalized d) in Standford sleepiness scores after 8-h recovery sleep, 30 min-nap + 8-h recovery sleep, or 10-h recovery sleep at 1 p.m. (A) and after 8-h recovery sleep and 10-h recovery sleep at 3 p.m. (B). Values are expressed as mean (SEM).  indicates p < 0.05 between conditions.

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of artery calcification, a marker of the atheromatous plaque, the main process involved in the development of cardiovascular diseases. Results reported that shorter measured sleep length was associated with a higher incidence of coronary artery calcification in a healthy middle-aged population of 495 participants (King et al., 2008). Our data suggest that leukocyte recovery can be improved by having a nap prior to recovery sleep; this has a stressreleasing effect as shown by the decrease in cortisol levels immediately after the nap and could explain why midday napping in healthy individuals has been reported to be inversely associated with coronary mortality, particularly among working men, after controlling for potential confounders, e.g., physical activity, diet, co morbidity (Naska et al., 2007). Similarly, longer recovery sleep may also have a stress-reducing effect. This would suggest that the increase in leukocyte count is secondary to autonomic activation and not a direct effect of sleep restriction on the immune system. However, it is difficult to determine whether the immune alterations are associated with activation of a non-specific immune response or a stress response that occurs post-sleep deprivation (Meerlo et al., 2008). To our knowledge, other sleep restriction studies have not reported cortisol variations during the period we assessed, i.e., the end of the morning and afternoon. However, several studies have reported increased cortisol levels during the evening and the early morning after total or partial sleep deprivation (Leproult et al., 1997; Vgontzas et al., 2004). With regard to the effect of napping on cortisol release, one could hypothesize that SWS within the nap could inhibit the hypothalamic-pituaryadrenal axis and cortisol release as previously described as well as the elevated release of catecholamines by the sympathoadrenal system observed following partial sleep deprivation (Späth-Schwalbe et al., 1993; Vgontzas et al., 2007; Irwin et al., 1999). Indeed, catecholamines could contribute to vascular neutrophil mobilization because neutrophils and leukocyte subsets with cytotoxic effector functions can be swept into the circulation after epinephrine administration (Brohee et al., 1990; Davis et al., 1991; Dimitrov et al., 2010). Further experiments are needed to better understand the mechanism(s) involved in the effect of additional sleep on immune cells and its relation to the neuroendocrine system. In summary, the current study reports for the first time that when young healthy subjects are sleep deprived, modulating sleep recovery by having a short nap during the day before the recovery night or by extending the duration of the recovery night can improve alertness and aid the return of normal cellular immune function. However, larger samples of subjects need to be investigated to better understand whether leukocytosis induced by sleep restriction – and its reversal by sleep extension – are involved in cardiovascular disease pathogenesis. Acknowledgments We would like to thank Geneviève Turci (CHU Charleroi), the study nurse, Benita Middleton (University of Surrey) for cortisol assays, Karen Pickett for English-language editing and also Alain Bosseloir (Zentech), Didier Serteijn and Ginette Deby-Dupont (Université de Liège). Sources of Funding: European Union Grant MCRTN-CT-2004-512362 and Scientific Research Fund of the ISPPC-CHU de Charleroi and Institut de Recherche en Pathologie et en Génétique (IRSPG), Gosselies.

Fig. 7. Saliva cortisol concentrations at baseline, after sleep restriction, and after 8h recovery sleep in the no-nap and nap conditions. The solid black line on the abscissa indicates the nap period. Values are expressed as mean (SEM).  indicates p < 0.05 between no-nap and nap conditions.

(data not shown). Accordingly, measurement of objective sleep duration by actimetry has been investigated in relation to the level

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