Physiology & Behavior 155 (2016) 66–76
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Effects of sleep restriction during pregnancy on the mother and fetuses in rats Grace Violeta Espinoza Pardo, Jéferson Ferraz Goularte, Ana Lúcia Hoefel, Alexandre Luz de Castro, Luiz Carlos Kucharski, Alex Sander da Rosa Araujo, Aldo Bolten Lucion ⁎ Departamento de Fisiologia, Instituto de Ciências Básicas da Saúde (ICBS), Universidade Federal do Rio Grande do Sul (UFRGS), Rua Sarmento Leite 500, Porto Alegre, RS, 90050-170, Brazil
H I G H L I G H T S • Sleep restriction in pregnancy increases plasma prolactin and oxytocin • Sleep restriction in pregnancy reduces body weight gain • Sleep restriction in pregnancy increases BDNF in the hippocampus of the fetuses
a r t i c l e
i n f o
Article history: Received 4 September 2015 Received in revised form 29 November 2015 Accepted 30 November 2015 Available online 2 December 2015 Keywords: Maternal behavior BDNF ROS Prolactin Oxytocin Corticosterone
a b s t r a c t The present study aimed to analyze the effects of sleep restriction (SR) during pregnancy in rats. The following three groups were studied: home cage (HC pregnant females remained in their home cage), Sham (females were placed in tanks similar to the SR group but with sawdust) and SR (females were submitted to the multiple platform method for 20 h per day from gestational days (GD) 14 to 20). Plasma corticosterone after 6 days of SR was not different among the groups. However, the relative adrenal weight was higher in the SR group compared with the HC group, which suggests possible stress impact. SR during pregnancy reduces the body weight of the female but no changes in liver glycogen, cholesterol and triglycerides, and muscle glycogen were detected. On GD 20, the fetuses of the females submitted to SR exhibited increased brain derived neurotrophic factor (BDNF) in the hippocampus, which indicates that sleep restriction of mothers during the ﬁnal week of gestation may affect neuronal growth factors in a fetal brain structure, in which active neurogenesis occurs during the deprivation period. However, no changes in the total reactive oxygen species (ROS) in the cortex, hippocampus, or cerebellum of the fetuses were detected. SR females showed no major change in the maternal behavior, and the pups' preference for the mother's odor on postpartum day (PPD) 7 was not altered. On GD 20, the SR females exhibited increased plasma prolactin (PRL) and oxytocin (OT) compared with the HC and Sham groups. The negative outcomes of sleep restriction during delivery could be related, in part, to this hormonal imbalance. Sleep restriction during pregnancy induces different changes compared with the changes described in males and affects both the mother and offspring. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Disturbances in sleep, such as a reduction in the duration, poor sleep quality and insomnia, have increased in the general population and have become a health concern [1,2]. In pregnant women, fragmentation and/or sleep disruption increase [3–6]. The impact of pregnancy on sleep is more pronounced during the third trimester, which leads to
⁎ Corresponding author. E-mail addresses: [email protected]
(G.V.E. Pardo), [email protected]
(J.F. Goularte), [email protected]
(A.L. Hoefel), [email protected]
(A.L. de Castro), [email protected]
(L.C. Kucharski), [email protected]
(A.S. da Rosa Araujo), [email protected]
http://dx.doi.org/10.1016/j.physbeh.2015.11.037 0031-9384/© 2015 Elsevier Inc. All rights reserved.
increased episodes of nighttime awakenings and, as a consequence, a reduction in sleep efﬁciency. These alterations may remain until the ﬁrst month following labor [4, and for a review 7]. The physiological changes during pregnancy, which are intensiﬁed by environmental and lifestyle changes, may be related to the alterations in sleep patterns [5,8,9]. An important consequence of sleep disturbances during pregnancy is the impact on the fetus. Sleep deprivation is a risk factor for poor fetal growth and preterm delivery [3,10–12]. The mother-infant relationship may also be affected by sleep deprivation during pregnancy. Sleep disturbances have been related to maternal depressive symptoms during the post-partum period, which can affect the infant [13,14]. A healthy emotional state during the postpartum period is important for the establishment of mother-child bonding, which provides a secure
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basis for the child's development . A previous study has demonstrated that SR in pregnant females is causally related to reduced ultrasonic calls in the offspring, which indicates a disturbance in cognitive development . Although several epidemiological studies have reported that sleep disturbances affect the mother and fetus, the mechanisms remain to be analyzed. The causes and consequences of sleep deprivation have been less studied in females compared with males. In animal models, REM sleep deprivation may cause alterations in metabolic [17, 18], immune , and hormonal [20,21] parameters in male rats. In cycling females, previous studies have demonstrated that SR alters the estrous cycle, plasma levels of corticosterone and progesterone  and cognitive performance . In pregnant rats, SR has been related to an increase in the adrenal gland weight on GD 20 , which indicates that this procedure may activate the hypothalamic–pituitary–adrenal (HPA) axis in pregnant females, despite its hyporesponsiveness during this period [reviewed in 25]. Pregnancy is characterized by a delicate balance of several hormones, and the hypothalamic–hypophyseal–ovarian axis and the HPA axis are essential for a successful pregnancy, delivery and offspring development [reviewed in 26]. Prolactin (PRL) and oxytocin (OT) are also important for fetal development, labor, lactation and the organization of maternal behavior [27,28]. OT and PRL increase during pregnancy, and at the end of pregnancy, OT plays a crucial role in delivery, whereas PRL plays a role in lactation and the onset of maternal behavior. Moreover, hormones may respond differently in pregnancy vs. nonpregnancy states. It has been well established that the HPA axis exhibits a reduced response to a wide range of physical and psychological stressors during gestation [reviewed in 29]. This hyporesponsiveness appears necessary for the maintenance of pregnancy, maternal behavior, and the development of several fetal organs that are highly sensitive to maternal glucocorticoids [reviewed in 30]. The present study aimed to analyze the effects of sleep restriction (SR) from gestational day (GD) 14 to 20, using the multiple platform method, in the pregnant females and their fetuses, as well as the mother and offspring following birth in rats. The effects of SR were analyzed after 6 sessions of partial sleep deprivation for 20 h per day per session. This experimental sequence was adopted to mimic a more realistic situation considering that sleep disturbances are typically repeated. In the females, we analyzed the effects of SR on the plasma corticosterone, PRL, progesterone and OT on GD 20 and subsequently during lactation on postpartum day (PPD) 7. Previous studies have identiﬁed a decrease in body weight in male  and pregnant female rats submitted to SR ; thus, the levels of triglycerides, glycogen and cholesterol were analyzed in the liver and the soleus muscle on GD 20 to investigate the potential causes of weight loss. Considering the period of synaptogenesis during the ﬁnal week of gestation in the rat [reviewed in 32], and that pre-natal stress may affect brain development in the offspring [reviewed in 33, 34], we measured brain-derived neurotrophic factor (BDNF) in the fetuses on GD20. Reactive oxygen species (ROS) were simultaneously analyzed to assess the potential cellular damage by oxidants. Following birth, maternal behavior was assessed to investigate the effects of SR on the mother-offspring relationship. Moreover, in the 7-day-old male and female pups, the behaviors of the pups were evaluated using the nest odor preference test. 2. Materials and methods 2.1. Subjects Female (113) and male (24) rats approximately 70 days old from the Centre for Reproduction and Animal Experimentation Laboratory of the Universidade Federal do Rio Grande do Sul, UFRGS were used. The animals were group-housed (4 per cage of 40 × 33 × 17 cm) in a temperature (21 ± 1 °C) and humidity-controlled (60%) animal facility. The rats were maintained on a 12-h light/dark schedule (lights on at 06:00 h) with access to water and rodent chow (Nuvilab Cr2,
Colombo, Brazil) ad libitum. The experimental protocol was approved by the Ethics Committee in Use of Animals (CEUA) of the UFRGS (No. 23948/2013). The experimental procedures were performed following the Guidelines for Animal Care and Use of Laboratory Animals of the National Institutes of Health (2011). 2.2. Sleep restriction (SR) SR was induced using the multiple platform method, according to the protocol described in [23,24]. The paradigm consisted of placing 4 rats of the same group in a 90 × 50 × 50 cm tank ﬁlled with water. Ten cylinder platforms (7.5 cm in diameter and 10 cm in height) were placed in the water tank and were arranged at distances of 11 cm from one another. These platforms protruded 2 cm above the water surface, which enabled the animals to move from one platform to another. To better isolate the sleep restriction variable, one group of animals was utilized to control for the environmental parameters of the multiple platform method. In this Sham group, tanks with the same characteristics and dimensions as the tanks used to induce SR were used; however, instead of water, the ﬂoor was covered with sawdust. These tanks also contained the 10 platforms of equal size, and the animals had to climb to a platform to reach food and water. Pregnant females were submitted to sleep deprivation or exposed to the new environment for 20 h per day (14:00 to 10:00 h next day) from GD 14 to 20. In the present sleep restriction protocol, the animals could recover sleep; thus, the design comprised partial sleep deprivation. 2.3. Experimental procedure After 4 regular estrous cycles, 90-day-old virgin females (n = 74) were mated with males on the evening of proestrus. The presence of spermatozoids in the vaginal smear the following morning was considered a positive indicator of pregnancy (n = 64), and this day was designated GD 0. The pregnancy was monitored daily, and the body weight of each pregnant female was measured using an electronic balance (SHIMADZU BL3200H, Tokyo, Japan) between 10:00–10:30 h every day. On GD 1, the animals were randomly assigned to the following groups: home cage (HC, n = 26), Sham (n = 17), and sleep restriction (SR, n = 21). On GDs 12 and 13, at 14:00 h, the pregnant rats in the 3 groups were transported to a room with the tanks for sleep restriction, which was maintained at similar conditions to the animal facility. The SR rats were placed together on platforms inside the tanks (4 animals from the same cage per tank) for 30 min. The same procedure was adopted for the Sham group. The HC rats were left in their home cages (40 × 33 × 17 cm) in the same room for the same period as the Sham and SR rats. The purpose of this procedure was to habituate the animals to the new environment of the tanks with the platforms. On GD 14 at 14:00 h, the HC, Sham and SR rats were again transported to the experimental room. The SR rats were placed on the platforms in the water tank, where they remained until 10:00 h of the next day. The Sham rats were placed in tanks that contained shavings and were arranged with multiple platforms. The HC rats were maintained in their home cages without manipulation in the same room as the other groups (Sham and SR). On the following day at 10:00 h, the SR and Sham rats were removed from the tanks and placed back in their respective home cages. The 3 groups were returned to the animal facility where they remained for 4 h (10:00 to 14:00 h), when the animals could recover sleep. This procedure was repeated daily until GD 20. During the sleep restriction period, food and water were offered ad libitum. Food pellets were left on a compartment in the grid (similar to the home cage) above the platforms. On GD 20 at 10:00 h, following the conclusion of the SR period, the pregnant rats were returned to the animal facility and weighed. Immediately afterwards, females from each group (HC = 10, Sham = 6 and SR = 7) were randomly selected; they were decapitated, and blood was collected. The fetuses were subsequently removed via
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hysterectomy, measured, and weighed, and cerebral tissue samples were obtained. The visceral adipose tissue, adrenal glands, soleus muscle, and liver were also obtained. The remaining pregnant rats were individually housed in Plexiglas transparent cages (44 × 20 × 21 cm) covered with shavings. The day of birth was considered day 0. On the morning of PPD 1, the litters were standardized to 8 pups (4 males and 4 females). Some females from the original sample required removal from the analysis because of problems in the parturition or because they delivered less than 8 pups (Table 5). For the postpartum studies, the HC, Sham, and SR groups comprised 14, 8, and 8 animals, respectively. Maternal behavior was recorded from PPD 1 to 6. On the morning of PPD 7 (between 10:00 and 12:00 h), the mothers were weighed and decapitated for blood collection, and their adrenal glands were collected. Following the olfactory preference test, the 7-day-old pups were also weighed and decapitated for blood collection.
2.4. Maternal hormonal and metabolic analysis 2.4.1. Hormonal determination Plasma hormones were measured in pregnant (GD 20) females following the completion of the ﬁnal SR session, between 10:00 and 12:00 h, as well as in the lactating females (PPD 7) at approximately the same time. Trunk blood samples were collected in tubes that contained 25 μL of EDTA (0.5 M). The blood was immediately centrifuged (4 °C, 1000 g 15 min), and the plasma was stored at − 80 °C until hormone assay analysis. All hormones were measured in duplicate via the ELISA method according to the manufacturer's instructions. The plasma prolactin concentration was measured using a SPIBio kit (Montigny Le Bretonneux, France); the plasma oxytocin concentration was measured using an Enzo Life Sciences kit (Farmingdale, NY, USA); the plasma progesterone concentration was measured using a Cayman Chemical Progesterone kit (Ann Arbor, MI, USA); and the plasma corticosterone concentration was measured using an Arbor Assays kit (Ann Arbor, MI, USA).
2.4.2. Glycogen, triglycerides and cholesterol Glycogen, triglycerides and cholesterol were measured in the livers of the pregnant rats on GD 20. Glycogen was also analyzed in the soleus muscle. Following decapitation, the liver and soleus muscle were dissected and stored at − 20 °C. The glycogen concentration was determined according to the method of . The triglyceride and cholesterol concentrations in the liver were measured according to the protocol described in .
2.5. Analysis of fetal brains (GD20) 2.5.1. BDNF concentration The BDNF concentration in the brain tissue was determined via the ELISA method using the BDNF ChemiKine Sandwich kit (CTY306, Millipore, MA, USA), according to the manufacturer's instructions with the adaptations proposed in  for fetal brains. On GD 20, the females were decapitated, and the fetuses were extracted by cesarean section. The fetuses (n = 6–7 per group) were decapitated, and the cortex, hippocampus and cerebellum were immediately dissected in cold saline solution, frozen in liquid nitrogen, and stored at −80 °C. The cerebral cortex, hippocampus, and cerebellum were homogenized in 10–50 volumes of cold lysis buffer that contained 100 mM Tris–HCl, pH 7.4, 0.6 M NaCl, 0.5% bovine serum albumin, 1 mM EDTA, 0.2% Triton X-10, 0.1 mM PMSF, and 0.1 mM pepstatin. The extracts were centrifuged at 14,000 × g for 30 min at 4 °C. The BDNF concentrations in the tissue were analyzed from the supernatants following centrifugation. Male fetuses were used (one fetus per litter), and only the BDNF concentration was processed for these brains.
2.5.2. Total reactive oxygen species (ROS) After defrosting, the fetal cortex, hippocampus, and cerebellum were weighed and homogenized in 5 volumes (by weight of tissue) of 20 mmol sodium phosphate buffer, 120 mmol KCl, and 1:100 PMSF; the samples were centrifuged at 1000 ×g and 4 °C for 10 min. The supernatants were used to determine the concentrations of ROS according to the method in . Only male fetuses were used (one fetus per litter), and only ROS measurements were processed for these brains. 2.6. Behavioral analysis 2.6.1. Maternal behavior Maternal behavior was recorded 4 times per day, including 3 periods during the light cycle phase (09:00, 12:00, 15:00 h) and one time at the beginning of the dark cycle (18:00 h), from PPD 1 to 6. Within each observation session, the behavior of each female at that speciﬁc moment was scored every 3 min (25 observations in 4 periods per day = 100 total observations/mother/day). The following behaviors were recorded: high crouch posture (mother nursing pups in an arched-back posture); low crouch posture (mother nursing pups in a “blanket” low arched back posture); supine posture (a passive posture in which the mother is lying on her back or side while the pups nurse); licking the pups (licking the surface of their bodies and their anogenital regions); nest building (piling sawdust were the pups laid); mother off the nest (the lactating female is out of the nest); and self-grooming (mother grooms herself). The maternal care score was calculated by summing the behaviors directed towards the pups (licking + breastfeeding postures) in all recording periods. The data are reported as the number of observations that the behavior was recorded (maximum of 600 times) [39–41] and were averaged among the females within the groups. A detailed description of the maternal care score in each period (time of the day) and day from PPD 1 to 6 was also performed, using a subset of the females. 2.6.2. Olfactory preference test for familiar odors in the offspring The olfactory preference test for the odor of the home cage sawdust (mother and littermates) was based on previous studies, and it was performed according to a previously described protocol . The test comprised a two-odor choice between areas with nest or fresh bedding. A Plexiglas box (40 × 34 × 24 cm) was divided, using a tape glued on the ﬂoor, into two halves of 19 cm each. A portion of nest bedding (approximately 300 mL) was placed in one corner (right to the middle line), and a similar portion of fresh sawdust was placed at the other corner (left to the middle line). On postnatal day (PND) 7, one male and one female pup from each litter were randomly removed from the nest. Each pup was placed on the middle line of the testing box (neutral zone) opposite to the side where the olfactory stimuli were, with their heads facing the area where the sawdust was placed at a distance of approximately 17 cm from the target areas. Their behaviors were videotaped. The test was performed in 5 consecutive sessions, which lasted 1 min each with a 2-min interval between each session. At the end of each session, the box was cleaned with 70% alcohol, and the positions of the nest bedding and fresh bedding were switched. 2.7. Statistical analyses The data were analyzed using a one-way or two-way ANOVA followed by Tukey or Bonferroni multiple comparison post hoc tests, respectively. For the one-way ANOVA, if the Bartlett test for equality of variances was signiﬁcant, the analysis was performed using a KruskalWallis test followed by a Dunn's multiple comparison tests. Data are expressed as the mean ± SEM or the median (interquartile interval, IQ). Differences were considered signiﬁcant at p b 0.05. Statistical analyses were performed using the software GraphPad Prism 5 (San Diego, CA, USA).
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3. Results 3.1. Maternal hormones on GD 20 and PPD 7 In the mothers, we analyzed the effects of SR on the plasma corticosterone, PRL, progesterone and OT on GD 20 and subsequently during lactation on postpartum day PPD 7. Table 1 shows that the plasma corticosterone was not different among the groups (F 2,17 = 0.62) on GD 20. The plasma PRL was increased in the SR compared with the HC and Sham groups (F 2,16 = 9.92; p b 0.05, followed by Tukey test). Moreover, the plasma OT was increased in the SR group compared with the HC group, but not the Sham group (F 2,17 = 4.39; p b 0.05, followed by Tukey test). As a result of technical problems, the plasma progesterone was not measured on GD 20. Table 1 also shows that on PPD 7, the plasma corticosterone was not signiﬁcantly different among the groups (F 2,17 = 1.74). Similarly, the plasma progesterone (F 2,17 = 0.04) and oxytocin (F 2,17 = 2.97) were not different among the groups. As a result of technical problems, the plasma prolactin was not measured on PPD 7.
Fig. 1. Effects of sleep restriction during pregnancy on the percentage of body weight gain. Points with vertical bars represent the mean ± SEM percentage of the weight gain in relation to gestational day (GD) 2 (100%). The arrow marks the initiation of sleep restriction. ⁎Different from the HC. #Different from the Sham. Home cage (HC n = 26), Sham (n = 19), and Sleep restriction (SR n = 19).
3.2. Adrenal gland weight in females on GD 20 and PPD 7 On GD 20, the absolute weight (g) of the adrenal was not different among the groups (mean ± SEM for HC 0.096 ± 0.008, Sham 0.112 ± 0.006, SR 0.115 ± 0.008, one-way ANOVA, F 2,22 = 1.83; n = 6–7 per group). In contrast, the relative adrenal weight (total weight of the left plus the right adrenal glands/body weight in grams normalized by 100 g) in the SR group was increased compared with the HC group, but not the Sham group (mean ± SEM for HC 0.026 ± 0.002, Sham 0.032 ± 0.001, SR 0.035 ± 0.003, one-way ANOVA F 2,22 = 4.431, p b 0.05, followed by Tukey test; n = 6–7 per group). On PPD 7, the absolute weight (g) of the adrenal glands was not different among the groups (mean ± SEM for HC 0.089 ± 0.002, Sham 0.082 ± 0.003, SR 0.088 ± 0.004, one-way ANOVA F 2,29 = 1.65; n = 8–14 per group). The relative adrenal weight did not attain a statistically reliable difference among the groups (mean ± SEM for HC 0.029 ± 0.001, Sham 0.082 ± 0.003, SR 0.031 ± 0.002, one-way ANOVA F 2,29 = 2.53, p = 0.10, n = 8–14 per group). 3.3. Body weight in females on GD 20 and PPD 7 Fig. 1 presents the mean (±SEM) percentage of body weight gain during pregnancy, considering GD 2 as 100%. The results showed a reduction in the body weight gain in the pregnant rats submitted to SR compared with the HC and Sham groups (two-way ANOVA, interaction of groups and days of pregnancy, F 14,43 = 16.61, p b 0.01). Moreover, a post hoc Bonferroni analysis demonstrated that the body weight gain of the SR group was signiﬁcantly lower compared with the HC group on GD 16, 18 and 20 and the Sham group on GD 18 and GD 20. In addition, the Sham group exhibited a signiﬁcantly lower body weight gain compared with the HC group on GD 16, 18 and 20.
The mean of the absolute body weight (g) of the SR group on GD 20 (322.40 ± 5.68, n = 26) was signiﬁcantly lower compared with the HC group (357.10 ± 5.18, n = 19); however, it was not different from the Sham group (339.35 ± 6.04, n = 19) (one way ANOVA F2,63 = 9.97; p b 0.05, followed Tukey test). At all gestational days after the initiation of sleep restriction, the mean body weight of the SR group was lower than the HC. Although differences in the body weight gain were identiﬁed between the Sham and HC groups, the absolute weight in the Sham group was not different than the HC group at any time point. On PPD 7, no signiﬁcant difference in the mean body weights (g) of the mothers was identiﬁed among the groups: home cage (n = 14) = 305.1 ± 3.3; Sham (n = 8) = 303.5 ± 4.7; and SR (n = 8) = 289.9 ± 7.1; one-way ANOVA F 2,29 = 2.20. 3.4. Metabolic analysis of pregnant females on GD 20 Table 2 shows the visceral adipose tissue weight; the triglyceride and cholesterol concentrations in the liver; and the glycogen concentration in the liver and soleus muscle in pregnant females on GD 20. The visceral adipose tissue weight did not attain a statistically reliable difference among the 3 groups (one-way ANOVA, F 2,18 = 2.69; p = 0.09); however, there was a tendency towards an increase in the HC group. The triglyceride concentration in the liver was not different among the groups (F 2,18 = 2.137). Similarly, the hepatic cholesterol concentration was not signiﬁcantly different among the groups (F 2,17 = 0.28; Table 2). The glycogen concentration in the liver was not signiﬁcantly different among the groups on GD 20 (F 2,17 = 0.65). A similar result was identiﬁed for the glycogen concentration in the soleus muscle (F 2,18 = 2.17, Table 2).
Table 1 Effects of sleep restriction on the plasma levels of corticosterone, progesterone, PRL, and OT in female rats on GD 20 and PPD 7. Hormones
Corticosterone (ng/mL) Progesterone (ng/mL) Prolactin (ng/mL) Oxytocin (pg/mL)
Gestational day 20
Postpartum day 7
264.5 ± 41.02 n.m. 1.94 ± 0.67 17.70 ± 1.58
247.6 ± 22.82 n.m. 4.73 ± 1.86 30.91 ± 7.17
316.6 ± 63.35 n.m. 13.04 ± 2.25a,b 37.67 ± 4.11a
318.9 ± 52.16 374.5 ± 57.55 n.m. 25.86 ± 5.54
376.7 ± 41.38 400.5 ± 94.29 n.m. 39.05 ± 4.02
426.2 ± 23.40 236.4 ± 45.93 n.m. 26.29 ± 3.14
The data are represented as the mean ± SEM. n.m. (not measured). Parentheses contain the number of female rats per group. Home cage group (HC); Sham group (Sham), sleep restriction group (SR). a Different from the HC group. b Different from the Sham group.
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Table 2 Effects of sleep restriction during pregnancy on the visceral adipose tissue weight; triglyceride and cholesterol levels in the liver; and glycogen in the liver and soleus muscle in females on GD 20. Morpho-physiological parameters
Weight of visceral adipose tissue (g) Triglycerides liver (μg/100 g tissue) Cholesterol liver (μg/g tissue) Glycogen liver (μg glucose/g tissue) Glycogen soleus (μg glucose/g tissue)
7.648 ± 2.50 936.4 ± 84.51 173.6 ± 28.64 492.3 ± 53.70 31.83 ± 6.87
5.591 ± 0.51 1162 ± 65.09 190.8 ± 34.64 449.6 ± 40.24 33.91 ± 6.89
5.853 ± 0.38 1098 ± 80.17 211.1 ± 41.65 548.5 ± 82.58 17.14 ± 5.45
The data are represented as the mean ± SEM. The parentheses contain the number of female rats. Home cage group (HC), Sham group (Sham), and Sleep restriction group (SR).
3.5. Number, body weight and length of fetuses on GD 20 Table 3 shows that the number of fetuses identiﬁed on GD 20 was lower in the SR animals compared with the Home cage group, but not the Sham group (Kruskal Wallis test, K = 7.34, p b 0.05, followed by Dunn multiple comparison test). The average body weight of the fetuses was not signiﬁcantly different among the groups (F 2,18 = 1.92). In addition, the average size of the fetuses was not signiﬁcantly different among the groups (F 2,16 = 1.63, Table 3). 3.6. Total ROS and BDNF concentrations in the brains of male fetuses on GD 20 Table 4 indicates that there was no signiﬁcant difference in the total ROS concentration in the cerebral cortex among the groups (F 2,18 = 0.60). Similar results were identiﬁed in the cerebellum (F 2,18 = 0.29) and hippocampus (F 2,16 = 1.29). Table 4 also shows that the hippocampal BDNF concentration in the male fetuses whose mothers were submitted to sleep restriction was increased compared with the HC group (F 2,16 = 4.10, p b 0.05, followed by Tukey test). The difference between the Sham and HC groups did not reach statistical signiﬁcance. No signiﬁcant difference was identiﬁed among the groups for the BDNF concentration in the fetal cortex (F 2,16 = 1.96) or cerebellum (F 2,18 = 0.45, Table 4).
increased number of licking the pups while in the high crouch position, which was increased in the Sham group compared with the HC; in contrast, the SR was not signiﬁcantly different compared with the HC. Table 7 shows the Maternal Care Score, which was the sum of high crouching, low crouching, supine and licking. The Score was discriminated by time of the day and by postpartum day. On day 1 at 12:00 h, the Sham group showed a decrease in the score. On that same day at 15:00 h, sleep restricted females also showed a signiﬁcant reduction. In other periods and days, no signiﬁcant changes were detected on that cumulative index of pup directed behaviors. 3.9. Olfactory preference of pups The olfactory preference of the pups was assessed based on the amount of time they spent in the nest bedding area. Fig. 2 shows that in the male pups, a two-way ANOVA revealed a main effect of time (F 1,27 = 12.47; p b 0.05) but no difference among the groups (F 2,27 = 0.45) or interaction of the factors (F 2,27 = 1.55). A similar result was identiﬁed for the olfactory preference of the female offspring. Two-way ANOVA indicated a main effect of time on area (F 1,27 = 21.77; p b 0.05) but no difference among the groups (F 2,27 = 3.29) or interaction of the factors (F 2,27 = 1.20). 4. Discussion 4.1. Effects of SR on plasma corticosterone and adrenal weight in pregnant females
3.7. Pregnancy outcomes Table 5 shows that the median duration of pregnancy, the median number of pups per litter, and the percentage of litters with more than 8 pups were not different among the 3 groups (Kruskal Wallis test, K = 1.41). The daily observation of the rats showed that on GD 16 (2 days after the onset of sleep restriction), 2 miscarriages occurred in the females of the SR group. Moreover, in 2 females in the SR and 2 females in the Sham groups, labor began on GD 21 and lasted until 23. Some of these pups were already dead when born. On PPD 7, the mean body weight (g) of the litter (HC group = 15.16 ± 0.27, n = 14; Sham group = 14.28 ± 0.60, n = 8; and SR group = 14.32 ± 0.47, n = 8) was not different among the groups (F 2,29 = 1.85). 3.8. Maternal behavior Table 6 shows the behaviors of the lactating females during the ﬁrst 6 days after delivery. The only difference among the groups was the Table 3 Effects of sleep restriction during pregnancy on the fetuses on GD 20. Characteristics
Number of fetuses Body weight of fetuses (g) Length of fetuses (cm)
13 (12–14) 3.571 ± 0.038 3.210 ± 0.027
12 (12–13) 3.668 ± 0.067 3.391 ± 0.062
11 (9–12)a 3.445 ± 109 3.310 ± 0.098
Parametric data are expressed as the mean ± SEM. Non-parametric data are represented as the median (IQ). Parentheses next to the groups contain the number of litters. Home cage group (HC), Sham group (Sham), and Sleep restriction group (SR). a Different from the HC.
This study evaluated the effects of sleep restriction, induced by the multiple platform method, 6 days after the initiation of the intervention. We focused on the sustained changes because the repetitive deprivation is more clearly related to the expected sleep disruption during pregnancy in women. In the present protocol, the plasma corticosterone on GD 20 (immediately after the end of the 6th sleep restriction session) and after parturition on PPD 7 were not altered. However, the mean values in the sleep deprived females, both on GD 20 and PPD 7, were slightly increased compared with the other groups but did not reach signiﬁcance (the standard error is high). In contrast, previous studies in male rats have shown that sleep deprivation increases plasma ACTH and corticosterone [20 and for a review 43]. The most direct explanation for the absence of a change in corticosterone in our experiment is likely the pregnancy. It is well known that peripheral corticosterone during pregnancy is maintained with minimal changes in response to several stressors, which indicates a strong inhibition of the HPA, mainly by the hippocampus [for reviews 26, 29]. In addition to the sex differences and the peculiarities of pregnancy, another variable that could explain the discrepancies in the results is the sleep restriction protocol. In most experiments with males, the animals were continuously deprived of REM sleep for several days (typically 4), whereas in the present study, the females were deprived for 20 h per day (4 h in the home cage) repeatedly for 6 days. Thus, the females had the opportunity to recover sleep every day. In both protocols, the animals were basically deprived of REM sleep; however, in the case of males, this deprivation was more prolonged than the present protocol. It is also possible to imagine that corticosterone had increased after the initial deprivation sessions,
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Table 4 Effects of sleep restriction during pregnancy on the total ROS levels and BDNF concentrations in the fetal cortex, cerebellum, and hippocampus on GD 20. Structures
Cortex Cerebellum Hippocampus
Total ROS (pmol/mg)
7.10 ± 1.02 5.71 ± 0.75 6.55 ± 0.76
7.08 ± 1.12 6.69 ± 0.76 5.99 ± 1.01
5.98 ± 0.28 5.68 ± 1.35 4.74 ± 0.79
47.83 ± 11.43 70.47 ± 10.47 29.26 ± 8.135
102.70 ± 26.08 85.42 ± 13.72 73.91 ± 15.00
81.99 ± 16.45 88.20 ± 16.22 94.47 ± 23.21a
Data are represented as the mean ± SEM. Parentheses contain the number of fetuses. Home cage group (HC), Sham group (Sham), and Sleep restriction group (SR). a Different from the HC.
and following repetition, the HPA axis would adapt. However, previous study has demonstrated an increase in plasma corticosterone in male rats submitted to a sleep restriction regime of 18 h every day during 21 days, which indicates persistent activation of the HPA axis . Nevertheless, present ﬁnding demonstrated that repeated sleep restriction in female rats during the particular neuroendocrine period of pregnancy did not induce a sustained HPA axis activation, at least as measured by plasma corticosterone. Another result related to the HPA axis activation was the adrenal gland weight. There appears to be a relationship between the increase in the relative adrenal weight induced by paradoxical sleep deprivation and the plasma ACTH . A previous study in pregnant females, using the same sleep restriction protocol, has demonstrated an increased relative adrenal gland weight in SR females compared with females that remained in their home cages . Consistent with this ﬁnding, we showed that on GD 20, the relative adrenal gland weight in the females submitted to sleep restriction was higher than the females that remained in their home cages. However, they were not different than the females submitted to the novel environment (Sham group), which suggests that the environmental change may have an effect per se and is an important variable to be considered. On PPD 7, the effect on the adrenal weight was no longer present. It remains to be determined whether the noradrenergic system is stimulated in response to sleep restriction in pregnant rats, or if this system would also be hyporesponsive to the intervention as the HPA during this speciﬁc period (ﬁnal week of gestation). Male rats submitted to a 24-h, 48-h or 96-h sleep deprivation protocol exhibit a temporal effect, in which an increase in plasma noradrenaline is identiﬁed only following the 96 h deprivation protocol. However, during recovery, the levels are lower than the home cage group . The effects of sleep restriction on the HPA axis are complex and appear to depend on several factors, including the of sleep restriction protocol; the interval between the last intervention and the blood sample; the gender of the animal; and the interaction with other hormones, such as gonadal hormones. 4.2. Effects of sleep restriction on body weight gain and metabolic parameters in pregnant females The results demonstrated that the body weight of the pregnant females submitted to the SR was lower than the females that remained in the home cage and the sham group. The SR reduced body weight
Table 5 Effects of sleep restriction during pregnancy on the duration of gestation, number of pups per litter, and percentage of litters with 8 or more pups. HC (16)
Duration of gestation (days)
Number of live pups born per litter
11 (9.25–12) 87.5
22 (21–23) 9 (6–12)
Percentage of litters with 8 or more pups
9.5 (7.25–11) 66.7
Data are represented as the median (IQ). Parentheses next to the groups contain the number of litters. Home cage group (HC), Sham group (Sham), and Sleep restriction group (SR).
gain beginning on the 2nd day of the deprivation until the end of the protocol (GD 20). In the sham group, the weight gain was also lower than the HC, but the absolute body weight was not signiﬁcantly different between these groups. These ﬁndings are consistent with previous studies in pregnant female  and male rats [17,45] that have also identiﬁed a reduction in body weight gain, which is more pronounced in the initial days after the sleep deprivation . In males, sleep deprivation increases food intake; does not induce changes in blood glucose; reduces the plasma levels of insulin and leptin; and increases leptin receptors in the hypothalamus . Furthermore, sleep abnormalities have been causally related to impairments in glucose homeostasis, metabolic syndrome and type 2 diabetes mellitus . In the analogy with males, we reasoned that the sleep restriction protocol would be a stressful event for pregnant females, and stress could be an explanation for the reduction in body weight. However, at least at the end of the 6-day deprivation period, the change in plasma corticosterone did not reach statistical signiﬁcance. Other mechanisms involved with metabolism may be affected by the sleep restriction. In male rats, REM sleep deprivation increases hypocretin-1 in the cerebrospinal ﬂuid . Moreover, prolonged sleep restriction (96 h) may affect hormones, such as leptin and orexin, which have been implicated in metabolism, potentially via inﬂammatory changes [18,31,48]. The analysis of the metabolic pathways would be an important approach to demonstrate the causes for the body weight reduction induced by the sleep restriction. Present results showed no signiﬁcant differences in the adipose tissue weight, cholesterol, triglyceride and glycogen concentrations in the liver or glycogen concentration in the soleus muscle on GD 20. To our knowledge, this is the ﬁrst study to assess sleep restriction with metabolic changes during pregnancy in an animal model. On the other hand, in males, a reduction in liver glycogen has been demonstrated 24 h after sleep deprivation, which was associated with the activation of catabolic processes that promote the breakdown of glycogen and fat reserves, thereby increasing cholesterol and plasma triglycerides . It is possible that during pregnancy, efﬁcient compensatory mechanisms are activated to counterbalance the energy demand that results from the loss of body weight. 4.3. Effects of sleep restriction on plasma levels of oxytocin (OT) and prolactin (PRL) in pregnant female We measured PRL and OT, which are hormones with several functions in pregnancy, delivery and lactation that also respond to several stressors [for reviews 28, 49]. Results showed that the sleep restriction protocol increased the plasma PRL on GD 20, at the end of the 6 days of intervention, compared with the females that remained in the home cage and the sham group. OT also increased in response to SR on GD 20 (after the end of the ﬁnal restriction session) compared with the home cage group but not the sham group, which suggests that the novel environment (different cage) may impact this neurohormone. The increase in plasma OT shows that the repeated sleep restriction activated the hypothalamic paraventricular nucleus (PVN). To our knowledge, the current ﬁndings provide the ﬁrst evidence of OT changes induced by sleep restriction in pregnant females. In male rats, REM sleep deprivation does not change plasma OT . It is possible that the differences in the experimental protocols could explain the
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Table 6 Effects of sleep restriction during pregnancy on the maternal behavior. Behavioral parameters (number of observations)
HC (n = 14)
Sham (n = 8)
SR (n = 8)
F 2,29 or K
Total maternal care score High crouch posture Low crouch posture Supine posture High crouch posture and licking Licking Nest building Self-grooming
313.1 ± 11.89 224.4 ± 11.69 39 (32.25–48) 24 (10.25–48) 35.5 ± 2.33 50.79 ± 3.71 29.36 ± 4.18 64.86 ± 3.94
305.4 ± 13.27 232.4 ± 12.47 38.5 (27.75–49.75) 16 (2–18.5) 52.25 ± 4.36a 66.38 ± 4.06 22.38 ± 3.67 56 ± 3.06
304.3 ± 22.63 216.1 ± 18.90 44 (29–82) 19.5 (4.5–30.25) 42.63 ± 6.63 59 ± 7.897 37.50 ± 6.72 60.50 ± 5.00
0.90 0.77 0.70 0.22 0.02 0.10 0.17 0.33
0.11 0.27 0.72 2.99 4.29 2.49 1.92 1.16
Data represent the accumulated occurrence of the behaviors from postpartum days 1 to 6, averaged by the females in each group. Parametric data are represented as the mean ± SEM. Non-parametric data are represented as the median (IQ). One-way ANOVA was performed, followed by Tukey post hoc test when appropriate. If the Bartlett test for equality of variances was signiﬁcant, the analysis was performed using a Kruskal–Wallis test followed by a Dunn's multiple comparison test. Parentheses contain the number of litters. Home cage group (HC), Sham group (Sham), Sleep restriction group (SR). a Different from HC.
discrepancies in the results. In this previous study, the deprivation was obtained by stroking the animals with a brush every time they began REM sleep for 2 days. Alternatively, because of the peculiarities in OT secretion in pregnant females compared with males, sleep restriction may increase the secretion of this neurohormone. OT is secreted by magnocellular neurons in the PVN and stored in the posterior pituitary. Several factors, such as hormones and stimuli, induce OT release in the blood stream. Plasma OT increases in response to stressors via noradrenaline stimulation of the PVN. However, at the end of pregnancy, OT magnocellular neurons in the PVN are hyporesponsive to the afferents that typically induce secretion into the blood [as reviewed in 49, 51, 52]. Despite this hyporesponsiveness, results showed that sleep restriction using the multiple platform method during pregnancy increased plasma OT via a mechanism that remains to be determined. As a future working hypothesis, we suggest that the sustained increase in plasma OT may be caused by an increase in the excitatory noradrenergic activity on the OT neurons, which would exceed the inhibitory tone on these neurons. It is noteworthy that the sleep deprivation protocol activates the PVN, as previous studies have demonstrated for other peptides, such as CRH and orexin [17,18].
Also related to the hypothalamus, the results showed that SR increased the plasma PRL, which is mainly produced in the lactogens in the anterior pituitary. PRL secretion is tonically inhibited by hypothalamic tuberoinfundibular dopamine (TIDA) neurons. These neurons receive serotonergic and noradrenergic inhibitory inputs that decrease dopamine activity and, as a consequence, increase PRL secretion. OT may also stimulate PRL secretion [reviewed in 53]. During pregnancy, lactotrophs increase PRL synthesis; however, its secretion is inhibited; at the end of pregnancy, previous to parturition, there is a PRL surge that is mediated by opioids that reduces TIDA neuronal activity . In addition to the peculiarities of the role of PRL during pregnancy, several studies have identiﬁed a close relationship between PRL and sleep. PRL administration increases the duration of REM sleep [55–57]. Moreover, rodents that are genetically deﬁcient in PRL exhibit less REM sleep, and exogenous PRL injections increase their amount of REM sleep  apparently via cholinergic pathway activation in the brainstem. In male rats, 96 h of sleep deprivation increases plasma PRL, and after 24 h of sleep recovery, the levels return to the basal levels . In contrast, prolonged sleep deprivation (15 days) decreases plasma PRL and other hormones, such growth hormone, insulin-like growth factor,
Table 7 Effects of sleep restriction during pregnancy on the total maternal care score (sum of nursing behaviors) during 4 periods per day. Postpartum day
Period of observation
HC (n = 12)
Sham (n = 8)
SR (n = 8)
F 2,27 or K
09:00 h 12:00 h 15:00 h 18:00 h 09:00 h 12:00 h 15:00 h 18:00 h 09:00 h 12:00 h 15:00 h 18:00 h 09:00 h 12:00 h 15:00 h 18:00 h 09:00 h 12:00 h 15:00 h 18:00 h 09:00 h 12:00 h 15:00 h 18:00 h
21.58 ± 0.10 21.54 ± 0.77 20.38 ± 1.00 7.85 ± 2.19 20.92 ± 0.96 22 (16.75–25) 15.83 ± 1.68 3.50 (0.25–5) 14.42 ± 1.29 16 ± 1.55 13.42 ± 1.311 1.5 (1–5) 16 (14–18) 14.33 ± 1.96 12.5 (9.25–14.75) 2.5 (1.25–7) 16 ± 1.86 14.58 ± 1.95 10.92 ± 1.72 0 (0–4.75) 12.33 ± 1.53 11 ± 1.86 8.67 ± 1.61 1.5 (0–3)
23.13 ± 0.88 14.88 ± 2.38a 20.38 ± 1.75 14.5 ± 2.01 21.25 ± 1.57 19 (17.25–21) 14.13 ± 1.81 7 (1.75–10.75) 15 ± 3.01 11.5 ± 2.07 9.25 ± 1.82 5.5 (1–8.75) 17 (8.5–20.75) 14.25 ± 2.12 8 (6.25–11) 5.5 (0.25–9.25) 16 ± 1.56 13.25 ± 2.33 11.25 ± 1.16 0 (0–6.75) 16.13 ± 1.90 9.5 ± 2.76 10.38 ± 1.31 0 (00–3.75)
21.5 ± 1.41 21.13 ± 1.53b 14.5 ± 2.04a,b 11.63 ± 2.25 20 ± 1.99 18.50 (16.25–23.50) 13.38 ± 4.72 6.5 (1.25–11) 18.5 ± 2.15 18.38 ± 2.41 12.63 ± 1.40 2 (1–5) 17 (10–23.25) 14.88 ± 3.52 12.50 (9.25–21.25) 0.5 (0–4,75) 13 ± 3.35 12 ± 2.78 9 ± 1.54 2.5 (1.25–6.25) 14.75 ± 2.64 14.6 ± 2.60 11.5 ± 2.39 1 (0–4)
0.55 0.01 0.02 0.11 0.84 0.25 0.80 0.28 0.35 0.08 0.14 0.16 0.87 0.98 0.06 0.21 0.59 0.72 0.60 0.44 0.37 0.33 0.52 0.76
0.62 5.94 4.63 2.36 0.18 2.76 0.22 2.54 1.09 2.79 2.09 1.99 0.43 0.02 5.76 3.13 0.52 0.33 0.52 1.67 1.02 1.16 0.67 0.56
Data represent the maternal care score (number of high crouch + low crouch + supine + licking) on 4 periods (time of the day) from PPD 1 to 6. Parametric data are represented as the mean ± SEM and non-parametric as the median (IQ). One-way ANOVA was performed, followed by Tukey post hoc test when appropriate. If the Bartlett test for equality of variances was signiﬁcant, the analysis was performed using a Kruskal–Wallis test followed by a Dunn's multiple comparison test. Parentheses next to the groups contain the number of litters. a Different from HC. b Different from Sham.
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Fig. 2. Effects of sleep restriction during pregnancy on the olfactory preference test of the male and female pups on postnatal day 7. The bars represent the mean ± SEM of the duration (s) that the pup spent in the areas. Home cage group (HC, n = 14), Sham group (Sham, n = 8), and sleep restriction group (SR, n = 8).
and leptin, in male rats . The current ﬁndings using a protocol of repetitive sleep restriction demonstrated an increase in plasma PRL 6 days after protocol initiation in pregnant females. Furthermore, this increase occurred on GD 20, approximately 24 to 48 h prior to parturition (in HC and SR animals, parturition was observed between GD 21 and 22). Regarding the hormones measured in this study, it is possible that PRL had systematically changed after every session of SR or, alternatively, that the increase only occurred in response to the ﬁnal session. This is important because the restriction in sleep would anticipate the PRL surge that physiologically occurs immediately prior to parturition (GD 21 and 22) . The causes for this increase remain to be determined. Since sleep restriction, using the multiple platform method, appears to primarily affect hypothalamic and pituitary functions, we suggest that the intervention affects the delicate hormonal milieu and neurotransmitters that would interfere on the secretion of both PRL and OT. The imbalance in these hormones could impact the development of gestation and parturition. Results suggested that hormones such as PRL and OT would be important, besides cortisol, to be checked during pregnancy in women with sleep disturbances. Changes in those hormones may in turn increase susceptibility to develop mood disorders, when facing natural challenges and possible stressors after delivery.
of 21 pregnant females. On GD 20, the average number of fetuses was lower in the SR group compared with the HC group but not the Sham group. The duration of pregnancy was similar in all 3 groups (21 to 22 days); however, 2 rats in the SR group and 2 rats in the Sham group started labor and delivered on the 23rd day of pregnancy, with subsequent consequences to the fetuses. We did not detect infanticide. The number of pups reported corresponds to the pups that were reared by their mothers. It is not known whether the body weight of the pups was different among the groups at birth. We chose not to measure this parameter to reduce interference with the initiation of the mother-offspring relationship. Based on the hypothesis that sleep restriction could affect fetal development, we analyzed parameters related to brain development. It has been well established that BDNF is an important neurotrophin for the formation of synaptic connections, synaptic plasticity, development, and the maturation of neural circuits following neurogenesis [37,61]. The BDNF increase in the developing hippocampus may lead to modiﬁcations in the number and speciﬁcity of synaptic contacts of the basic mechanisms that underlie learning and memory in the postnatal period. The current results showed a signiﬁcant increase in the concentration of hippocampal BDNF on GD 20 in the SR group compared with the HC group. Accordingly, increased BDNF in the hippocampus has been identiﬁed in rat offspring exposed to stressors in the second  and ﬁnal weeks of gestation [63,64], as well as in response to the exposure to high levels of proinﬂammatory cytokines [37,65]. BDNF also increases in the developing hippocampus following exposure to bacterial infection, which affects the development of cognitive performance in rats . Moreover, in hippocampal cell cultures, BDNF affects AMPA glutamate receptors . However, it is not known whether the effects identiﬁed in the hippocampus also occur in the postnatal period, as demonstrated in other models of prenatal stress [34,68]. No signiﬁcant differences were detected in the BDNF concentrations in the cerebellum and cerebral cortex of the fetuses on GD 20. Considering that females were submitted to sleep restriction from GD 14 to 20, it is possible the fetal cerebellum and cerebral cortex were not affected because the neurogenesis period starts in the second week of gestation in these structures, whereas in the hippocampus in the third week , coincidently with the intervention. It remains unknown whether other neurotrophins in the cortex and cerebellum were also altered. The total ROS concentrations in the cortex, cerebellum, and hippocampus of the fetuses on GD 20 were not different among the groups. We initially hypothesized that the offspring of the females submitted to sleep restriction during gestation would have brain damage. The overproduction of ROS and the consequent cellular damage by oxidants have been related to increases in maternal glucocorticoids, which affect glucose utilization and the excitatory activity of glutamate in the fetal brain . However, the current ﬁndings showed no signiﬁcant increase in the plasma corticosterone of the pregnant rats, at least on GD 20, which is consistent with the absence of changes in the ROS concentration in the fetal brain structures. On the other hand, considering that sleep restriction during pregnancy increased hippocampal BDNF in the fetuses, an increase in the total amount of ROS in this region was not expected. BDNF in developing neurons stimulates antioxidant proteins and reduces ROS in response to oxidative stress . On the other hand, the measurement of antioxidant markers would provide another interesting perspective on this point.
4.4. Effects of sleep restriction on fetuses
4.5. Effects of sleep restriction on maternal behavior and olfactory preference in the pups
Another central hypothesis of the study was that the sleep restriction during pregnancy would affect the offspring. Human studies [10, 11,59,60] have related the mother sleep pattern with offspring wellbeing. The current ﬁndings demonstrated that repetitive sleep restriction altered pregnancy progression and fetal development. After 2 days of sleep restriction (GD 16), 2 miscarriages occurred of a total
Our protocol of sleep restriction using the multiple platform method induced minor change in the maternal behavior during the neonatal period, more speciﬁcally on a time point in the ﬁrst day after delivery. No signiﬁcant change on the structure of the behavior was detected. The repeated changes in the environment during the same period in the Sham group also did not signiﬁcantly alter maternal behavior. Other protocols
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for pre-natal stress have demonstrated a disruption of the motherinfant relationship . Furthermore, the performance of the offspring on the olfactory preference test on the 7th PND was not affected by maternal sleep restriction. Interestingly, the olfactory preference in rat pups was related to maternal behavior. The absence of major continued alterations in maternal behavior is consistent with the behavior of the pups. The appropriate behaviors of the mother and the offspring were maintained, despite SR during pregnancy. A previous study, using a protocol of sleep restriction during the entire period of gestation, has reported that behaviors directed to the offspring are also not affected; however, a decrease in defensive behaviors and the inhibition of anxiolytic behaviors are identiﬁed . Another study using a quite different model to induce sleep deprivation (handling the pregnant females for 5 h per day) has identiﬁed hyperactivity and high risk behavior later in the offspring . Epidemiological studies have demonstrated that pregnant women exposed to periods of sleep restriction are at a high risk of developing mood disorders during the postpartum period, with potential consequences on the mother-child relationship [7,12,14,15]. We may conclude that the repeated periods with sleep restriction per se does not induce a major change on the mother-infant relationship. It would important to analyze how lactating females that were previously deprived of sleep during pregnancy would react to an unfavorable or stressful environment. It is possible that previous sleep deprivation would increase the susceptibility to postpartum complications.
4.6. Sleep restriction protocol The multiple platform method protocol involved at least two casual variables: repeated exposure to a new environment and sleep restriction. The sleep restriction protocol is based on increased muscle relaxation, which has been related to REM sleep. However, we did not record EEGs in the present experiment. Nevertheless, this protocol has been extensively used to induce paradoxical sleep restriction [73,74]. An important variable in the protocol is the change in the environment. Thus, we had two control groups: one group in which the pregnant females remained in their home cage; and a second group in which the pregnant females were removed from their home cage and placed in a novel environment similar to the environment used to induce sleep restriction (Sham group). In the Sham group, the females were exposed to most variables of the SR group, with the exception of water on the ﬂoor. The environment was larger than the home cage, and drinking water and food could only be reached by climbing the platforms. In this environment, animals were exposed to more stimuli than their standard home cage. Whether the environment to which the Sham group was exposed to was stressful is an open question. For example, the environment was more demanding to reach water and food. Moreover, in the Sham group, the females were handled every day and exposed to a novel environment because the tanks were cleaned and the sawdust was changed daily. In a larger environment, the Sham females would have more opportunities to exhibit social behaviors compared with the relatively small environment of the home cage. In the SR and Sham groups, the females were placed together in the environments in groups of 4, and they were always the same during the repetition of the interventions. In the SR group, the opportunity to interact socially was greatly reduced because the females had to remain on the platforms one at a time. We do not know whether the environment in the Sham group also induced some level of sleep restriction because there were physical and social stimuli that could induce alertness. However, previous studies [74,75] using a different apparatus, in which male rats could walk on a grid that covered the tank ﬂoor, have demonstrated minimal change in sleep. Considering the results of present study, the use of a group in which the pregnant females were exposed to an environment different from their home cage while similar to the SR group appears to be an important control group.
5. Conclusions The current ﬁndings demonstrate that sleep restriction during pregnancy, using the multiple platform method (20 h per day, from GD 14 to 20), affects hypothalamic hormones, such as PRL and OT, which are important in gestational development. Increases in the plasma PRL and OT were identiﬁed following sleep restriction period (GD 20). We would suggest that the negative outcomes of sleep restriction during delivery may be related, in part, to this hormonal imbalance. In contrast, no statistically signiﬁcant change in the plasma corticosterone was identiﬁed after 6 days of sleep restriction. This study also conﬁrms that sleep restriction during pregnancy reduces the body weight of females, but does not change the levels of liver triglycerides, glycogen and cholesterol or muscle glycogen, which indicates that other metabolic routes may be affected by this intervention. Results also showed increase in BDNF in the hippocampus in fetuses on GD 20, which suggests that maternal sleep restriction during the ﬁnal week of gestation may affect the development of that structure. In contrast to our expectation, no change in ROS in the litter brains was detected. Sleep restriction during pregnancy induced a speciﬁc reduction in pup-directed behaviors on a time point on PPD 1 with no persistent change of the maternal behavior. No change on the pups' preference for the mother's odor on PPD 7. Compared with the ﬁndings described in males, sleep restriction in pregnant females induces different changes and may affect both the mother and offspring. Acknowledgments This work was supported by CAPES and FAPERGS (10/0018-3 CAPES/PROAP). We are very grateful to Thiago Pereira Henriques, Rafael Cáceres Corrêa, Cátia Nunes Corrêa, Amanda Brondani Mucellini, Ana Carla Araújo da Cunha, and Enver Oruro Puma for their technical assistance in data collection. We thank Professor Gilberto Sanvitto for his important suggestions for the development of this work. References  S. Banks, D.F. Dinges, Behavioral and physiological consequences of sleep restriction, J. Clin. Sleep Med. 3 (5) (2007) 519–528 http://www.ncbi.nlm.nih.gov/pubmed/ 17803017.  C. Hublin, M. Partinen, M. Koskenvuo, J. Kaprio, Sleep and mortality: a populationbased 22-year followup study, Sleep 30 (10) (2007) 1245–1253, http://www.ncbi. nlm.nih.gov/pmc/articles/PMC2266277.  J.J. Chang, G.W. Pien, S.P. Duntley, G.A. Macones, Sleep deprivation during pregnancy and maternal and fetal outcomes: is there a relationship? Sleep Med. Rev. 14 (2) (2010) 107–114, http://dx.doi.org/10.1016/j.smrv.2009.05.001.  C. Hedman, T. Pohjasvaara, U. Tolonen, A.S. Suhonen-Malm, V.V. Myllylä, Effects of pregnancy on mothers' sleep, Sleep Med. 3 (2002) 37–42, http://dx.doi.org/10. 1016/S1389-9457(01)00130-7.  M.L. Moline, L. Broch, R. Zak, Sleep in women across the life cycle from adulthood through menopause, Med. Clin. N. Am. 88 (2004) 705–736, http://dx.doi.org/10. 1016/j.mcna.2004.01.009.  J.A. Mindell, R.A. Cook, J. Nikolovski, Sleep patterns and sleep disturbances across pregnancy, Sleep Med. 16 (2015) 483–488, http://dx.doi.org/10.1016/j.sleep.2014. 12.006.  L.E. Ross, B.J. Murray, M. Steiner, Sleep and perinatal mood disorders: a critical eview, J. Psychiatry Neurosci. 30 (2005) 247–256, http://www.ncbi.nlm.nih.gov/ pmc/articles/PMC1160560.  D. Oyiengo, M. Louis, B. Hott, G. Bourjeily, Sleep disorders in pregnancy, Clin. Chest Med. 35 (3) (2014) 571–587, http://dx.doi.org/10.1016/j.ccm.2014.06.012.  K.A. Lee, Alterations in sleep during pregnancy and postpartum: a review of 30 years of research, Sleep Med. Rev. 2 (4) (1998) 231–242.  K. Micheli, I. Komninos, E. Bagkeris, T. Roumeliotaki, A. Koutis, M. Kogevinas, et al., Sleep patterns in late pregnancy and risk of preterm birth and fetal growth restriction, Epidemiology 22 (2011) 738–744, http://dx.doi.org/10.1097/EDE. 0b013e31822546fd.  M.L. Okun, C.D. Schetter, L.M. Glynn, Poor sleep quality is associated with preterm birth, Sleep 34 (2011) 1493–1498, http://dx.doi.org/10.5665/sleep.1384.  L. Palagini, A. Gemignani, S. Banti, M. Manconi, M. Mauri, D. Riemann, Chronic sleep loss during pregnancy as a determinant of stress: impact on pregnancy outcome, Sleep Med. 15 (8) (2014) 853–859, http://dx.doi.org/10.1016/j.sleep.2014.02.013.  S.K. Dørheim, G.T. Bondevik, M. Eberhard-Gran, B. Bjorvatn, Sleep and depression in postpartum women: a population-based study, Sleep 32 (2009) 847–855 http:// www.ncbi.nlm.nih.gov/pmc/articles/PMC2704916.
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