Sleep/Wake Behaviors in Mice During Pregnancy and Pregnancy-Associated Hypertensive Mice

Sleep/Wake Behaviors in Mice During Pregnancy and Pregnancy-Associated Hypertensive Mice Abstract Study Objectives In humans and other mammals, sleep is altered during pregnancy. However, no studies have been conducted on sleep/wakefulness during pregnancy in mice. In this study, we examined sleep/wakefulness in female C57BL/6 mice during pregnancy. We also examined sleep/wake behaviors in an animal model of preeclampsia, pregnancy-associated hypertensive (PAH) mice, in which increased angiotensin causes hypertension. Methods Sleep/wake behaviors of female C57BL/6 and PAH mice were examined based on electroencephalogram (EEG) or electromyogram recordings before, during, and after pregnancy. To examine whether high blood pressure disrupts the integrity of the blood–brain barrier in PAH mice, Evans blue dye was injected intravenously. Angiotensin II receptor blocker (olmesartan)-administered PAH mice and female Tsukuba hypertensive mice were also examined. Results C57BL/6 mice showed a decreased total wake time and increased nonrapid eye movement (NREM) sleep time during late pregnancy. Rapid eye movement (REM) sleep time did not change during the course of pregnancy. PAH mice exhibited a general slowing of EEG during late pregnancy and subsequently returned to apparently normal sleep/wakefulness after delivery. All PAH mice exhibited multiple focal leakages of Evans blue dye in the brain. Spike-and-wave discharges were observed in 50% of PAH mice. Olmesartan-administered PAH mice did not show general slowing of EEG. Tsukuba hypertensive mice showed a normal time spent in wakefulness and NREM sleep and a decreased total REM sleep time. Conclusions This study showed pregnant-stage-specific changes in sleep/wakefulness in C57BL/6 mice. Furthermore, PAH mice may be useful as an animal model for eclampsia. pregnancy, hypertension, animal models, blood pressure Statement of Significance Despite the broad use of mice for preclinical studies, to the best of our knowledge, there have been no studies on sleep/wakefulness during pregnancy in mice. Total nonrapid eye movement sleep time was increased during the course of pregnancy. Pregnancy-associated hypertensive (PAH) mice exhibited a general slowing of electroencephalogram during late pregnancy. All PAH mice exhibited disruption of the blood–brain barrier, and half of the PAH mice showed spike-and-wave discharges. The current findings showed similar changes during pregnancy between humans and mice, which provides a basis for preclinical studies for human diseases during pregnancy. PAH mice may be a useful model for the development of therapeutic intervention for eclampsia. Introduction Sleep is altered during pregnancy, and different sleep disturbances are associated with each trimester, likely reflecting a wide range of physiological and hormonal changes [1–5]. However, few sleep studies of pregnant women using polysomnography have been reported [6–8]. Mice are suitable model species for examining sleep/wakefulness during pregnancy because they have a gestation period of less than 3 weeks. Furthermore, many gene-modified mice have been developed as animal models of human diseases, including preeclampsia [9] and the sleep disorder narcolepsy [10], which enables the examination of how pregnancy affects the phenotypes of animal models. To the best of our knowledge, however, there have been no published studies on sleep/wakefulness during pregnancy in mice. Continued progress in genetic engineering, including genome editing, has provided rapid progress in the genetics of sleep [11]. By using optogenetic and pharmacogenetic approaches, the understanding of neural circuitries regulating sleep/wake behaviors has rapidly progressed [12]. Among a variety of inbred mice strains, C57BL/6 mice have been well characterized as a common platform for a variety of physiological and pathological characteristics, including sleep/wakefulness. Thus, a large-scale gene knockout program has been developed as a resource using C57BL/6 mice [13], and the subsequent phenomics program has continued to uncover the phenotypes of gene knockout mice [14]. Although most behavioral studies have been conducted using male C57BL/6 mice, male-biased research results may hinder the characterization of sexually dimorphic effects, which may lead to an underestimation of risks for females [15]. Indeed, those few studies on sleep/wakefulness in female mice did show sex differences in sleep [16–18]. Preeclampsia is a pregnancy complication characterized by hypertension and proteinuria after 20 weeks of gestation in patients without hypertension prior to pregnancy [19–22]. Eclampsia is a severe phase of preeclampsia with the occurrence of convulsions and/or disturbed consciousness [20, 23]. The World Health Organization Multicounty Survey reported that preeclampsia and eclampsia develop in 2%–3% and 0.3% of pregnancies, respectively [24]. Although women with preeclampsia have a 3.7-fold increased risk of death compared with pregnant women without preeclampsia, women with eclampsia have a 42-fold increase [24]. As an animal model of preeclampsia, pregnancy-associated hypertensive (PAH) mice have been developed by crossing female mice carrying the human angiotensinogen transgene with male mice carrying the human renin transgene [9]. PAH mice utilize the “species barrier” between humans and mice in terms of angiotensinogen processing by renin. Human renin cannot process murine angiotensinogen, whereas mouse renin cannot cleave human angiotensinogen into angiotensin I. Thus, only when human renin exits, human angiotensinogen is processed into angiotensin I. Since the amino acid sequence of angiotensin I is the same between human and mouse, angiotensin I in mice is subsequently processed into angiotensin II via the removal of two amino acids at the carboxyl terminus by angiotensin-converting enzyme [25], resulting in high blood pressure. In PAH mice, when a female angiotensinogen transgenic mouse conceives embryos carrying the human renin transgene, the pregnant mouse shows hypertension and proteinuria in late pregnancy because the human renin produced from the placenta processes human angiotensinogen into angiotensin I [9] and subsequently angiotensin II. Increased angiotensin II causes acute and severe hypertension with proteinuria in PAH mice [9, 25], which can be alleviated by olmesartan, an angiotensin II receptor blocker, from 150–160 to 120 mm Hg in late pregnancy, which was still higher than that of normal pregnant mice, 100–110 mm Hg [26, 27]. As eclampsia patients often exhibit hyperintensity on T2-weighted brain MRI indicative of vasogenic edema [28–31], acute severe hypertension in PAH mice may disrupt the blood–brain barrier (BBB) [32], but PAH mice have not been examined for the integrity of the BBB. Another line of hypertension model mice, called Tsukuba hypertensive mice (THM), carries both human renin and human angiotensinogen transgenes and suffers from moderate and chronic hypertension, 20–30 mm Hg higher than that of wild-type mice [33, 34]. Since the systolic blood pressure of THM is similar to that of olmesartan-administered PAH mice, the sleep/wake behaviors of these mice allow us to examine how mild-to-moderate hypertension affects sleep. In the present study, we evaluated sleep/wake behaviors during pregnancy in C57BL/6 mice by analyzing the electroencephalogram (EEG) or electromyogram (EMG), which provides a basis for future studies on the mechanism of sleep regulation during pregnancy. We also examined the sleep/wake behaviors of PAH mice and nonpregnant female THM. PAH mice showed abnormal sleep/wakefulness frequently accompanied by spike-wave discharges during late pregnancy, which was alleviated by the administration of olmesartan, implicating PAH mice as an animal model of eclampsia. Methods Animals Female C57BL/6J mice (CLEA Japan) were used in this study. PAH mice and nonpregnant THM were maintained on a C57BL/6 background. Mice were housed under a 12:12-hr light/dark cycle. Food and water were delivered ad libitum. A water gel pack (Napa Nector, 8 oz., System Engineering Lab Group Inc.) was used for a water source because this pack allows both pregnant wild-type and PAH mice to intake water easily. All procedures were approved by the Institutional Animal Care and Use Committee of the University of Tsukuba. EEG or EMG Electrode Implantation Surgery At 8–12 weeks of age (19–23 g), female wild-type, THM, and PAH mice were anesthetized using isoflurane (3%–4% for induction and 1%–2% for maintenance). Each electrode has four electrode pins and two wires. Four electrode pins were lowered to the dura under stereotaxic control, and two flexible wires for EMG recording were inserted in the neck muscles and subsequently attached to the skull using dental cement. The electrodes for EEG signals were positioned over the frontal and occipital cortices (anteroposterior: 0.5 mm, mediolateral: 1.3 mm, dorsoventral: −1.3 mm and anteroposterior: −4.5 mm, mediolateral: 1.3 mm, dorsoventral: −1.3 mm). After recovery from anesthesia, the mice were housed individually and tethered to a counterbalanced arm (Instech Laboratories) that enabled the free movement and exerted minimal weight. All mice were allowed at least 4 days of recovery from surgery and habituation to the recording conditions for at least 3 days. EEG or EMG Recording Schedule To average the possible variability in sleep during the estrous cycle, we analyzed for 4 consecutive days to assess sleep/wake behaviors before pregnancy. After recording, the female mice were housed with a male mouse. For PAH mice, female mice carrying the human angiotensinogen transgene were mated with a male mouse carrying the human renin transgene. When the vaginal plug was observed, the male mouse was removed from the cage. The day of vaginal plug detected was designated pregnant day 0. EEG or EMG was continuously recorded from PD0 through the postpartum period. Consistent with the sleep analysis before pregnancy, we evaluated 4 days for early pregnancy (pregnant days 1–4), mid pregnancy (pregnant days 8–11), and late pregnancy (pregnant days 15–18). For postpartum, we evaluated the third and fourth days after delivery because sleep/wake behaviors during the first 2 days after delivery varied widely depending on the time of delivery and the number of pups. In most cases, the mice gave birth on day 19. Consistent with a previous report on high mortality [9], 3 out of 10 PAH mice died after delivery. We excluded the sleep/wake data for mice that died or were sick during pregnancy or postpartum. Since PAH mice did not raise newborn pups and ignored them, pups of PAH mice were killed by decapitation. Administration of Angiotensin II Receptor Blocker Olmesartan (ChemScene) was dissolved in sterile water containing 0.01% NaHCO3 and 0.01% KHCO3 and administered at a dose of 15 mg/L (equivalent to 3 mg/kg BW) in drinking water from pregnant day 13 to day 19 to PAH mice. This dosage of olmesartan decreases the systolic blood pressure of PAH mice to approximately 120 mm Hg [26, 27]. Sleep Data Analysis Sleep/wakefulness was analyzed as previously described with some modifications [35]. EEG or EMG signals were amplified using a bioamplifier (#AB-611J, Nihon Koden), filtered (EEG: 0.3–300 Hz; EMG: 30–300 Hz), digitized at a sampling rate of 250 Hz, and displayed using LabView (National Instruments)-based custom-made software. The sleep/wakefulness in each 20-s epoch was classified as nonrapid eye movement (NREM) sleep (NREMS), rapid eye movement (REM) sleep (REMS), or wakefulness using MatLab (MathWorks)-based semiautomated staging system followed by visual inspection. The scoring criteria for wakefulness include high amplitude and variable EMG. NREMS was scored based on high amplitude EEG and low muscular activity. REMS is characterized by θ (6–9 Hz)-dominant EEG and low amplitude of EMG. Total time spent in wakefulness, NREMS, and REMS was derived by summing the total number of 20-s epochs in each state. Mean episode durations were determined by dividing the total time spent in each state by the number of episodes of that state. Epochs containing movement artifacts were included in the state totals but excluded from subsequent spectral analysis. EEG signals were subjected to a fast Fourier transform analysis from 1 to 30 Hz with a 1-Hz bin using MATLAB (MathWorks)-based custom software. The EEG power density in each frequency bin was expressed as a percentage of the mean total EEG power over all frequency bins and sleep/wake states. Hourly δ density during NREMS or all sleep/wake states indicates hourly averages of δ density as a ratio of the δ power (1–4 Hz) to the total EEG power (1–30 Hz) at each 20-s NREMS epoch or all epochs. EEG spectrogram was drawn using multitaper method [36] (time-half bandwidth 3, number of tapers 5, window size 30 s, overlap 0.5) implemented in the Chronux tool box [37] for MATLAB. Evaluation of BBB Permeability To examine the integrity of the BBB of PAH mice in late pregnancy, Evans blue solution was injected via the tail vein. Evans blue (WAKO, Japan) was dissolved to 2% (wt/vol) in 0.9% NaCl and passed through a 0.45 μm PES syringe filter (Starlab Scientific). Subsequently, 4 mL/kg of Evans blue solution was injected into the tail vein of PAH and normotensive control mice under anesthesia using 1.5% isoflurane in late pregnancy. Each treatment was completed within 5 min. One hour later, the mice were transcardially perfused with cold phosphate-buffered saline and then 4% paraformaldehyde/phosphate-buffered saline. The brain was removed and fixed in 4% paraformaldehyde/phosphate-buffered saline overnight at 4°C. As a normotensive control, we used pregnant human angiotensinogen transgenic mice mated with male human angiotensin transgenic mice. Since Evans blue leakages were generally very light without an apparent boundary, we counted the number of dye leakages from the brain surface, which provided more reliable and reproducible evaluations than on brain slices. We could not evaluate the size of each leakage because of its vague boundary and multiple overlaps. Statistics Total time, episode duration, episode number, EEG power density, and δ density of wild-type and PAH mice were compared during the course of pregnancy using one-way repeated measures ANOVA (analysis of variance) followed by post hoc Tukey’s test. For group comparisons among PAH, olmesartan-administered PAH, and THM, total δ density and REMS parameters were analyzed using one-way ANOVA followed by post hoc Tukey’s test. Sleep parameters of THM and wild-type mice were compared using Student’s t-test. Analyses were carried out using two-sided tests and a statistical significance level of 0.05. All analyses were performed using SPSS software version 22 (IBM, Chicago, IL). Results Sleep/Wakefulness During Pregnancy in C57BL/6 Mice Pregnant and postpartum mice showed moderate amplitude, fast EEG, and variable muscle activities during wakefulness, high amplitude, and slow waves during NREMS, and homogenous θ waves and muscle atonia during REMS, consistent with the sleep behavior observed before pregnancy (Figure 1A). During the course of pregnancy, the EEG spectrogram showed a polyphasic sleep pattern mainly during the light phase, similar to that before pregnancy. However, the difference in sleep/wake behavior between the light and dark phases was less marked in postpartum mice that cared for newborn mice (Figure 1B). Figure 1. View largeDownload slide EEG and EMG during pregnancy in wild-type and PAH mice. (A) Representative 8-s EEG and EMG during wake, NREMS, and REMS during mid and late pregnancy and postpartum in a wild-type mouse. (B) Representative daily EEG spectrogram during mid and late pregnancy and postpartum in a wild-type mouse. (C) Representative 8-s EEG and EMG during wake, NREMS, and REMS during mid and late pregnancy and postpartum in a PAH mouse. (D) Representative daily EEG spectrogram during mid and late pregnancy and postpartum in a PAH mouse. Figure 1. View largeDownload slide EEG and EMG during pregnancy in wild-type and PAH mice. (A) Representative 8-s EEG and EMG during wake, NREMS, and REMS during mid and late pregnancy and postpartum in a wild-type mouse. (B) Representative daily EEG spectrogram during mid and late pregnancy and postpartum in a wild-type mouse. (C) Representative 8-s EEG and EMG during wake, NREMS, and REMS during mid and late pregnancy and postpartum in a PAH mouse. (D) Representative daily EEG spectrogram during mid and late pregnancy and postpartum in a PAH mouse. Before pregnancy, the daily total wake time of female mice was 868 ± 29.2 min, consistent with a previous report and much longer than that of male mice [38]. During the course of pregnancy, the total daily wake time tended to decrease and was shortest during late pregnancy (710 ± 24.4 min), which was significantly shorter than that before and during early pregnancy (F = 6.622, df = 4, p < .0001; Figure 2A). Similarly, the total wake time during the dark phase during late pregnancy was significantly shorter than that before and during early pregnancy (F = 5.949, df = 4, p = .001; Figure 2A). In contrast, the total wake time during the light phase was similar to that before and during pregnancy. After delivery, the daily total wake time lengthened to levels similar to those before pregnancy. However, postpartum mice showed significantly longer total wake time during the light phase than before pregnancy (F = 3.271, df = 4, p = .025; Figure 2A). Figure 2. View largeDownload slide Sleep/wake behaviors during the course of pregnancy in wild-type mice. (A–C) Total time spent in wake (A), wake episode duration (B), and wake episode number (C) before, during, and after pregnancy in wild-type mice. (D–F) Total time spent in NREMS (D), NREMS episode duration (E), and NREMS episode number (F) before, during, and after pregnancy. (G–I) Total time spent in REMS (G), REMS episode duration (H), and REMS episode number (I) before, during, and after pregnancy. n = 8; *p < .05, **p < .01, ***p < .001. One-way repeated measures ANOVA followed by Tukey’s test. The data from individual mice are presented as the group mean ± SEM. Figure 2. View largeDownload slide Sleep/wake behaviors during the course of pregnancy in wild-type mice. (A–C) Total time spent in wake (A), wake episode duration (B), and wake episode number (C) before, during, and after pregnancy in wild-type mice. (D–F) Total time spent in NREMS (D), NREMS episode duration (E), and NREMS episode number (F) before, during, and after pregnancy. (G–I) Total time spent in REMS (G), REMS episode duration (H), and REMS episode number (I) before, during, and after pregnancy. n = 8; *p < .05, **p < .01, ***p < .001. One-way repeated measures ANOVA followed by Tukey’s test. The data from individual mice are presented as the group mean ± SEM. The wake episode duration before pregnancy was significantly longer than that during mid, late, and after pregnancy (F = 7.429, df = 4, p < .0001; Figure 2B). The wake episode duration during the light phase during late pregnancy was shorter than that before and after pregnancy (F = 4.270, df = 4, p = .008; Figure 2B). During the dark phase, the wake episode duration before pregnancy was significantly longer than that during mid and late pregnancy and after pregnancy (F = 4.898, df = 4, p = .004; Figure 2B). The number of wake episodes during late pregnancy was higher than that during any other periods (F = 10.408, df = 4, p < .0001; Figure 2C). An increased number of wake episodes during late pregnancy was observed during both the light phase (F = 6.540, df = 4, p < .0001; Figure 2C) and dark phase (F = 10.806, df = 4, p < .0001; Figure 2C). Consistent with the short total wake time during late pregnancy, the daily time spent in NREMS during late pregnancy was longer than that before and during early pregnancy (F = 8.620, df = 4, p = .0001; Figure 2D). The total NREMS time during the light phase was similar to that before, during, and after pregnancy (Figure 2D). The total NREMS time during the dark phase during late pregnancy was significantly longer than that before and during early pregnancy (F = 6.751, df = 4, p < .0001; Figure 2D). Despite the longer total NREMS time during late pregnancy, the NREMS episode duration during late pregnancy was shorter than that before and during early pregnancy (F = 5.168, df = 4, p = .003; Figure 2E). The NREMS episode duration during the light phase during late pregnancy was shorter than that before pregnancy (F = 3.796, df = 4, p = .014; Figure 2E). During the dark phase, the duration of the NREMS episode during late pregnancy was significantly shorter than that before and during early pregnancy (F = 3.456, df = 4, p = .020; Figure 2E). The number of NREMS episodes during late pregnancy was higher than that during any other periods during both light phase (F = 4.795, df = 4, p = .004) and dark phase (F = 8.697, df = 4, p < .0001; Figure 2F). Thus, an increased NREMS episode number may contribute to increased time spent in NREMS by overcoming the short NREMS episode duration. Total REMS time was not significantly changed before, during, and after pregnancy (Figure 2G). The duration of the REMS episode decreased during mid and late pregnancy and postpartum compared with that before pregnancy (F = 13.474, df = 4, p < .0001; Figure 2H). The REMS episode duration during the light phase before pregnancy was longer than that during mid, late, and after pregnancy (F = 8.652, df = 4, p < .0001; Figure 2H). During the dark phase, there was no difference in the REMS episode duration (Figure 2H). Although the number of REMS episodes was similar among pregnancy groups for 24 hr and during the light phase, the number of REMS episodes during the dark phase was larger during late pregnancy compared with that before pregnancy (F = 3.625, df = 4, p = .017; Figure 2I). The EEG spectral analysis of the wake state revealed that the power density at 4 and 5 Hz during late pregnancy was higher than that before pregnancy (F = 7.964, df = 4, p < .0001 for 4 Hz; F = 10.754, df = 4, p <.0001 for 5 Hz), whereas the power density at 10 and 11 Hz during late pregnancy was lower than that before pregnancy (F = 12.378, df = 4, p < .0001, for 10 Hz; F = 12.418, df = 4, p < .0001 for 11 Hz; Figure 3A). For NREMS, power density at 4 Hz during late pregnancy was higher than that before pregnancy and during early pregnancy (F = 13.384, df = 4, p < .0001; Figure 3B). For REMS, the power density at 4 and 5 Hz during late pregnancy was higher than that before pregnancy (F = 4.865, df = 4, p = .004 for 4 Hz; F = 10.561, df = 4, p < .0001 for 5 Hz; Figure 3C). There was no significant difference in NREMS δ density before, during, and after pregnancy (Figure 3D). Figure 3. View largeDownload slide EEG spectrum analysis during the course of pregnancy in wild-type mice. (A) Power density during wake before, during, and after pregnancy in wild-type mice. (B) Power density during NREMS before, during, and after pregnancy. (C) Power density during REMS before, during, and after pregnancy. (D) NREMS δ density before, during, and after pregnancy. n = 8; *p < .05. One-way repeated measures ANOVA followed by Tukey’s test. The data are presented as the mean ± SEM. Figure 3. View largeDownload slide EEG spectrum analysis during the course of pregnancy in wild-type mice. (A) Power density during wake before, during, and after pregnancy in wild-type mice. (B) Power density during NREMS before, during, and after pregnancy. (C) Power density during REMS before, during, and after pregnancy. (D) NREMS δ density before, during, and after pregnancy. n = 8; *p < .05. One-way repeated measures ANOVA followed by Tukey’s test. The data are presented as the mean ± SEM. Abnormal Sleep/Wakefulness of PAH Mice The systolic blood pressure of PAH mice was 100–110 mm Hg which is similar to that of wild-type mice until pregnant day 12, started to increase at pregnant day 13, up to 160 mm Hg just prior to delivery. After delivery, blood pressure started to decrease and returned to 100–110 mm Hg by the third day after delivery [9, 25]. Consistent with pregnancy-stage-specific hypertension, PAH mice showed frequent spontaneous locomotion for foraging and exploration and exhibited normal EEG and EMG signals during wakefulness, NREMS, and REMS in early and mid pregnancy (Figure 1C). In late pregnancy, all PAH mice showed much fewer and slower spontaneous locomotion but they were still able to move in response to external stimuli, such as air puff and walked to obtain food and drink. PAH mice in late pregnancy continuously exhibited abnormal EEG and EMG with increased slower activity in EEG and lower variability in EMG during wakefulness, and lower amplitude EEG and continuous EMG activities during NREMS (Figure 1C). Because of these changes, we could not determine wakefulness or NREMS for many epochs. The EEG spectrum of PAH mice in late pregnancy showed a decrease in high frequency power (>8 Hz) (Figure 1D), lacked a clear difference between the light and dark phases, and lacked any stretch of wake or sleep epochs (Figure 1D). A general slowing of EEG in PAH mice during late pregnancy was confirmed by the high total δ density during late pregnancy compared with that during mid pregnancy (Figure 4A). Figure 4. View largeDownload slide Periodic discharges of EEG during late pregnancy in PAH mice. (A) δ-Densities of all epochs during mid, late, and after pregnancy. n = 6. One-way repeated measures ANOVA followed by Tukey’s test. The data are presented as the mean ± SEM. (B and C) Representative EEG and EMG recordings showing the periodic spike-and-wave discharges of two PAH mice. (D) Enlarged spike-and-wave discharges indicated in (B and C). (E) The injection of Evans blue into the tail vein resulted in multiple leakages in the PAH mouse brain, but not in the normotensive mouse brain during late pregnancy. Figure 4. View largeDownload slide Periodic discharges of EEG during late pregnancy in PAH mice. (A) δ-Densities of all epochs during mid, late, and after pregnancy. n = 6. One-way repeated measures ANOVA followed by Tukey’s test. The data are presented as the mean ± SEM. (B and C) Representative EEG and EMG recordings showing the periodic spike-and-wave discharges of two PAH mice. (D) Enlarged spike-and-wave discharges indicated in (B and C). (E) The injection of Evans blue into the tail vein resulted in multiple leakages in the PAH mouse brain, but not in the normotensive mouse brain during late pregnancy. Surprisingly, the prominent abnormalities in EEG and EMG of PAH mice quickly disappeared within 2 days from delivery (Figure 1C and D). In parallel, the spontaneous activities of PAH mice increased, with a clear resting-active pattern consistent with the light-dark phases. The total δ density of postpartum PAH mice was reduced to a level similar to that of PAH mice in mid pregnancy (Figure 4A). Three out of six PAH mice showed periodic abnormal discharges on EEG signals during late pregnancy (Figure 4B, C, and D). Typically, periodic spike-and-wave discharges that were clearly different from background EEG activity occurred during the NREMS-like state and accompanied phasic EMG activities with jerky body movements (video in Supplementary Material), which typically lasted 10–15 min and repeated 4–6 times during the 24-hr period. To examine whether severe hypertension disrupted the integrity of the BBB of PAH mice, we injected Evans blue dye into the tail vein during late pregnancy. We observed multiple and very light Evans blue leakages ranging in diameter from less than 0.4 mm to larger than 2 mm in the cerebral cortex and cerebellum in all PAH mice as reported in acute hypertensive rats [32], but did not find any leakage in normotensive control mice during late pregnancy (Figure 4E). In PAH mice, the number of dye leakages was 4.5 ± 0.96 (mean ± SEM). Since there were many epochs that we could not properly classify into wake or NREMS during late pregnancy, we evaluated the sleep/wakefulness of PAH mice except for those in late pregnancy. The total wake time was similar among before, early, mid, and after pregnancy (Figure 5A). There was no difference in the wake episode duration (Figure 5B) and wake episode number (Figure 5C). The NREMS time was also similar before, early, mid, and after pregnancy (Figure 5D). The NREMS episode duration and episode number were similar before, early, mid, and after pregnancy (Figure 5E and F). The daily time spent in REMS of postpartum PAH mice was longer than that during early and mid pregnancy (F = 9.058, df = 3, p = .002; Figure 5G), likely reflecting a rebound phenomenon after the drastically reduced REMS time during late pregnancy (see below). An increased total REMS during postpartum was observed during the light phase (F = 11.141, df = 3, p = .0008; Figure 5G), but not during the dark phase. We observed no difference in the REMS episode duration (Figure 5H) or REMS episode number (Figure 5I). Figure 5. View largeDownload slide Sleep/wake behaviors during the course of pregnancy in PAH mice, except for during late pregnancy. (A–C) Total time spent in wake (A), wake episode duration (B), and wake episode number (C) before, during early and mid pregnancy, and postpartum in PAH mice. (D–F) Total time spent in NREM sleep (NREMS) (D), NREMS episode duration (E), and NREMS episode number (F) before, during early and mid-pregnancy, and postpartum in PAH mice. (G–I) Total time spent in REM sleep (REMS) (G), REMS episode duration (H), and REMS episode number (I) before, during early and mid pregnancy, and postpartum in PAH mice. NREMS during late pregnancy is not presented because there are many intermediate epochs that are not classified into NREMS or wakefulness. n = 6. *p < .05. One-way repeated measures ANOVA followed by Tukey’s test. The data from individual mice are presented as the group mean ± SEM. Figure 5. View largeDownload slide Sleep/wake behaviors during the course of pregnancy in PAH mice, except for during late pregnancy. (A–C) Total time spent in wake (A), wake episode duration (B), and wake episode number (C) before, during early and mid pregnancy, and postpartum in PAH mice. (D–F) Total time spent in NREM sleep (NREMS) (D), NREMS episode duration (E), and NREMS episode number (F) before, during early and mid-pregnancy, and postpartum in PAH mice. (G–I) Total time spent in REM sleep (REMS) (G), REMS episode duration (H), and REMS episode number (I) before, during early and mid pregnancy, and postpartum in PAH mice. NREMS during late pregnancy is not presented because there are many intermediate epochs that are not classified into NREMS or wakefulness. n = 6. *p < .05. One-way repeated measures ANOVA followed by Tukey’s test. The data from individual mice are presented as the group mean ± SEM. We further examined the EEG power spectrum of PAH mice, except for late pregnancy. For wakefulness, the power density at 3 Hz during mid pregnancy was higher than that during postpartum (F = 5.783, df = 3, p = .011) and the power density at 6, 7, 9, 12, 13, and 14 Hz after pregnancy was higher than that before pregnancy (Figure 6A). The power density of PAH mice did not exhibit any significant difference during NREMS (Figure 6B). For REMS, the power density during mid pregnancy was lower at 6 Hz (F = 10.101, df = 3, p = .001) and 7 Hz (F = 23.156, df = 3, p < .0001), and higher at 9 Hz (F = 14.724, df = 3, p < .0001) and 10 Hz (F = 9.019, df = 3, p = .002 for 10 Hz) compared with that before pregnancy (Figure 6C). The NREMS δ density tended to be lower in postpartum PAH mice (Figure 6D). Figure 6. View largeDownload slide EEG spectrum analysis during the course of pregnancy in PAH mice, except for during late pregnancy. (A) Power density during wake before, during early and mid pregnancy, and postpartum in PAH mice. (B) Power density during NREMS before, during early and mid pregnancy, and postpartum. (C) Power density during REMS before, during early and mid-pregnancy, and postpartum. (D) NREMS δ density before, during early and mid pregnancy, and postpartum. n = 6; *p < .05. One-way ANOVA followed by Tukey’s test. The data are presented as the mean ± SEM. Figure 6. View largeDownload slide EEG spectrum analysis during the course of pregnancy in PAH mice, except for during late pregnancy. (A) Power density during wake before, during early and mid pregnancy, and postpartum in PAH mice. (B) Power density during NREMS before, during early and mid pregnancy, and postpartum. (C) Power density during REMS before, during early and mid-pregnancy, and postpartum. (D) NREMS δ density before, during early and mid pregnancy, and postpartum. n = 6; *p < .05. One-way ANOVA followed by Tukey’s test. The data are presented as the mean ± SEM. Olmesartan Alleviated Abnormal Sleep/Wakefulness of PAH Mice Olmesartan-administered PAH mice had a systolic blood pressure of approximately 120 mm Hg, which does not affect the integrity of the BBB [39]. In late pregnancy, olmesartan-administered PAH mice showed spontaneous locomotion similar to pregnant wild-type mice. The EEG or EMG signaling and daily EEG spectrogram of olmesartan-administered PAH mice did not show the abnormalities (Figure 7A) observed in PAH mice (Figure 1D). Olmesartan treatment also decreased the total δ density of PAH mice to that of nonpregnant PAH (human angiotensinogen–transgenic) mice (Figure 7B). Figure 7. View largeDownload slide EEGs and sleep/wakefulness of olmesartan-administered PAH mice. (A) Representative daily EEG spectrogram during late pregnancy in a PAH mouse that was administered olmesartan. (B) δ-Densities of all epochs of nonpregnant PAH (normotensive) mice (n = 6), PAH mice (n = 6), and olmesartan-administered PAH mice (n = 5) during late pregnancy. (C–E) Total time spent in REMS (C), REMS episode duration (D), and REMS episode number (E) in nonpregnant PAH mice, olmesartan-administered PAH mice, and THM. **p < .01, ***p < .001. One-way ANOVA followed by Tukey’s test. The data are presented as the group mean ± SEM. Figure 7. View largeDownload slide EEGs and sleep/wakefulness of olmesartan-administered PAH mice. (A) Representative daily EEG spectrogram during late pregnancy in a PAH mouse that was administered olmesartan. (B) δ-Densities of all epochs of nonpregnant PAH (normotensive) mice (n = 6), PAH mice (n = 6), and olmesartan-administered PAH mice (n = 5) during late pregnancy. (C–E) Total time spent in REMS (C), REMS episode duration (D), and REMS episode number (E) in nonpregnant PAH mice, olmesartan-administered PAH mice, and THM. **p < .01, ***p < .001. One-way ANOVA followed by Tukey’s test. The data are presented as the group mean ± SEM. Since we unequivocally identified REMS in PAH mice during late pregnancy based on the appearance of the θ wave (6–9 Hz) and muscle atonia (Figure 1C), we examined how the administration of olmesartan affects REMS in PAH mice. The total REMS time of PAH mice was only 4.3 ± 1.9 min (mean ± SEM), but olmesartan-administered PAH mice showed a significantly longer REMS time (F = 17.230, df = 2, p < .0001, Figure 7C). Olmesartan-administered PAH mice showed a REMS episode duration similar to PAH mice (Figure 7D) and increased numbers of REMS episodes (F = 25.613, df = 2, p < .0001, Figure 7E). Thus, an increased number of REMS episodes contributes to the increased total REMS time of olmesartan-administered PAH mice. Olmesartan treatment did not alter the total time, episode duration, or episode number of wakefulness, NREMS, or REMS in male wild-type mice (data not shown). Sleep/Wakefulness of THM Lastly, we examined the sleep/wakefulness of nonpregnant female THM. The total wake time of female THM was similar to that of female wild-type mice (Figure 8A). There was no difference in the wake episode duration (Figure 8B) or the wake episode number (Figure 8C). The total NREMS time of female THM was similar to that of female wild-type mice (Figure 8D). There was no difference in the NREMS episode duration (Figure 8E) or the NREMS episode number (Figure 8F). The total REMS time of female THM was shorter than that of female wild-type mice (Figure 8G). A decreased time spent in REMS was associated with a reduced REMS episode duration (Figure 8H), but not with significant changes in the REMS episode number (Figure 8I). Total REMS time, REMS episode duration, and REM episode number of THM were similar to those of olmesartan-administered PAH mice (Figure 7C, D, and E). Figure 8. View largeDownload slide Sleep/wake behaviors of THM. (A–C) Total time spent in wake (A), wake episode duration (B), and wake episode number (C) of THM and wild-type mice. (D–F) Total time spent in NREM sleep (NREMS) (D), NREMS episode duration (E), and NREMS episode number (F). (G–I) Total time spent in REM sleep (REMS) (G), REMS episode duration (H), and REMS episode number (I). Five THM and eight wild-type mice. *p < .05, **p < .01, ***p < .001. Two-tailed t-test. The data from individual mice are presented as the group mean ± SEM. Figure 8. View largeDownload slide Sleep/wake behaviors of THM. (A–C) Total time spent in wake (A), wake episode duration (B), and wake episode number (C) of THM and wild-type mice. (D–F) Total time spent in NREM sleep (NREMS) (D), NREMS episode duration (E), and NREMS episode number (F). (G–I) Total time spent in REM sleep (REMS) (G), REMS episode duration (H), and REMS episode number (I). Five THM and eight wild-type mice. *p < .05, **p < .01, ***p < .001. Two-tailed t-test. The data from individual mice are presented as the group mean ± SEM. Discussion To the best of our knowledge, the present study is the first report on sleep/wakefulness during the course of pregnancy in mice. This study also proposes that PAH mice may be an animal model of eclampsia. Sleep/Wakefulness During and After Pregnancy The daily time spent in wakefulness decreased and the total NREMS time increased during late pregnancy, primarily reflecting increased NREMS during the dark phase, consistent with the long NREMS in pregnant rats [40, 41]. Approaching delivery, an increased physical load such as the weight of embryos, extended uterus, and increasing abdominal pressure, may necessitate more sleep and rest in pregnant mice. Sleep/wakefulness during late pregnancy was also characterized by frequent switches between wakefulness and NREMS, resulting in an increased episode number and short episode duration of both wakefulness and NREMS during both the light and dark phases. These findings are consistent with previous reports on pregnant humans and rats [42]. Fragmented sleep may be due to spontaneous movements of the embryos in the uterus and/or the pressure of the growing uterus on neighboring tissues and organs. Pregnancy affected REMS differently from wakefulness and NREMS. The daily time spent in REMS and the daily number of REMS episodes were not altered during the course of pregnancy, which is consistent with previously reported for pregnant rats [40, 43]. A short REMS episode duration was recognized from mid pregnancy through postpartum. Altered REMS, independent of NREMS changes, supports the idea that the regulatory system for REMS functions differently from that for NREMS [44]. The EEG spectrum analysis of REMS showed pregnancy-induced changes at 6 and 7 Hz from the early stages of pregnancy, although the physiological significance of this finding is not clear. In addition to the physical effects of the growing uterus and embryos during pregnancy, hormonal changes may affect sleep/wake behaviors during pregnancy. Estrogen and progesterone increase as pregnancy progresses and then abruptly decrease after parturition [45]. Estrogen and progesterone generally increase wakefulness and decrease sleep, as indicated by the increased wakefulness of ovariectomized mice replaced with estradiol alone or with both estradiol and progesterone [4, 18, 46–49]. Consistently, receptors for estrogen are abundant in the preoptic area, which is involved in sleep regulation [50]. Thus, increased estrogen and progesterone during pregnancy may work to enhance wakefulness. However, this interpretation could be too simple since progesterone has also been reported to reduce wakefulness [51]. Importantly, the sleep/wake behaviors of postpartum mice were similar to nonpregnant mice, despite lactation and increased prolactin and oxytocin. Mice that showed multiple altered parameters in wakefulness, NREMS, and REMS during late pregnancy quickly returned to normal sleep/wakefulness, excluding the decreased episode duration of wakefulness and REMS. There have been several reports on increased REMS by prolactin [52], but peptide hormones may be less permeable to the brain and less influential on brain functions compared with gonadal steroid hormones. PAH Mice as an Animal Model for Eclampsia The present study also supports PAH mice as an animal model for eclampsia, based on seizures, altered sleep/wakefulness, fewer spontaneous activities, and high perinatal mortality. There is no known animal model for eclampsia, although there have been pharmacological and genetic interventions to develop an animal model for preeclampsia [53–55], including PAH mice. Angiotensin II is a major factor inducing hypertension in the renin–angiotensin–aldosterone system [56]. Consistently, angiotensin II type-1a receptor is required for the high blood pressure in PAH mice [57]. PAH mice exhibit elevated blood pressure with severe proteinuria during late pregnancy, which are diagnostic criteria for human preeclampsia [19, 20]; however, these animals have not been examined for neuropsychiatric phenotypes. The present study showed that PAH mice exhibited markedly abnormal vigilance states with a generalized slowing of EEG during late pregnancy. Although PAH mice still slowly responded to external stimuli and obtained food and water during late pregnancy, these animals rarely showed spontaneous and swift movements, staying in the nest area almost all day. PAH mice showed normal REMS characterized by θ wave and muscle atonia during late pregnancy, but the total REMS time drastically decreased compared with PAH mice during mid pregnancy and pregnant wild-type mice. We also observed that half of the PAH mice showed spike-and-wave discharges during late pregnancy, based on EEG or EMG recording supplemented with video recording. Surprisingly, postpartum PAH mice quickly recovered from the general slowing of EEG and had a normal time spent in wakefulness and NREMS. Similarly, the most commonly observed EEG abnormality in human eclampsia is generalized or focal slowing of EEG [28, 58, 59], and this abnormal EEG normalizes after the release from hypertension [59]. Thus, PAH mice bear a symptomatic resemblance to human eclampsia. Postpartum PAH mice showed a longer total REMS time, suggesting that the homeostatic mechanism may compensate for the diminished total REMS during late pregnancy. Since PAH mice do not raise newborn pups and ignore them, we removed the newborn pups. It is unlikely that the removal of pups largely altered the sleep/wake behavior because PAH mice simply neglected the pups and did not show any interest in them. All PAH mice examined herein showed disruption of the BBB, as assessed by the intravenous injection of Evans blue dye during late pregnancy. Brain circulation has an autoregulatory mechanism to maintain stable cerebral blood flow between mean arterial pressures of 60 and 120 mm Hg [39]. However, blood pressures higher than the upper limit of cerebral vascular autoregulation can lead to disruption of the BBB and vasogenic edema, eventually resulting in hypertensive encephalopathy [39]. An acute increase in arterial blood pressure by 80 mm Hg disrupts the BBB integrity in rats [60]. Four hours of intra-abdominal hypertension resulted in the leakage of Evans blue dye into the brain parenchyma, which was not observed 1 hr after release from abdominal hypertension [61]. In addition, the administration of olmesartan alleviated sleep/wake behavior abnormalities of PAH mice during late pregnancy such as general slowing of EEG, loss of the circadian change in sleep/wakefulness, and a drastic reduction in REMS. Thus, the acute and severe increase in blood pressure may solely be responsible for the BBB disruption in PAH mice and subsequently lead to abnormal sleep/wake behaviors. Neuroimaging studies of eclampsia patients revealed posterior reversible encephalopathy syndrome, which refers to reversible vasogenic cerebral edema in the posterior region of the cerebral cortex accompanied by acute neuropsychiatric symptoms [28–31]. Thus, PAH mice exhibit a pathophysiologic mechanism similar to human eclampsia during late pregnancy. Sleep/Wakefulness of THM THM showed a reduced REMS time and normal NREMS time compared with wild-type mice, suggesting that chronic and moderate hypertension suppresses REMS and does not affect NREMS. Interestingly, the REMS time of THM was similar to that of olmesartan-administered PAH mice. Since THM and olmesartan-administered PAH mice showed similar levels of hypertension, increased blood pressure may result in decreased REMS time, although further studies are necessary to elucidate the mechanistic link between hypertension and REMS regulation. We cannot deny the possibility that angiotensin produced in the brain affects REMS since both the human renin transgene and human angiotensinogen transgene are weakly or mildly expressed in the brain [62, 63] where angiotensin-converting enzymes and angiotensin II receptors are expressed [64, 65]. Limitations Although various kinds of preeclampsia model mice, including PAH mice, have been reported, none of them fully reproduce human preeclampsia in terms of pathogenesis, symptoms, and responses to pharmacological intervention. To evaluate the validity of PAH mice as a model of eclampsia, further studies using magnesium sulfate, the most potent anticonvulsant for eclampsia patients [22], may be necessary. Furthermore, we cannot exclude the possibility that humoral factors are involved in the altered sleep/wakefulness of PAH mice independently of high blood pressure. Consistent with abnormal placentation and increased secretion of angiogenic factors in human preeclampsia [21], PAH mice exhibit abnormal placental angiogenesis and intrauterine growth retardation [57, 66], which may increase humoral factors and reactive oxygen species to disrupt the BBB [67]. Indeed, modification of the Keap1-Nrf2 pathway, which functions as a defense system for oxidative stress, enhances placental angiogenesis and survival rates of PAH mice and their fetuses independently of high blood pressure [68]. Another limitation of the present study was the modest number of mice examined. Major findings, such as long NREMS time during late pregnancy in wild-type mice and the abnormal sleep/wakefulness of PAH mice during late pregnancy, were sufficiently robust with small interindividual differences, which can be replicated with good reproducibility. In addition, we failed to obtain pregnant female THM and thus could not examine sleep/wake behaviors of pregnant THM compared with PAH mice. Although we did not examine the fertility rate of THM in detail, we conjecture that chronic hypertension or enhanced renin–angiotensin system may decrease the fertility of THM. In summary, the present study revealed a similarity in the sleep changes during pregnancy between humans and mice [6, 7], which provides a basis for future studies on sleep regulation in animal models for human diseases during pregnancy. We also suggest that PAH mice may be a useful model for the development of therapeutic intervention for eclampsia. Supplementary Material Supplementary material is available at SLEEP online. Funding This work was supported by the World Premier International Research Center Initiative from MEXT (to MY), JSPS KAKENHI (Grant Number 17H06095 to MY and HF; 16K15187 to HF; 26507003 to CM and HF; 15K18966), MEXT KAKENHI (Grant Number 15H05935 to MY and HF), Funding Program for World-Leading Innovative R&D on Science and Technology (FIRST program) from JSPS (to MY), Research grant from Uehara Memorial Foundation research grant (to MY), and Research grant from Takeda Science Foundation research grant (to MY). Disclosure Statement This was not an industry-supported study. MY is a former Investigator of the Howard Hughes Medical Institute. Acknowledgments We thank all Yanagisawa/Funato lab and IIIS members for discussion and comments on this manuscript and Go Taniguchi for his comments on spike-wave seizures. References 1. Lee KA. 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Abstract

Abstract Study Objectives In humans and other mammals, sleep is altered during pregnancy. However, no studies have been conducted on sleep/wakefulness during pregnancy in mice. In this study, we examined sleep/wakefulness in female C57BL/6 mice during pregnancy. We also examined sleep/wake behaviors in an animal model of preeclampsia, pregnancy-associated hypertensive (PAH) mice, in which increased angiotensin causes hypertension. Methods Sleep/wake behaviors of female C57BL/6 and PAH mice were examined based on electroencephalogram (EEG) or electromyogram recordings before, during, and after pregnancy. To examine whether high blood pressure disrupts the integrity of the blood–brain barrier in PAH mice, Evans blue dye was injected intravenously. Angiotensin II receptor blocker (olmesartan)-administered PAH mice and female Tsukuba hypertensive mice were also examined. Results C57BL/6 mice showed a decreased total wake time and increased nonrapid eye movement (NREM) sleep time during late pregnancy. Rapid eye movement (REM) sleep time did not change during the course of pregnancy. PAH mice exhibited a general slowing of EEG during late pregnancy and subsequently returned to apparently normal sleep/wakefulness after delivery. All PAH mice exhibited multiple focal leakages of Evans blue dye in the brain. Spike-and-wave discharges were observed in 50% of PAH mice. Olmesartan-administered PAH mice did not show general slowing of EEG. Tsukuba hypertensive mice showed a normal time spent in wakefulness and NREM sleep and a decreased total REM sleep time. Conclusions This study showed pregnant-stage-specific changes in sleep/wakefulness in C57BL/6 mice. Furthermore, PAH mice may be useful as an animal model for eclampsia. pregnancy, hypertension, animal models, blood pressure Statement of Significance Despite the broad use of mice for preclinical studies, to the best of our knowledge, there have been no studies on sleep/wakefulness during pregnancy in mice. Total nonrapid eye movement sleep time was increased during the course of pregnancy. Pregnancy-associated hypertensive (PAH) mice exhibited a general slowing of electroencephalogram during late pregnancy. All PAH mice exhibited disruption of the blood–brain barrier, and half of the PAH mice showed spike-and-wave discharges. The current findings showed similar changes during pregnancy between humans and mice, which provides a basis for preclinical studies for human diseases during pregnancy. PAH mice may be a useful model for the development of therapeutic intervention for eclampsia. Introduction Sleep is altered during pregnancy, and different sleep disturbances are associated with each trimester, likely reflecting a wide range of physiological and hormonal changes [1–5]. However, few sleep studies of pregnant women using polysomnography have been reported [6–8]. Mice are suitable model species for examining sleep/wakefulness during pregnancy because they have a gestation period of less than 3 weeks. Furthermore, many gene-modified mice have been developed as animal models of human diseases, including preeclampsia [9] and the sleep disorder narcolepsy [10], which enables the examination of how pregnancy affects the phenotypes of animal models. To the best of our knowledge, however, there have been no published studies on sleep/wakefulness during pregnancy in mice. Continued progress in genetic engineering, including genome editing, has provided rapid progress in the genetics of sleep [11]. By using optogenetic and pharmacogenetic approaches, the understanding of neural circuitries regulating sleep/wake behaviors has rapidly progressed [12]. Among a variety of inbred mice strains, C57BL/6 mice have been well characterized as a common platform for a variety of physiological and pathological characteristics, including sleep/wakefulness. Thus, a large-scale gene knockout program has been developed as a resource using C57BL/6 mice [13], and the subsequent phenomics program has continued to uncover the phenotypes of gene knockout mice [14]. Although most behavioral studies have been conducted using male C57BL/6 mice, male-biased research results may hinder the characterization of sexually dimorphic effects, which may lead to an underestimation of risks for females [15]. Indeed, those few studies on sleep/wakefulness in female mice did show sex differences in sleep [16–18]. Preeclampsia is a pregnancy complication characterized by hypertension and proteinuria after 20 weeks of gestation in patients without hypertension prior to pregnancy [19–22]. Eclampsia is a severe phase of preeclampsia with the occurrence of convulsions and/or disturbed consciousness [20, 23]. The World Health Organization Multicounty Survey reported that preeclampsia and eclampsia develop in 2%–3% and 0.3% of pregnancies, respectively [24]. Although women with preeclampsia have a 3.7-fold increased risk of death compared with pregnant women without preeclampsia, women with eclampsia have a 42-fold increase [24]. As an animal model of preeclampsia, pregnancy-associated hypertensive (PAH) mice have been developed by crossing female mice carrying the human angiotensinogen transgene with male mice carrying the human renin transgene [9]. PAH mice utilize the “species barrier” between humans and mice in terms of angiotensinogen processing by renin. Human renin cannot process murine angiotensinogen, whereas mouse renin cannot cleave human angiotensinogen into angiotensin I. Thus, only when human renin exits, human angiotensinogen is processed into angiotensin I. Since the amino acid sequence of angiotensin I is the same between human and mouse, angiotensin I in mice is subsequently processed into angiotensin II via the removal of two amino acids at the carboxyl terminus by angiotensin-converting enzyme [25], resulting in high blood pressure. In PAH mice, when a female angiotensinogen transgenic mouse conceives embryos carrying the human renin transgene, the pregnant mouse shows hypertension and proteinuria in late pregnancy because the human renin produced from the placenta processes human angiotensinogen into angiotensin I [9] and subsequently angiotensin II. Increased angiotensin II causes acute and severe hypertension with proteinuria in PAH mice [9, 25], which can be alleviated by olmesartan, an angiotensin II receptor blocker, from 150–160 to 120 mm Hg in late pregnancy, which was still higher than that of normal pregnant mice, 100–110 mm Hg [26, 27]. As eclampsia patients often exhibit hyperintensity on T2-weighted brain MRI indicative of vasogenic edema [28–31], acute severe hypertension in PAH mice may disrupt the blood–brain barrier (BBB) [32], but PAH mice have not been examined for the integrity of the BBB. Another line of hypertension model mice, called Tsukuba hypertensive mice (THM), carries both human renin and human angiotensinogen transgenes and suffers from moderate and chronic hypertension, 20–30 mm Hg higher than that of wild-type mice [33, 34]. Since the systolic blood pressure of THM is similar to that of olmesartan-administered PAH mice, the sleep/wake behaviors of these mice allow us to examine how mild-to-moderate hypertension affects sleep. In the present study, we evaluated sleep/wake behaviors during pregnancy in C57BL/6 mice by analyzing the electroencephalogram (EEG) or electromyogram (EMG), which provides a basis for future studies on the mechanism of sleep regulation during pregnancy. We also examined the sleep/wake behaviors of PAH mice and nonpregnant female THM. PAH mice showed abnormal sleep/wakefulness frequently accompanied by spike-wave discharges during late pregnancy, which was alleviated by the administration of olmesartan, implicating PAH mice as an animal model of eclampsia. Methods Animals Female C57BL/6J mice (CLEA Japan) were used in this study. PAH mice and nonpregnant THM were maintained on a C57BL/6 background. Mice were housed under a 12:12-hr light/dark cycle. Food and water were delivered ad libitum. A water gel pack (Napa Nector, 8 oz., System Engineering Lab Group Inc.) was used for a water source because this pack allows both pregnant wild-type and PAH mice to intake water easily. All procedures were approved by the Institutional Animal Care and Use Committee of the University of Tsukuba. EEG or EMG Electrode Implantation Surgery At 8–12 weeks of age (19–23 g), female wild-type, THM, and PAH mice were anesthetized using isoflurane (3%–4% for induction and 1%–2% for maintenance). Each electrode has four electrode pins and two wires. Four electrode pins were lowered to the dura under stereotaxic control, and two flexible wires for EMG recording were inserted in the neck muscles and subsequently attached to the skull using dental cement. The electrodes for EEG signals were positioned over the frontal and occipital cortices (anteroposterior: 0.5 mm, mediolateral: 1.3 mm, dorsoventral: −1.3 mm and anteroposterior: −4.5 mm, mediolateral: 1.3 mm, dorsoventral: −1.3 mm). After recovery from anesthesia, the mice were housed individually and tethered to a counterbalanced arm (Instech Laboratories) that enabled the free movement and exerted minimal weight. All mice were allowed at least 4 days of recovery from surgery and habituation to the recording conditions for at least 3 days. EEG or EMG Recording Schedule To average the possible variability in sleep during the estrous cycle, we analyzed for 4 consecutive days to assess sleep/wake behaviors before pregnancy. After recording, the female mice were housed with a male mouse. For PAH mice, female mice carrying the human angiotensinogen transgene were mated with a male mouse carrying the human renin transgene. When the vaginal plug was observed, the male mouse was removed from the cage. The day of vaginal plug detected was designated pregnant day 0. EEG or EMG was continuously recorded from PD0 through the postpartum period. Consistent with the sleep analysis before pregnancy, we evaluated 4 days for early pregnancy (pregnant days 1–4), mid pregnancy (pregnant days 8–11), and late pregnancy (pregnant days 15–18). For postpartum, we evaluated the third and fourth days after delivery because sleep/wake behaviors during the first 2 days after delivery varied widely depending on the time of delivery and the number of pups. In most cases, the mice gave birth on day 19. Consistent with a previous report on high mortality [9], 3 out of 10 PAH mice died after delivery. We excluded the sleep/wake data for mice that died or were sick during pregnancy or postpartum. Since PAH mice did not raise newborn pups and ignored them, pups of PAH mice were killed by decapitation. Administration of Angiotensin II Receptor Blocker Olmesartan (ChemScene) was dissolved in sterile water containing 0.01% NaHCO3 and 0.01% KHCO3 and administered at a dose of 15 mg/L (equivalent to 3 mg/kg BW) in drinking water from pregnant day 13 to day 19 to PAH mice. This dosage of olmesartan decreases the systolic blood pressure of PAH mice to approximately 120 mm Hg [26, 27]. Sleep Data Analysis Sleep/wakefulness was analyzed as previously described with some modifications [35]. EEG or EMG signals were amplified using a bioamplifier (#AB-611J, Nihon Koden), filtered (EEG: 0.3–300 Hz; EMG: 30–300 Hz), digitized at a sampling rate of 250 Hz, and displayed using LabView (National Instruments)-based custom-made software. The sleep/wakefulness in each 20-s epoch was classified as nonrapid eye movement (NREM) sleep (NREMS), rapid eye movement (REM) sleep (REMS), or wakefulness using MatLab (MathWorks)-based semiautomated staging system followed by visual inspection. The scoring criteria for wakefulness include high amplitude and variable EMG. NREMS was scored based on high amplitude EEG and low muscular activity. REMS is characterized by θ (6–9 Hz)-dominant EEG and low amplitude of EMG. Total time spent in wakefulness, NREMS, and REMS was derived by summing the total number of 20-s epochs in each state. Mean episode durations were determined by dividing the total time spent in each state by the number of episodes of that state. Epochs containing movement artifacts were included in the state totals but excluded from subsequent spectral analysis. EEG signals were subjected to a fast Fourier transform analysis from 1 to 30 Hz with a 1-Hz bin using MATLAB (MathWorks)-based custom software. The EEG power density in each frequency bin was expressed as a percentage of the mean total EEG power over all frequency bins and sleep/wake states. Hourly δ density during NREMS or all sleep/wake states indicates hourly averages of δ density as a ratio of the δ power (1–4 Hz) to the total EEG power (1–30 Hz) at each 20-s NREMS epoch or all epochs. EEG spectrogram was drawn using multitaper method [36] (time-half bandwidth 3, number of tapers 5, window size 30 s, overlap 0.5) implemented in the Chronux tool box [37] for MATLAB. Evaluation of BBB Permeability To examine the integrity of the BBB of PAH mice in late pregnancy, Evans blue solution was injected via the tail vein. Evans blue (WAKO, Japan) was dissolved to 2% (wt/vol) in 0.9% NaCl and passed through a 0.45 μm PES syringe filter (Starlab Scientific). Subsequently, 4 mL/kg of Evans blue solution was injected into the tail vein of PAH and normotensive control mice under anesthesia using 1.5% isoflurane in late pregnancy. Each treatment was completed within 5 min. One hour later, the mice were transcardially perfused with cold phosphate-buffered saline and then 4% paraformaldehyde/phosphate-buffered saline. The brain was removed and fixed in 4% paraformaldehyde/phosphate-buffered saline overnight at 4°C. As a normotensive control, we used pregnant human angiotensinogen transgenic mice mated with male human angiotensin transgenic mice. Since Evans blue leakages were generally very light without an apparent boundary, we counted the number of dye leakages from the brain surface, which provided more reliable and reproducible evaluations than on brain slices. We could not evaluate the size of each leakage because of its vague boundary and multiple overlaps. Statistics Total time, episode duration, episode number, EEG power density, and δ density of wild-type and PAH mice were compared during the course of pregnancy using one-way repeated measures ANOVA (analysis of variance) followed by post hoc Tukey’s test. For group comparisons among PAH, olmesartan-administered PAH, and THM, total δ density and REMS parameters were analyzed using one-way ANOVA followed by post hoc Tukey’s test. Sleep parameters of THM and wild-type mice were compared using Student’s t-test. Analyses were carried out using two-sided tests and a statistical significance level of 0.05. All analyses were performed using SPSS software version 22 (IBM, Chicago, IL). Results Sleep/Wakefulness During Pregnancy in C57BL/6 Mice Pregnant and postpartum mice showed moderate amplitude, fast EEG, and variable muscle activities during wakefulness, high amplitude, and slow waves during NREMS, and homogenous θ waves and muscle atonia during REMS, consistent with the sleep behavior observed before pregnancy (Figure 1A). During the course of pregnancy, the EEG spectrogram showed a polyphasic sleep pattern mainly during the light phase, similar to that before pregnancy. However, the difference in sleep/wake behavior between the light and dark phases was less marked in postpartum mice that cared for newborn mice (Figure 1B). Figure 1. View largeDownload slide EEG and EMG during pregnancy in wild-type and PAH mice. (A) Representative 8-s EEG and EMG during wake, NREMS, and REMS during mid and late pregnancy and postpartum in a wild-type mouse. (B) Representative daily EEG spectrogram during mid and late pregnancy and postpartum in a wild-type mouse. (C) Representative 8-s EEG and EMG during wake, NREMS, and REMS during mid and late pregnancy and postpartum in a PAH mouse. (D) Representative daily EEG spectrogram during mid and late pregnancy and postpartum in a PAH mouse. Figure 1. View largeDownload slide EEG and EMG during pregnancy in wild-type and PAH mice. (A) Representative 8-s EEG and EMG during wake, NREMS, and REMS during mid and late pregnancy and postpartum in a wild-type mouse. (B) Representative daily EEG spectrogram during mid and late pregnancy and postpartum in a wild-type mouse. (C) Representative 8-s EEG and EMG during wake, NREMS, and REMS during mid and late pregnancy and postpartum in a PAH mouse. (D) Representative daily EEG spectrogram during mid and late pregnancy and postpartum in a PAH mouse. Before pregnancy, the daily total wake time of female mice was 868 ± 29.2 min, consistent with a previous report and much longer than that of male mice [38]. During the course of pregnancy, the total daily wake time tended to decrease and was shortest during late pregnancy (710 ± 24.4 min), which was significantly shorter than that before and during early pregnancy (F = 6.622, df = 4, p < .0001; Figure 2A). Similarly, the total wake time during the dark phase during late pregnancy was significantly shorter than that before and during early pregnancy (F = 5.949, df = 4, p = .001; Figure 2A). In contrast, the total wake time during the light phase was similar to that before and during pregnancy. After delivery, the daily total wake time lengthened to levels similar to those before pregnancy. However, postpartum mice showed significantly longer total wake time during the light phase than before pregnancy (F = 3.271, df = 4, p = .025; Figure 2A). Figure 2. View largeDownload slide Sleep/wake behaviors during the course of pregnancy in wild-type mice. (A–C) Total time spent in wake (A), wake episode duration (B), and wake episode number (C) before, during, and after pregnancy in wild-type mice. (D–F) Total time spent in NREMS (D), NREMS episode duration (E), and NREMS episode number (F) before, during, and after pregnancy. (G–I) Total time spent in REMS (G), REMS episode duration (H), and REMS episode number (I) before, during, and after pregnancy. n = 8; *p < .05, **p < .01, ***p < .001. One-way repeated measures ANOVA followed by Tukey’s test. The data from individual mice are presented as the group mean ± SEM. Figure 2. View largeDownload slide Sleep/wake behaviors during the course of pregnancy in wild-type mice. (A–C) Total time spent in wake (A), wake episode duration (B), and wake episode number (C) before, during, and after pregnancy in wild-type mice. (D–F) Total time spent in NREMS (D), NREMS episode duration (E), and NREMS episode number (F) before, during, and after pregnancy. (G–I) Total time spent in REMS (G), REMS episode duration (H), and REMS episode number (I) before, during, and after pregnancy. n = 8; *p < .05, **p < .01, ***p < .001. One-way repeated measures ANOVA followed by Tukey’s test. The data from individual mice are presented as the group mean ± SEM. The wake episode duration before pregnancy was significantly longer than that during mid, late, and after pregnancy (F = 7.429, df = 4, p < .0001; Figure 2B). The wake episode duration during the light phase during late pregnancy was shorter than that before and after pregnancy (F = 4.270, df = 4, p = .008; Figure 2B). During the dark phase, the wake episode duration before pregnancy was significantly longer than that during mid and late pregnancy and after pregnancy (F = 4.898, df = 4, p = .004; Figure 2B). The number of wake episodes during late pregnancy was higher than that during any other periods (F = 10.408, df = 4, p < .0001; Figure 2C). An increased number of wake episodes during late pregnancy was observed during both the light phase (F = 6.540, df = 4, p < .0001; Figure 2C) and dark phase (F = 10.806, df = 4, p < .0001; Figure 2C). Consistent with the short total wake time during late pregnancy, the daily time spent in NREMS during late pregnancy was longer than that before and during early pregnancy (F = 8.620, df = 4, p = .0001; Figure 2D). The total NREMS time during the light phase was similar to that before, during, and after pregnancy (Figure 2D). The total NREMS time during the dark phase during late pregnancy was significantly longer than that before and during early pregnancy (F = 6.751, df = 4, p < .0001; Figure 2D). Despite the longer total NREMS time during late pregnancy, the NREMS episode duration during late pregnancy was shorter than that before and during early pregnancy (F = 5.168, df = 4, p = .003; Figure 2E). The NREMS episode duration during the light phase during late pregnancy was shorter than that before pregnancy (F = 3.796, df = 4, p = .014; Figure 2E). During the dark phase, the duration of the NREMS episode during late pregnancy was significantly shorter than that before and during early pregnancy (F = 3.456, df = 4, p = .020; Figure 2E). The number of NREMS episodes during late pregnancy was higher than that during any other periods during both light phase (F = 4.795, df = 4, p = .004) and dark phase (F = 8.697, df = 4, p < .0001; Figure 2F). Thus, an increased NREMS episode number may contribute to increased time spent in NREMS by overcoming the short NREMS episode duration. Total REMS time was not significantly changed before, during, and after pregnancy (Figure 2G). The duration of the REMS episode decreased during mid and late pregnancy and postpartum compared with that before pregnancy (F = 13.474, df = 4, p < .0001; Figure 2H). The REMS episode duration during the light phase before pregnancy was longer than that during mid, late, and after pregnancy (F = 8.652, df = 4, p < .0001; Figure 2H). During the dark phase, there was no difference in the REMS episode duration (Figure 2H). Although the number of REMS episodes was similar among pregnancy groups for 24 hr and during the light phase, the number of REMS episodes during the dark phase was larger during late pregnancy compared with that before pregnancy (F = 3.625, df = 4, p = .017; Figure 2I). The EEG spectral analysis of the wake state revealed that the power density at 4 and 5 Hz during late pregnancy was higher than that before pregnancy (F = 7.964, df = 4, p < .0001 for 4 Hz; F = 10.754, df = 4, p <.0001 for 5 Hz), whereas the power density at 10 and 11 Hz during late pregnancy was lower than that before pregnancy (F = 12.378, df = 4, p < .0001, for 10 Hz; F = 12.418, df = 4, p < .0001 for 11 Hz; Figure 3A). For NREMS, power density at 4 Hz during late pregnancy was higher than that before pregnancy and during early pregnancy (F = 13.384, df = 4, p < .0001; Figure 3B). For REMS, the power density at 4 and 5 Hz during late pregnancy was higher than that before pregnancy (F = 4.865, df = 4, p = .004 for 4 Hz; F = 10.561, df = 4, p < .0001 for 5 Hz; Figure 3C). There was no significant difference in NREMS δ density before, during, and after pregnancy (Figure 3D). Figure 3. View largeDownload slide EEG spectrum analysis during the course of pregnancy in wild-type mice. (A) Power density during wake before, during, and after pregnancy in wild-type mice. (B) Power density during NREMS before, during, and after pregnancy. (C) Power density during REMS before, during, and after pregnancy. (D) NREMS δ density before, during, and after pregnancy. n = 8; *p < .05. One-way repeated measures ANOVA followed by Tukey’s test. The data are presented as the mean ± SEM. Figure 3. View largeDownload slide EEG spectrum analysis during the course of pregnancy in wild-type mice. (A) Power density during wake before, during, and after pregnancy in wild-type mice. (B) Power density during NREMS before, during, and after pregnancy. (C) Power density during REMS before, during, and after pregnancy. (D) NREMS δ density before, during, and after pregnancy. n = 8; *p < .05. One-way repeated measures ANOVA followed by Tukey’s test. The data are presented as the mean ± SEM. Abnormal Sleep/Wakefulness of PAH Mice The systolic blood pressure of PAH mice was 100–110 mm Hg which is similar to that of wild-type mice until pregnant day 12, started to increase at pregnant day 13, up to 160 mm Hg just prior to delivery. After delivery, blood pressure started to decrease and returned to 100–110 mm Hg by the third day after delivery [9, 25]. Consistent with pregnancy-stage-specific hypertension, PAH mice showed frequent spontaneous locomotion for foraging and exploration and exhibited normal EEG and EMG signals during wakefulness, NREMS, and REMS in early and mid pregnancy (Figure 1C). In late pregnancy, all PAH mice showed much fewer and slower spontaneous locomotion but they were still able to move in response to external stimuli, such as air puff and walked to obtain food and drink. PAH mice in late pregnancy continuously exhibited abnormal EEG and EMG with increased slower activity in EEG and lower variability in EMG during wakefulness, and lower amplitude EEG and continuous EMG activities during NREMS (Figure 1C). Because of these changes, we could not determine wakefulness or NREMS for many epochs. The EEG spectrum of PAH mice in late pregnancy showed a decrease in high frequency power (>8 Hz) (Figure 1D), lacked a clear difference between the light and dark phases, and lacked any stretch of wake or sleep epochs (Figure 1D). A general slowing of EEG in PAH mice during late pregnancy was confirmed by the high total δ density during late pregnancy compared with that during mid pregnancy (Figure 4A). Figure 4. View largeDownload slide Periodic discharges of EEG during late pregnancy in PAH mice. (A) δ-Densities of all epochs during mid, late, and after pregnancy. n = 6. One-way repeated measures ANOVA followed by Tukey’s test. The data are presented as the mean ± SEM. (B and C) Representative EEG and EMG recordings showing the periodic spike-and-wave discharges of two PAH mice. (D) Enlarged spike-and-wave discharges indicated in (B and C). (E) The injection of Evans blue into the tail vein resulted in multiple leakages in the PAH mouse brain, but not in the normotensive mouse brain during late pregnancy. Figure 4. View largeDownload slide Periodic discharges of EEG during late pregnancy in PAH mice. (A) δ-Densities of all epochs during mid, late, and after pregnancy. n = 6. One-way repeated measures ANOVA followed by Tukey’s test. The data are presented as the mean ± SEM. (B and C) Representative EEG and EMG recordings showing the periodic spike-and-wave discharges of two PAH mice. (D) Enlarged spike-and-wave discharges indicated in (B and C). (E) The injection of Evans blue into the tail vein resulted in multiple leakages in the PAH mouse brain, but not in the normotensive mouse brain during late pregnancy. Surprisingly, the prominent abnormalities in EEG and EMG of PAH mice quickly disappeared within 2 days from delivery (Figure 1C and D). In parallel, the spontaneous activities of PAH mice increased, with a clear resting-active pattern consistent with the light-dark phases. The total δ density of postpartum PAH mice was reduced to a level similar to that of PAH mice in mid pregnancy (Figure 4A). Three out of six PAH mice showed periodic abnormal discharges on EEG signals during late pregnancy (Figure 4B, C, and D). Typically, periodic spike-and-wave discharges that were clearly different from background EEG activity occurred during the NREMS-like state and accompanied phasic EMG activities with jerky body movements (video in Supplementary Material), which typically lasted 10–15 min and repeated 4–6 times during the 24-hr period. To examine whether severe hypertension disrupted the integrity of the BBB of PAH mice, we injected Evans blue dye into the tail vein during late pregnancy. We observed multiple and very light Evans blue leakages ranging in diameter from less than 0.4 mm to larger than 2 mm in the cerebral cortex and cerebellum in all PAH mice as reported in acute hypertensive rats [32], but did not find any leakage in normotensive control mice during late pregnancy (Figure 4E). In PAH mice, the number of dye leakages was 4.5 ± 0.96 (mean ± SEM). Since there were many epochs that we could not properly classify into wake or NREMS during late pregnancy, we evaluated the sleep/wakefulness of PAH mice except for those in late pregnancy. The total wake time was similar among before, early, mid, and after pregnancy (Figure 5A). There was no difference in the wake episode duration (Figure 5B) and wake episode number (Figure 5C). The NREMS time was also similar before, early, mid, and after pregnancy (Figure 5D). The NREMS episode duration and episode number were similar before, early, mid, and after pregnancy (Figure 5E and F). The daily time spent in REMS of postpartum PAH mice was longer than that during early and mid pregnancy (F = 9.058, df = 3, p = .002; Figure 5G), likely reflecting a rebound phenomenon after the drastically reduced REMS time during late pregnancy (see below). An increased total REMS during postpartum was observed during the light phase (F = 11.141, df = 3, p = .0008; Figure 5G), but not during the dark phase. We observed no difference in the REMS episode duration (Figure 5H) or REMS episode number (Figure 5I). Figure 5. View largeDownload slide Sleep/wake behaviors during the course of pregnancy in PAH mice, except for during late pregnancy. (A–C) Total time spent in wake (A), wake episode duration (B), and wake episode number (C) before, during early and mid pregnancy, and postpartum in PAH mice. (D–F) Total time spent in NREM sleep (NREMS) (D), NREMS episode duration (E), and NREMS episode number (F) before, during early and mid-pregnancy, and postpartum in PAH mice. (G–I) Total time spent in REM sleep (REMS) (G), REMS episode duration (H), and REMS episode number (I) before, during early and mid pregnancy, and postpartum in PAH mice. NREMS during late pregnancy is not presented because there are many intermediate epochs that are not classified into NREMS or wakefulness. n = 6. *p < .05. One-way repeated measures ANOVA followed by Tukey’s test. The data from individual mice are presented as the group mean ± SEM. Figure 5. View largeDownload slide Sleep/wake behaviors during the course of pregnancy in PAH mice, except for during late pregnancy. (A–C) Total time spent in wake (A), wake episode duration (B), and wake episode number (C) before, during early and mid pregnancy, and postpartum in PAH mice. (D–F) Total time spent in NREM sleep (NREMS) (D), NREMS episode duration (E), and NREMS episode number (F) before, during early and mid-pregnancy, and postpartum in PAH mice. (G–I) Total time spent in REM sleep (REMS) (G), REMS episode duration (H), and REMS episode number (I) before, during early and mid pregnancy, and postpartum in PAH mice. NREMS during late pregnancy is not presented because there are many intermediate epochs that are not classified into NREMS or wakefulness. n = 6. *p < .05. One-way repeated measures ANOVA followed by Tukey’s test. The data from individual mice are presented as the group mean ± SEM. We further examined the EEG power spectrum of PAH mice, except for late pregnancy. For wakefulness, the power density at 3 Hz during mid pregnancy was higher than that during postpartum (F = 5.783, df = 3, p = .011) and the power density at 6, 7, 9, 12, 13, and 14 Hz after pregnancy was higher than that before pregnancy (Figure 6A). The power density of PAH mice did not exhibit any significant difference during NREMS (Figure 6B). For REMS, the power density during mid pregnancy was lower at 6 Hz (F = 10.101, df = 3, p = .001) and 7 Hz (F = 23.156, df = 3, p < .0001), and higher at 9 Hz (F = 14.724, df = 3, p < .0001) and 10 Hz (F = 9.019, df = 3, p = .002 for 10 Hz) compared with that before pregnancy (Figure 6C). The NREMS δ density tended to be lower in postpartum PAH mice (Figure 6D). Figure 6. View largeDownload slide EEG spectrum analysis during the course of pregnancy in PAH mice, except for during late pregnancy. (A) Power density during wake before, during early and mid pregnancy, and postpartum in PAH mice. (B) Power density during NREMS before, during early and mid pregnancy, and postpartum. (C) Power density during REMS before, during early and mid-pregnancy, and postpartum. (D) NREMS δ density before, during early and mid pregnancy, and postpartum. n = 6; *p < .05. One-way ANOVA followed by Tukey’s test. The data are presented as the mean ± SEM. Figure 6. View largeDownload slide EEG spectrum analysis during the course of pregnancy in PAH mice, except for during late pregnancy. (A) Power density during wake before, during early and mid pregnancy, and postpartum in PAH mice. (B) Power density during NREMS before, during early and mid pregnancy, and postpartum. (C) Power density during REMS before, during early and mid-pregnancy, and postpartum. (D) NREMS δ density before, during early and mid pregnancy, and postpartum. n = 6; *p < .05. One-way ANOVA followed by Tukey’s test. The data are presented as the mean ± SEM. Olmesartan Alleviated Abnormal Sleep/Wakefulness of PAH Mice Olmesartan-administered PAH mice had a systolic blood pressure of approximately 120 mm Hg, which does not affect the integrity of the BBB [39]. In late pregnancy, olmesartan-administered PAH mice showed spontaneous locomotion similar to pregnant wild-type mice. The EEG or EMG signaling and daily EEG spectrogram of olmesartan-administered PAH mice did not show the abnormalities (Figure 7A) observed in PAH mice (Figure 1D). Olmesartan treatment also decreased the total δ density of PAH mice to that of nonpregnant PAH (human angiotensinogen–transgenic) mice (Figure 7B). Figure 7. View largeDownload slide EEGs and sleep/wakefulness of olmesartan-administered PAH mice. (A) Representative daily EEG spectrogram during late pregnancy in a PAH mouse that was administered olmesartan. (B) δ-Densities of all epochs of nonpregnant PAH (normotensive) mice (n = 6), PAH mice (n = 6), and olmesartan-administered PAH mice (n = 5) during late pregnancy. (C–E) Total time spent in REMS (C), REMS episode duration (D), and REMS episode number (E) in nonpregnant PAH mice, olmesartan-administered PAH mice, and THM. **p < .01, ***p < .001. One-way ANOVA followed by Tukey’s test. The data are presented as the group mean ± SEM. Figure 7. View largeDownload slide EEGs and sleep/wakefulness of olmesartan-administered PAH mice. (A) Representative daily EEG spectrogram during late pregnancy in a PAH mouse that was administered olmesartan. (B) δ-Densities of all epochs of nonpregnant PAH (normotensive) mice (n = 6), PAH mice (n = 6), and olmesartan-administered PAH mice (n = 5) during late pregnancy. (C–E) Total time spent in REMS (C), REMS episode duration (D), and REMS episode number (E) in nonpregnant PAH mice, olmesartan-administered PAH mice, and THM. **p < .01, ***p < .001. One-way ANOVA followed by Tukey’s test. The data are presented as the group mean ± SEM. Since we unequivocally identified REMS in PAH mice during late pregnancy based on the appearance of the θ wave (6–9 Hz) and muscle atonia (Figure 1C), we examined how the administration of olmesartan affects REMS in PAH mice. The total REMS time of PAH mice was only 4.3 ± 1.9 min (mean ± SEM), but olmesartan-administered PAH mice showed a significantly longer REMS time (F = 17.230, df = 2, p < .0001, Figure 7C). Olmesartan-administered PAH mice showed a REMS episode duration similar to PAH mice (Figure 7D) and increased numbers of REMS episodes (F = 25.613, df = 2, p < .0001, Figure 7E). Thus, an increased number of REMS episodes contributes to the increased total REMS time of olmesartan-administered PAH mice. Olmesartan treatment did not alter the total time, episode duration, or episode number of wakefulness, NREMS, or REMS in male wild-type mice (data not shown). Sleep/Wakefulness of THM Lastly, we examined the sleep/wakefulness of nonpregnant female THM. The total wake time of female THM was similar to that of female wild-type mice (Figure 8A). There was no difference in the wake episode duration (Figure 8B) or the wake episode number (Figure 8C). The total NREMS time of female THM was similar to that of female wild-type mice (Figure 8D). There was no difference in the NREMS episode duration (Figure 8E) or the NREMS episode number (Figure 8F). The total REMS time of female THM was shorter than that of female wild-type mice (Figure 8G). A decreased time spent in REMS was associated with a reduced REMS episode duration (Figure 8H), but not with significant changes in the REMS episode number (Figure 8I). Total REMS time, REMS episode duration, and REM episode number of THM were similar to those of olmesartan-administered PAH mice (Figure 7C, D, and E). Figure 8. View largeDownload slide Sleep/wake behaviors of THM. (A–C) Total time spent in wake (A), wake episode duration (B), and wake episode number (C) of THM and wild-type mice. (D–F) Total time spent in NREM sleep (NREMS) (D), NREMS episode duration (E), and NREMS episode number (F). (G–I) Total time spent in REM sleep (REMS) (G), REMS episode duration (H), and REMS episode number (I). Five THM and eight wild-type mice. *p < .05, **p < .01, ***p < .001. Two-tailed t-test. The data from individual mice are presented as the group mean ± SEM. Figure 8. View largeDownload slide Sleep/wake behaviors of THM. (A–C) Total time spent in wake (A), wake episode duration (B), and wake episode number (C) of THM and wild-type mice. (D–F) Total time spent in NREM sleep (NREMS) (D), NREMS episode duration (E), and NREMS episode number (F). (G–I) Total time spent in REM sleep (REMS) (G), REMS episode duration (H), and REMS episode number (I). Five THM and eight wild-type mice. *p < .05, **p < .01, ***p < .001. Two-tailed t-test. The data from individual mice are presented as the group mean ± SEM. Discussion To the best of our knowledge, the present study is the first report on sleep/wakefulness during the course of pregnancy in mice. This study also proposes that PAH mice may be an animal model of eclampsia. Sleep/Wakefulness During and After Pregnancy The daily time spent in wakefulness decreased and the total NREMS time increased during late pregnancy, primarily reflecting increased NREMS during the dark phase, consistent with the long NREMS in pregnant rats [40, 41]. Approaching delivery, an increased physical load such as the weight of embryos, extended uterus, and increasing abdominal pressure, may necessitate more sleep and rest in pregnant mice. Sleep/wakefulness during late pregnancy was also characterized by frequent switches between wakefulness and NREMS, resulting in an increased episode number and short episode duration of both wakefulness and NREMS during both the light and dark phases. These findings are consistent with previous reports on pregnant humans and rats [42]. Fragmented sleep may be due to spontaneous movements of the embryos in the uterus and/or the pressure of the growing uterus on neighboring tissues and organs. Pregnancy affected REMS differently from wakefulness and NREMS. The daily time spent in REMS and the daily number of REMS episodes were not altered during the course of pregnancy, which is consistent with previously reported for pregnant rats [40, 43]. A short REMS episode duration was recognized from mid pregnancy through postpartum. Altered REMS, independent of NREMS changes, supports the idea that the regulatory system for REMS functions differently from that for NREMS [44]. The EEG spectrum analysis of REMS showed pregnancy-induced changes at 6 and 7 Hz from the early stages of pregnancy, although the physiological significance of this finding is not clear. In addition to the physical effects of the growing uterus and embryos during pregnancy, hormonal changes may affect sleep/wake behaviors during pregnancy. Estrogen and progesterone increase as pregnancy progresses and then abruptly decrease after parturition [45]. Estrogen and progesterone generally increase wakefulness and decrease sleep, as indicated by the increased wakefulness of ovariectomized mice replaced with estradiol alone or with both estradiol and progesterone [4, 18, 46–49]. Consistently, receptors for estrogen are abundant in the preoptic area, which is involved in sleep regulation [50]. Thus, increased estrogen and progesterone during pregnancy may work to enhance wakefulness. However, this interpretation could be too simple since progesterone has also been reported to reduce wakefulness [51]. Importantly, the sleep/wake behaviors of postpartum mice were similar to nonpregnant mice, despite lactation and increased prolactin and oxytocin. Mice that showed multiple altered parameters in wakefulness, NREMS, and REMS during late pregnancy quickly returned to normal sleep/wakefulness, excluding the decreased episode duration of wakefulness and REMS. There have been several reports on increased REMS by prolactin [52], but peptide hormones may be less permeable to the brain and less influential on brain functions compared with gonadal steroid hormones. PAH Mice as an Animal Model for Eclampsia The present study also supports PAH mice as an animal model for eclampsia, based on seizures, altered sleep/wakefulness, fewer spontaneous activities, and high perinatal mortality. There is no known animal model for eclampsia, although there have been pharmacological and genetic interventions to develop an animal model for preeclampsia [53–55], including PAH mice. Angiotensin II is a major factor inducing hypertension in the renin–angiotensin–aldosterone system [56]. Consistently, angiotensin II type-1a receptor is required for the high blood pressure in PAH mice [57]. PAH mice exhibit elevated blood pressure with severe proteinuria during late pregnancy, which are diagnostic criteria for human preeclampsia [19, 20]; however, these animals have not been examined for neuropsychiatric phenotypes. The present study showed that PAH mice exhibited markedly abnormal vigilance states with a generalized slowing of EEG during late pregnancy. Although PAH mice still slowly responded to external stimuli and obtained food and water during late pregnancy, these animals rarely showed spontaneous and swift movements, staying in the nest area almost all day. PAH mice showed normal REMS characterized by θ wave and muscle atonia during late pregnancy, but the total REMS time drastically decreased compared with PAH mice during mid pregnancy and pregnant wild-type mice. We also observed that half of the PAH mice showed spike-and-wave discharges during late pregnancy, based on EEG or EMG recording supplemented with video recording. Surprisingly, postpartum PAH mice quickly recovered from the general slowing of EEG and had a normal time spent in wakefulness and NREMS. Similarly, the most commonly observed EEG abnormality in human eclampsia is generalized or focal slowing of EEG [28, 58, 59], and this abnormal EEG normalizes after the release from hypertension [59]. Thus, PAH mice bear a symptomatic resemblance to human eclampsia. Postpartum PAH mice showed a longer total REMS time, suggesting that the homeostatic mechanism may compensate for the diminished total REMS during late pregnancy. Since PAH mice do not raise newborn pups and ignore them, we removed the newborn pups. It is unlikely that the removal of pups largely altered the sleep/wake behavior because PAH mice simply neglected the pups and did not show any interest in them. All PAH mice examined herein showed disruption of the BBB, as assessed by the intravenous injection of Evans blue dye during late pregnancy. Brain circulation has an autoregulatory mechanism to maintain stable cerebral blood flow between mean arterial pressures of 60 and 120 mm Hg [39]. However, blood pressures higher than the upper limit of cerebral vascular autoregulation can lead to disruption of the BBB and vasogenic edema, eventually resulting in hypertensive encephalopathy [39]. An acute increase in arterial blood pressure by 80 mm Hg disrupts the BBB integrity in rats [60]. Four hours of intra-abdominal hypertension resulted in the leakage of Evans blue dye into the brain parenchyma, which was not observed 1 hr after release from abdominal hypertension [61]. In addition, the administration of olmesartan alleviated sleep/wake behavior abnormalities of PAH mice during late pregnancy such as general slowing of EEG, loss of the circadian change in sleep/wakefulness, and a drastic reduction in REMS. Thus, the acute and severe increase in blood pressure may solely be responsible for the BBB disruption in PAH mice and subsequently lead to abnormal sleep/wake behaviors. Neuroimaging studies of eclampsia patients revealed posterior reversible encephalopathy syndrome, which refers to reversible vasogenic cerebral edema in the posterior region of the cerebral cortex accompanied by acute neuropsychiatric symptoms [28–31]. Thus, PAH mice exhibit a pathophysiologic mechanism similar to human eclampsia during late pregnancy. Sleep/Wakefulness of THM THM showed a reduced REMS time and normal NREMS time compared with wild-type mice, suggesting that chronic and moderate hypertension suppresses REMS and does not affect NREMS. Interestingly, the REMS time of THM was similar to that of olmesartan-administered PAH mice. Since THM and olmesartan-administered PAH mice showed similar levels of hypertension, increased blood pressure may result in decreased REMS time, although further studies are necessary to elucidate the mechanistic link between hypertension and REMS regulation. We cannot deny the possibility that angiotensin produced in the brain affects REMS since both the human renin transgene and human angiotensinogen transgene are weakly or mildly expressed in the brain [62, 63] where angiotensin-converting enzymes and angiotensin II receptors are expressed [64, 65]. Limitations Although various kinds of preeclampsia model mice, including PAH mice, have been reported, none of them fully reproduce human preeclampsia in terms of pathogenesis, symptoms, and responses to pharmacological intervention. To evaluate the validity of PAH mice as a model of eclampsia, further studies using magnesium sulfate, the most potent anticonvulsant for eclampsia patients [22], may be necessary. Furthermore, we cannot exclude the possibility that humoral factors are involved in the altered sleep/wakefulness of PAH mice independently of high blood pressure. Consistent with abnormal placentation and increased secretion of angiogenic factors in human preeclampsia [21], PAH mice exhibit abnormal placental angiogenesis and intrauterine growth retardation [57, 66], which may increase humoral factors and reactive oxygen species to disrupt the BBB [67]. Indeed, modification of the Keap1-Nrf2 pathway, which functions as a defense system for oxidative stress, enhances placental angiogenesis and survival rates of PAH mice and their fetuses independently of high blood pressure [68]. Another limitation of the present study was the modest number of mice examined. Major findings, such as long NREMS time during late pregnancy in wild-type mice and the abnormal sleep/wakefulness of PAH mice during late pregnancy, were sufficiently robust with small interindividual differences, which can be replicated with good reproducibility. In addition, we failed to obtain pregnant female THM and thus could not examine sleep/wake behaviors of pregnant THM compared with PAH mice. Although we did not examine the fertility rate of THM in detail, we conjecture that chronic hypertension or enhanced renin–angiotensin system may decrease the fertility of THM. In summary, the present study revealed a similarity in the sleep changes during pregnancy between humans and mice [6, 7], which provides a basis for future studies on sleep regulation in animal models for human diseases during pregnancy. We also suggest that PAH mice may be a useful model for the development of therapeutic intervention for eclampsia. Supplementary Material Supplementary material is available at SLEEP online. Funding This work was supported by the World Premier International Research Center Initiative from MEXT (to MY), JSPS KAKENHI (Grant Number 17H06095 to MY and HF; 16K15187 to HF; 26507003 to CM and HF; 15K18966), MEXT KAKENHI (Grant Number 15H05935 to MY and HF), Funding Program for World-Leading Innovative R&D on Science and Technology (FIRST program) from JSPS (to MY), Research grant from Uehara Memorial Foundation research grant (to MY), and Research grant from Takeda Science Foundation research grant (to MY). Disclosure Statement This was not an industry-supported study. MY is a former Investigator of the Howard Hughes Medical Institute. Acknowledgments We thank all Yanagisawa/Funato lab and IIIS members for discussion and comments on this manuscript and Go Taniguchi for his comments on spike-wave seizures. References 1. Lee KA. 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