Effects of circadian rhythm disorder on the hippocampus of SHR and WKY rats

Journal Pre-proofs Effects of Circadian Rhythm Disorder on the Hippocampus of SHR and WKY Rats YunLei Wang, YuGe Zhang, WenZhu Wang, Xu Liu, YaFei Chi, JianFeng Lei, BaoGui Zhang, Tong Zhang PII: DOI: Reference:

S1074-7427(19)30208-4 https://doi.org/10.1016/j.nlm.2019.107141 YNLME 107141

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Neurobiology of Learning and Memory

Received Date: Revised Date: Accepted Date:

26 May 2019 13 December 2019 13 December 2019

Please cite this article as: Wang, Y., Zhang, Y., Wang, W., Liu, X., Chi, Y., Lei, J., Zhang, B., Zhang, T., Effects of Circadian Rhythm Disorder on the Hippocampus of SHR and WKY Rats, Neurobiology of Learning and Memory (2019), doi: https://doi.org/10.1016/j.nlm.2019.107141

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Effects of Circadian Rhythm Disorder on the Hippocampus of SHR and WKY Rats YunLei Wang1,2,4, YuGe Zhang1,2,4, WenZhu Wang3, Xu Liu1,2,4, YaFei Chi5, JianFeng Lei5, BaoGui Zhang6, Tong Zhang1,2,4*

1. School of Rehabilitation Medicine, Capital Medical University, Beijing 100068, China 2. Lab of brain injury repair and rehabilitation, China Rehabilitation Science Institute, Beijing 100068, China 3. China Rehabilitation Medicine Institute, China Rehabilitation Science Institute, Beijing 100068, China 4. Department of Neurological Rehabilitation, Beijing Bo'ai Hospital, China Rehabilitation Research Center, Beijing, 100068, China 5. Capital Medical University, Beijing, 10069, China 6. Center for Advanced Imaging，Institute of Automation，the Chinese Academy of Sciences, Beijing, 100190, China

*Corresponding Author: Tong Zhang, School of Rehabilitation Medicine, Capital Medical University, Beijing Bo’ai Hospital, China Rehabilitation Research Center, No.10 North Road, Fengtai District, Beijing 100068, China (e-mail: [email protected])

Abstract The present study investigated the effects of circadian rhythm disorder (CRD) on the hippocampus of SHR and WKY rats. Male SHR rats (n = 27) and WKY rats (n = 27) were randomly divided into six groups: SHR and WKY normal (N)CR, SHR and WKY CRD 16/8 (CRD16/8), and SHR and WKY CRD 12/12 (CRD12/12). Activity patterns were adjusted using different photoperiods over 90 days and any changes were recorded. Rats were tested in the Morris water maze and in a novel object recognition experiment; serologic analysis, magnetic resonance imaging (diffusion tensor imaging + arterial spin labeling), hippocampal Nissl staining, Fluoro-Jade B staining, and immunohistochemistry were also performed. The results showed that both types of inverted photoperiod reduced CR amplitude and prolonged the circadian period. CRD and hypertension reduced memory performance and novel object recognition and preference. The decreases in memory and preference indices were greater in rats in the CRD12/12 group compared to the CRD16/8 group. CRD and hypertension decreased fractional anisotropy values, the number of neurons and astrocytes in the hippocampus, and the expression of brain-derived neurotrophic factor and synapsin 1; it also enhanced the degeneration of neurons and microglia and reduced blood flow in the hippocampus, and increased nuclear factor κB, caspase, neuron-specific enolase, and interleukin-6 levels. These findings reveal a biological basis for the link between CRD and cognitive decline, which has implications for CRD caused by shift work and other factors.

Key words: circadian rhythm disorder; hypertension; cognition; hippocampus

1.

Introduction Over the course of evolution, the circadian rhythm (CR) of organisms—which is an endogenous rhythm

with a cycle close to 24 h (Aschoff, 1981)—has remained highly consistent (Sahar & Sassone-Corsi, 2009). However, endogenous CRs are also affected by exogenous photic (Schmoll, 2011) or nonphotic (Hastings, 1998) entrainment. The activity rhythm of organisms is synchronized to the 24-h light/dark (LD) cycle, which is the normal (N)CR, but exogenous factors such as shift work or jetlag (Stevens, 2007; Zimmet, 2019) can desynchronize the body’s activity rhythm, resulting in CR disorder (CRD) (Bass, 2012). Two negative consequences of modern society’s rapid economic and technological development include increases in the prevalence of CRD (Taniyama et al., 2015) as well as hypertension. An epidemiologic survey conducted in China in 2015 showed that 27.9% of adults are hypertensive, representing a 50% increase compared to 2002 (Wang et al., 2018). Cognition includes functions such as memory, language, visuospatial processing, execution, calculation, and judgment. Many studies have demonstrated that CRD has adverse effects on cognitive functioning (Gumenyuk et al., 2010; Gibson, Wang, Tjho, Khattar, & Kriegsfeld, 2010; Shi et al., 2017; Sack et al., 2007); the same is true of hypertension (Foulquier et al., 2018), but no studies to date have examined the combined effects of CRD and hypertension on cognition. Cognitive decline is associated with decreased functioning of the hippocampus (Diamond, Fleshner, & Rose, 1999; John, 2002; Kreutzmann, Havekes, Abel, & Meerlo, 2015). The integrity of the hippocampus depends on the maintenance of the number and morphology of the constituent neurons (Xenia et al., 2008), while its plasticity is reflected in the dynamics of synapses and astrocytes (Woo et al., 2017; Borjkhani, Bahrami, & Janahmadi, 2018; Sofroniew, & Vinters, 2010). The suprachiasmatic nucleus of the hypothalamus regulates CR (Honma, 2018). The main factor controlling CR is illumination (Cho, et al., 2016; Fonken, Kitsmiller, Smale, & Nelson, 2012). CRD can be

induced by abnormal photoperiods (Emerson, Jeanne, Steven, & Christopher, 2017; Reid & Abbott, 2015) such as an inverted LD cycle and prolonged exposure to an artificial light source during the dark period (represented in this study by the CRD12/12 and CRD 16/8 models, respectively). In this study, we examined the effects of CRDs without and with hypertension on cognition, hippocampal structure, and plasticity in WKY and SHR rats, as well as the differences between these models.

2.

Methods

2.1. Animals A total of 27 adult male spontaneous hypertensive (SHR) rats and 27 homologous control rats (WKY) matched for age and body weight (approximately 220–260 g) were used for experiments. The animals were 10 weeks old at the time of purchase from Capital Medical University (Beijing, China) and were reared in the Animal Laboratory of Capital Medical University under specific-pathogen-free conditions. The rats were randomly assigned to the following groups using the statistical software program R v.3.5.0: SHR NCR (n = 9), SHR CRD 16/8 (n = 9), SHR CRD 12/12 (n = 9), WKY NCR (n = 9), WKY CRD16/8 (n = 9), and WKY CRD12/12 (n = 9). After grouping, the mean arterial pressure of each rat was measured with a six-channel noninvasive rat sphygmomanometer (Kent Scientific, Torrington, CT, USA). The mean arterial pressure of SHR and WKY rats differed significantly (P < 0.0001), but there were no significant differences between SHR or WKY rats with different CRs (P > 0.05) (Table 1). The most critical experiment was the Morris maze test as it requires a long time to complete (typically 6 days, as detailed in the Discussion section). To ensure that all rats were tested under the same phase and zeitgeber time (ZT), the experiments were conducted under a normal LD cycle on days 91–96 at 09:00 (ZT1). Given the 90-day training period, the phase of CRD rats was presumed to be the same as that of the NCR group

on days 91–93. However, phase differences in CRD rats began to emerge on days 94–96. We therefore added four groups of CRD rats (“9 pm”; n = 9 per group, for a total of 36 rats): SHR and WKY CRD16/8 (CRD16/8s), and SHR and WKY CRD12/12 (CRD12/12s). The modeling method used for the 9 pm groups was identical to that used for the main experimental groups of CRD rats, with the difference being that 9 pm groups were maintained under the appropriate abnormal LD conditions on days 94–96 and were tested in the Morris water maze under the same ZT (ZT1, 21:00) (Fig. 1).

2.2. Modeling of different CRs Rats were housed under normal LD cycle conditions for 2 weeks to allow acclimatization to the feeding environment. After 2 weeks, the CR of the rats was adjusted through exposure to the different photoperiods. NCR rats were exposed to light from 08:00–20:00 and to darkness from 20:00–08:00 (the following day). Rats in the CRD12/12 groups (Fig. 1A) were exposed to light from 08:00–20:00 and to darkness from 20:00–08:00 (the following day) for 3 days, and then to darkness from 08:00–20:00 and light from 20:00–08:00 (the following day) for 3 days. Rats in the CRD16/8 groups (Fig. 1B) were exposed to light from 08:00–20:00 and to darkness from 20:00–08:00 (the following day) for 3 days, and then to darkness from 12:00–20:00 and to light from 20:00–12:00 (the following day) for 3 days. Rats were subjected to the LD conditions for 90 days. The breeding room was illuminated by 100-W fluorescent lamps with a light intensity of 150–200 lx, and had a constant temperature of 20°C–24°C with an air humidity of 40%–70%. The rats had free access to food and water. This experiment was approved by the Ethics Committee of the Capital Medical University (AEEI2017-093). All animal experiments were conducted in accordance with the International Guiding Principles for Animal Biomedical Research of the World Health Organization.

2.3. Experimental equipment and methods 2.3.1. Behavioral analysis using ClockLab The base unit consisted of a rat cage (45 × 25 × 20 cm) with a metal runner (13 cm). Each experimental unit was connected to the US ClockLab (ACT-500) signal transducer, which transformed signals into data that were recorded by the operating system. The daily and nightly activity of rats was measured by recording the rat runner for 24 h each day and was analyzed by the system. Over the 60 days of the CR modeling process, we randomly selected six rats from each of the main experimental groups using R v.3.5.0. Each rat was subjected to behavioral analysis for 12 consecutive days. Activity rhythms were continuously recorded over 24 h with ClockLab (ACT-500) software. At the end of the experiment, we performed data analysis on the CR activity map with ClockLab (2.7.3) data package, which is based on MATLAB (MathWorks, Natick, MA, USA). The chi-square amplitude and rhythm period of each rat over 12 days were analyzed with the Chi-squared Periodogram data packet. The average rest time per day for each rat was analyzed using the Activity Profile data package.

2.4. Morris water maze All rats were tested in the Morris water maze on days 91–96 of the experimental period. The NCR group was tested at ZT1 (09:00) on days 91–96 (Fig. 1A), whereas the main experimental groups of CRD rats were tested under a normal LD cycle on days 91–96 at ZT1 (09:00). The 9 pm group of CRD rats was also tested under a normal LD cycle at ZT1 (09:00) on days 91–93. However, the 94–96-day test was performed under an abnormal LD cycle at ZT1 (21:00) (Fig. 1B, C). The Morris water maze consisted of a pool with a diameter of 120 cm and height of 40 cm; the water

depth was 25 cm, and water temperature was controlled at 22°C ± 2°C (XR-XM101). The water was rendered opaque by adding black food coloring. The pool, which was monitored from above with a digital camera, was divided into four quadrants, each with a unique marker. A hidden table (8 cm diameter) was placed 2.5 cm below the water surface in the middle of the third quadrant. The entire testing room was soundproofed, and all reference objects and light intensity were fixed. Each experiment was conducted between 08:30 and 11:30. The ANY-maze animal behavior analysis system (Shanghai Xinsoft, Shanghai, China) was used for data processing. A proportion of WKY rats float after being placed in the water and may be unresponsive to the Morris water maze. In addition, hyperresponsiveness to the maze caused by exercise-induced excitement is occasionally observed in SHR rats (Malkesman et al., 2006; Ferguson and Cada, 2003). Therefore, before the start of the test, rats were placed in a pool in order to identify those that were relatively slow or hyperresponsive. Based on the results, six rats per group for a total of 60 rats (main experimental group, n = 36 and supplementary experimental [9 pm] group, n = 24) were selected for behavioral testing.

2.5. Novel object recognition experiment Rats were subjected to a novel object recognition experiment after modeling (i.e., on days 91–93 of the experiment). Experiments for both the NCR and CRD groups were conducted under normal LD conditions at ZT2 (10:00) (Fig. 1). The experimental equipment was purchased from Shanghai Xinsoft and consisted of a test box, camera, and animal behavioral trajectory analysis system. The experimental site was soundproof with full artificial lowlight illumination at an intensity of 25–50 lx. The volume of the test cartridge was 40 × 40 × 20 cm. Rats were placed individually in the test cartridge. The experiment comprised familiarization, training, and test periods. The habituation period lasted for

1 day. Rats were placed in the test box for 5 min; at the end of the familiarization period, they were transferred to the box for the training session the following day. Before training, two identical objects were placed in each box; the rats were placed in the test box with their backs facing the objects. The experimenter immediately left the room and used video equipment to record the time spent and number of times the rat was in contact with the two objects. The training time was 5 min, and one training session was conducted. After 24 h, rats were subjected to the following test: one object in the test box was unchanged, while the other was replaced with a new object, although the spatial location was kept constant. The rats were placed in the test box with their backs facing the two objects, and time spent and number of times the rat was in contact with the two objects were recorded. Memory function was assessed by calculating the memory and preference indices of the rats with the following formulae: memory index = (exploration time of new object during test period − exploration time of old object) / (exploration time of new object + exploration time of old object); and preference index = exploration time of new object during test period / (exploration time of the new object + exploration time of the old object).

2.6. Magnetic resonance imaging (MRI) examination (diffusion tensor imaging [DTI] + arterial spin labeling [ASL]) After the water maze and novel object recognition tests (on day 97), we scanned the hippocampus of the rats using a 7.0 T MRI scanner (PharmaScan; Bruker Biospin, Rheinstetten, Germany). Five of the nine rats in each group were randomly selected for scanning; the sequences included T2 weighted imaging (T2WI) and DTI mapping. The RARE T2WI sequence was used in this study. The layer thickness was 0.8 mm, layer spacing was 0, and number of scanned layers was 30. The following parameters were used: repetition time (TR) = 3000 ms, echo time (TE) = 36 ms, fractional anisotropy (FA) = 179.1°, field of view (FOV) = 35 × 35 mm,

and matrix = 256 × 256, superimposed twice. A single-shot spin-plane echo sequence was used for DTI. The diffusion-sensitive gradient consisted of 30 non-collinear and non-planar gradient directions with b = 1000 s/mm2, and included six b values of 0 s/mm2. The layer thickness was 0.8 mm, layer spacing was 0, and number of scanned layers was 30; the parameters were as follows: TR = 6250 ms, TE = 22 ms, FA = 90°, FOV = 33 × 33 mm, matrix = 128 × 128, phase encoding direction from left to right, and average number of incentives = 4. The ASL sequence was also on a plane with a layer thickness of 2 mm, FOV = 30 × 30 mm, TE = 25 ms, TR = 18000 ms, and FA = 90°. After obtaining the images, original Digital Imaging and Communications in Medicine images were converted into three-dimensional Neuroimaging Informatics Technology Initiative format files (.nii) using dcm2niigui software. The spatial positions of the DTI map and T2 sequence images were registered with the “Coregister” function of the SPM12.0 package of MATLAB software. After registration, the images were further analyzed. The region of interest (ROI) in the rat hippocampus in this experiment was extracted by manual ticking, and DTI data were extracted and averaged by two MRI analysts. The layers with the largest hippocampus in T2 images and bilateral hippocampal ROIs were selected. FA values of the bilateral hippocampus ROIs, λ⊥, λ∥, and blood flow were recorded. Statistical analysis was performed with the average of the two-sided values.

2.7. Brain biopsy After MRI (on day 98), rats were sacrificed at ZT1 (09:00). The rats were anesthetized by intraperitoneal injection with pentobarbital sodium (40 mg/kg) and surgical scissors were used to cut the ribs and open the thoracic cavity. A perfusion syringe was used to penetrate the heart from the left ventricle, and 500

ml of normal saline was perfused into the whole blood vessel, followed by 200 ml of 4% paraformaldehyde. The brain was removed and fixed in 4% paraformaldehyde for 48 h, dehydrated, and embedded in paraffin. Five rats in each group were selected for Nissl and Fluoro-Jade (FJ)B staining and glial fibrillary acidic protein (GFAP), brain-derived neurotrophic factor (BDNF), and synapsin (Syn)-1 immunolabeling, and the other four rats were examined for ionized calcium binding adaptor molecule (Iba)-1, caspase, and nuclear factor (NF)-κB expression.

2.7.1. Nissl staining Paraffin-embedded tissue blocks were cut on a microtome into sections with an average thickness of 3–4 µm. The sections were collected on glass slides and deparaffinized in water, then sequentially incubated in the following reagents: xylene I (20 min), xylene II (20 min), anhydrous ethanol I (5 min), anhydrous ethanol II (5 min), and 75% alcohol (5 min). The slides were rinsed with tap water and stained with aniline blue. The sections were dried in an oven at 60°C, dehydrated, and sealed. Nissl staining was performed on rat brain biopsies for the quantification of neurons.

2.7.2. Immunohistochemistry Paraffin tissue blocks were sectioned on a microtome and deparaffinized in water. The sections were subjected to antigen retrieval in a microwave oven using a repair kit with citrate antigen retrieval buffer (pH 6.0). After cooling, one drop of 3% H2O2 was added to each section followed by incubation at room temperature for 25 min in the dark to quench peroxidase activity. The tissue was evenly covered with 3% bovine serum albumin and blocked at room temperature for 30 min. The blocking solution was removed and primary antibody diluted in phosphate-buffered saline (PBS) was added. Antibodies against Syn-1 (ab64581; Abcam, Cambridge,

MA, USA); BDNF (GB11096) and Iba-1 (GB11105) (both from Servicebio, Wuhan, China); GFAP (25699-1AP; Proteintech, Rosemont, IL, USA); and caspase (bs-0081R) and NF-κB (bs-0465R) (both from Bioss, Woburn, MA, USA) were used. After overnight incubation at 4°C in a humid chamber, the slides were placed in PBS (pH 7.4) and washed three times for 5 min each on a decolorizing shaker. When the sections had partially dried, the tissue was incubated with the appropriate horseradish peroxidase-conjugated secondary antibody for 50 min at room temperature. A drop of diaminobenzidine was added to each section to precipitate the colorimetric reaction. Nuclei were counterstained and the sections were dehydrated. Syn-1, BDNF, NF-κB, and caspase expression in the hippocampus was quantified using ImageJ software. For Nissl staining and GFAP and Iba-1 immunolabeling, the CA1 and CA3 areas and dentate gyrus (DG) of the hippocampus were examined at 20× magnification using TissueFAXS Viewer software (TissueGnostics, Vienna, Austria). The numbers of neurons, astrocytes, and microglia positive for each marker in each region were manually counted.

2.7.3. FJB staining Paraffin sections were deparaffinized in water and then sequentially incubated in the following reagents: xylene I (15 min), xylene II (15 min), anhydrous ethanol I (50 min), anhydrous ethanol II (5 min), 85% alcohol (5 min), and 75% alcohol (5 min). The slides were rinsed with distilled water. FJB working solution was prepared at 1:50 dilution in 0.1% glacial acetic acid and added to the tissue, followed by overnight incubation at 4°C. The sections were rinsed with water and then dried with a blower, then treated with xylene for 1 min and sealed with a neutral resin and observed and photographed under an inverted fluorescence microscope (Nikon, Tokyo, Japan), with excitation at 330–380 nm and emission at 420 nm for ultraviolet; 465–495 and 515–555 nm, respectively, for green fluorescence (fluorescein); and 510–560 and 590 nm, respectively, for red

fluorescence (Cy3). Fluorescein-labeled green or yellow cells were counted as immunopositive. For FJB staining, the CA1 and CA3 areas and dentate gyrus (DG) of the hippocampus were examined at 20× magnification using Case Viewer software (3DHISTECH, Budapest, Hungary). The number of degenerated neurons (FJB+ cells) in each region was manually counted.

2.7.4 Serologic analysis Analyses were performed at ZT 2 (10:00) on day 97. Four rats from each group were selected and 1 ml of blood was drawn from the tail of each rat. After allowing the samples to stand for 2 h, the cells were centrifuged and the supernatant was collected for enzyme-linked immunosorbent assay (ELISA) and serum biochemical testing. Commercial ELISA kits were used to determine the levels of neuron-specific enolase (NSE) (SEA537Ra) and interleukin (IL)-6 (ERC003.96) (both from USCN Life Science, Wuhan, China). The antibody was first diluted to a protein concentration of 1–10 μg/ml using carbonate coating buffer. Thereafter, 200 µl of blocking solution was added to each well, and the cells were incubated at 37°C for 1–2 h. The sample was added after washing. The plate was sealed with film and incubated at 37°C for 1–2 h and washed. After adding the antibody, the plate was re-incubated at 37°C for 1 h and washed. A pre-diluted enzyme conjugate working solution was added to each well followed by incubation for 30 min and washing. The chromogenic substrate was added; after terminating the reaction, the absorbance was measured. Four rats in each group were analyzed.

2.8. Statistical analysis Statistical analysis was performed using SPSS v.24.0 and R v.3.5.0 software. Statistical data were visualized with Prism 7.0 (GraphPad, La Jolla, CA, USA) and R v.3.5.0. Before statistical analysis, the data

were tested for normality, which is consistent with a two-factor factorial design. The rank sum test was used for non-normally distributed data, which are expressed as median and interquartile range. In the comparison of DTI and brain slice statistics, mean values of the bilateral hippocampus were analyzed. An independent samples t test was used to assess differences in quadrant memory time between the 9 pm and main groups. Statistical significance was defined at P < 0.05.

3. Results 3.1. Behavioral analysis in ClockLab 3.1.1. Period Both CRD models showed a prolonged circadian period compared to the NCR group (F = 23.966, P < 0.001) (Fig. 2A). The difference between CRD16/8 and CRD12/12 groups was not statistically significant (P > 0.05). Under the same LD conditions, there was no difference in CR period between SHR and WKY rats (F = 0.376, P > 0.05).

3.1.2. CR amplitude Both CRD models showed reduced CR amplitude (F = 10.234, P < 0.001) compared to the NCR group; however, the difference between the CRD16/8 and CRD12/12 groups was not statistically significant (P > 0.05) (Fig. 2B). Under the same LD conditions, there was no difference in CR amplitude between WKY and SHR rats (F = 1.047, P > 0.05).

3.1.3. Rest time Rest time was longer in the CRD16/8 group than in the CRD12/12 (P < 0.05) and NCR (P < 0.01)

groups (Fig. 2C). There was no difference in rest time between the CRD12/12 and NCR groups (P > 0.05). Under the same LD conditions, there was no difference in average rest time between SHR and WKY rats (F = 2.872, P > 0.05).

3.2. Analysis of cognitive function 3.2.1. Morris water maze test 3.2.1.1. Latency period The results from the 5-day training period for the Morris water maze in the main experiment are shown in Figure 3A. In the first 3 days of training, there was no difference in latency across CR groups. On days 4 and 5, the latencies were longer in SHR and WKY rats of both CRD groups than in the NCR group (P < 0.01). The CRD12/12 group showed a longer latency than the CRD16/8 group (P < 0.05). Under the same CR, latency was shorter for SHR rats than for WKY rats. The latency periods in the supplementary (9 pm) experiment were similar to those in the main experiment. In the first 3 days of training, there was no difference in the latency periods across CR groups (Fig. 4A). On days 4 and 5, the latencies were longer in SHR and WKY rats of both CRD groups than in the NCR group (P < 0.01) but unlike in the main experiment, the difference between CRD12/12 and CRD16/8 groups was non-significant (P > 0.05). Under the same LD conditions, the latency of SHR rats during the 5-day training was shorter than that of WKY rats.

3.2.1.2. Quadrant memory training In the main experiment, quadrant memory time was shorter in the CRD16/8 (P < 0.001) and CRD12/12 (P < 0.001) groups than in the NCR group (Fig. 3B). There was no difference in quadrant memory

time between the CRD16/8 and CRD12/12 groups (P > 0.05). The time was longer in WKY rats compared to SHR rats under the same LD conditions (F = 13.349, P < 0.001). A cluster analysis heatmap showed that quadrant memory time was longer in the WKY (NCR), WKY (CRD16/8), and SHR (NCR) groups than in the three other groups (Fig. 3C). In the 9 pm experiment, quadrant memory time was shorter in both groups of CRD rats than in NCR rats (P < 0.001) (Fig. 4B). Under the same LD conditions, quadrant memory time was shorter in SHR rats than in WKY rats (F = 10.662, P < 0.01). The bidirectional thermogram of the cluster analysis showed that the results were consistent with those obtained in the main experiment (Fig. 4C). There were no differences between WKY (CRD16/8) in the main group and WKY (CRD16/8) in the 9 pm group, SHR (CRD16/8) in the main group and SHR (CRD16/8) in the 9 pm group, WKY (CRD12/12) in the main group and WKY (CRD12/12) in the 9 pm group, and SHR (CRD12/12) in the main group and SHR (CRD12/12) in the 9 pm group in terms of quadrant memory time (P > 0.05; Fig. 4E). From the above results, we conclude that latency to find the platform in the Morris water maze decreased with increasing number of days in all groups. On days 4 and 5 of training, the search time was shorter in the NCR group than in the two CRD groups (both WKY and SHR rats), suggesting that CRD interfered with learning ability. Furthermore, in the quadrant memory training, the performance of CRD rats was worse than that of the NCR groups, although there was no difference between the CRD16/8 and CRD12/12 groups. In addition, under the same LD conditions, the memory performance of SHR rats was worse than that of WKY rats.

3.3. MRI (DTI + ASL) The FA values of the two CRD models were lower than that of the NCR group (F = 26.165, P < 0.001)

(Fig. 5A), but there was no difference in FA values between the CRD16/8 and CRD12/12 groups (P > 0.05). The hippocampal FA value was higher for WKY rats than for SHR rats under the same LD cycle (F = 40.89, P < 0.001). The λ⊥ values of both CRD groups were higher than those of NCR rats (F = 6.248, P < 0.01) (Fig. 5B). However, there was no difference between the CRD16/8 and CRD12/12 groups (P > 0.05), and the difference in λ⊥ between WKY and SHR rats under the same CR was not statistically significant (F = 0.558, P > 0.05). CR (F = 0.136, p > 0.05) and hypertension (F = 3.459, P > 0.05) models had similar λ‖ values (Fig. 5C). DTI revealed that both CRD models had reduced hippocampal FA and increased λ⊥ values, but there was no difference between CRD16/8 and CRD12/12 groups. Under the same CR, FA values of WKY rats were higher than those of SHR rats. There was no difference in λ⊥ between WKY and SHR rats. Hippocampal blood flow was lower in CRD models than in NCR rats (F = 10.905, P < 0.001) (Fig. 5D). There was no difference in blood flow between CRD16/8 and CRD12/12 groups (P > 0.05). Under the same CR, hippocampal blood flow was comparable between SHR and WKY rats (F = 0.075, P > 0.05).

3.4. Brain biopsy 3.4.1. Nissl staining Compared to the NCR condition, both CRD conditions decreased the number of neurons (Nissl+ cells) in the CA1 area (F = 10.069, P < 0.001) (Fig. 6A) although the difference between the two CRD conditions was non-significant (P > 0.05). Under the same CR conditions, the number of neurons (Nissl+ cells) in SHR rats was lower than that in WKY rats (F = 33.962, P < 0.001). Compared to the NCR groups, the number of neurons (Nissl+ cells) in the CA3 area of the two CRD models was significantly reduced (F = 44.055, P < 0.001) (Fig. 6B), but there was no difference between the two CRD models (P > 0.05). Under the same CR, there were fewer neurons (Nissl+ cells) in the CA3 area of SHR rats than WKY rats (F = 77.162, P < 0.001). The CR model (F =

0.849, P > 0.05) and blood pressure (F = 2.714, P > 0.05) did not affect the number of neurons (Nissl+ cells) in the DG (Fig. 6C). Nissl staining revealed that CRD had significant effects on the number of neurons in the CA1 and CA3 areas of the hippocampus in WKY and SHR rats. The effect was greater in SHR rats, which showed a significant reduction in the number of neurons in these two regions but not in the DG. The two CRD models did not differ in terms of the number of neurons.

3.4.2. GFAP The number of astrocytes (GFAP+ cells) was decreased in the CA1 area of both CRD models (F = 8.985, P < 0.01), but the difference between the two CRD conditions was not statistically significant (P > 0.05) (Fig. 6E). There were fewer astrocytes (GFAP+ cells) in SHR rats than in WKY rats under the same CR (F = 4.456, P < 0.05). CR had a statistically significant effect on the number of astrocytes (GFAP+ cells) in the CA3 area (F = 4.63, P < 0.05) (Fig. 6F), with a difference observed between NCR and CRD12/12 groups (P < 0.01) but not between CRD12/12 and CRD16/8 groups. There was no difference in the number of astrocytes (GFAP+ cells) in the CA3 area of the hippocampus between WKY and SHR rats under the same CR (P > 0.05). The CR model (F = 1.937, P > 0.05) and blood pressure (F = 0.909, P > 0.05) had no effect on the number of astrocytes (GFAP+ cells) in the DG (Fig. 6G). CRD reduced the number of astrocytes (GFAP+ cells) in the CA1 area in both WKY and SHR rats, which was more obvious in the latter. Rats in the CRD12/12 group had fewer astrocytes (GFAP+ cells) in the CA3 area whereas no difference was observed in the DG.

3.4.3. BDNF BDNF expression in the hippocampus was decreased by CRD (F = 8.206, P < 0.01) (Fig. 6I) but there

was no difference between the two CRD models (P > 0.05). Under the same CR, BDNF level in the hippocampus was lower in SHR rats than in WKY rats (F = 5.581, P < 0.05).

3.4.4. Syn-1 Syn-1 expression was downregulated under both CRD conditions compared to the NCR condition (F = 19.319, P < 0.001) (Fig. 6K), although the difference between the two CRD groups was non-significant (P > 0.05). Under the same CR, Syn-1 level in the hippocampus was lower in SHR rats than in WKY rats (F = 5.421, P < 0.05).

3.4.5 FJB staining CRD increased the number of degenerated neurons (FJB+ cells) in the CA1 (F = 8.057, P < 0.01) and CA3 (F = 7.823, P < 0.01) areas and DG (F = 4.443, P < 0.05) (Fig. 7A–C), whereas FJB staining did not differ between CRD12/12 and CRD16/8 in the CA1 (P > 0.05) or CA3 (P > 0.05) area or DG (P > 0.05). Under the same CR, there were no differences in the CA1 (F = 0.034, P > 0.05) and CA3 (F = 3.092, P > 0.05) areas of SHR and WKY rats, although the number of degenerated neurons (FJB+ cells) in the DG was higher in SHR rats than in WKY rats (F = 5.554, P < 0.05).

3.4.6. Iba-1 CRD caused an increase in the number of microglia (Iba-1+ cells) in the CA1 (F = 28.806, P < 0.001) and CA3 (F = 10.463, P < 0.001) areas and DG (F = 14.774, P < 0.001) (Fig. 8A–C). There was no difference between CRD12/12 and CRD16/8 groups in these areas (all P > 0.05). Under the same CR, there were no differences between SHR and WKY rats in terms of microglia (Iba-1+ cells) in the CA1 (F = 0.093, P > 0.05)

and CA3 (F = 2.017, P > 0.05) areas and DG (F = 0.038, P > 0.05).

3.4.7. Caspase Compared to the NCR condition, caspase was upregulated by CRD (F = 3.803, P < 0.05) (Fig. 8E) although the difference between the two CRD groups was non-significant (P > 0.05). Under the same CR, the caspase expression was higher in SHR rats than in WKY rats (F = 4.581, P < 0.05).

3.4.8. NF-κB NF-κB level was elevated in the CRD models compared to the NCR groups (F = 3.618, P < 0.05) (Fig. 8F), but there was no difference between the two CRD groups (P > 0.05). Under the same CR, there was no difference in NF-κB expression between SHR and WKY rats (F = 0.136, P > 0.05).

3.5. Serologic parameters NSE level was higher in CRD rats then in the NCR group (F = 5.974, P < 0.05) (Fig. 8I). However, there was no difference in NSE level between the CRD16/8 and CRD12/12 groups (P > 0.05). NSE level was higher in SHR rats than in WKY rats under the same CR (F = 15.964, P < 0.001). IL-6 level was higher in the CRD12/12 group than in the CRD16/8 (P < 0.01) and NCR (P < 0.01) groups (Fig. 8J), while no difference was observed between the CRD16/8 and NCR groups (P > 0.05). Under the same CR conditions, there was no difference in IL-6 level between SHR and WKY rats (F = 0.04, P > 0.05).

4. Discussion CRDs are associated with accumulation of amyloid β protein (Wang et al., 2016), perturbation of

neurotransmitter secretion (Hampp et al., 2008; Zhong, Naismith, Rogers, & Lewis, 2011), and abnormal expression of mitochondrial respiratory chain proteins in brain tissue (Ren et al., 2016), which have adverse effects on cognitive function. The expression of clock genes that play important roles in cognition and hippocampal plasticity may also be disrupted (Wardlaw, Phan, Saraf, Chen, & Storm, 2014; Snider et al,.2016; Morioka et al., 2016; Gao et al., 2010; Van der Zee et al,. 2008; Schnell et al., 2014). To date, this is the only study investigating the effects of CRD in a background of hypertension. In China, 250 million adults have hypertension (Mao & Jing, 2017) and owing to fast-paced lifestyles, many of these individuals will have CRD. The results of our study show that in rats with CRD, cognitive function and hippocampal neuron number and plasticity were reduced in SHR rats compared to WKY rats. In the Morris water maze test, SHR rats had a shorter latency to find the platform than WKY rats, although this was mainly due to the hyperactivity of the former group (Ferguson & Cada, 2003). These results merit further study as they suggest that CRD and hypertension synergize to exert more severe effects than either condition alone. CRD is known to decrease the amplitude of biorhythms, leading to the degradation of biologic functions through mechanisms that remain unclear (Baburski, Sokanovic, & Kostic, 2017; Gloston, Yoo, & Chen, 2017). Our experiments showed that the CR amplitude was reduced in both types of CRD rat compared to control NCR rats, which was accompanied by a cognitive decline. CRD12/12 and CRD16/8 have variable effects in organisms (Cui et al., 2016; Laura et al., 2010); this was confirmed by our observation that behavior was less affected by CRD16/8 than by CRD12/12. On the other hand, rest time was longer for CRD16/8 than for CRD12/12, suggesting that increasing rest time can reduce the adverse effects of CRD. DTI provides an accurate way to measure of hippocampal plasticity (Kantarci et al., 2011) and microstructural changes in the brain (Ding et al., 2013). Specifically, FA values represent the percentage of water molecules with different degrees of heterogeneity (Yoo et al., 2013); most experiments have shown that a

decrease in hippocampal FA values is associated with a decline in cognitive function (Nir et al., 2013). λ‖ and λ⊥ are important parameters in DTI and represent the direction of water molecules along the axon and perpendicular to the direction of axonal diffusion, respectively. An increase in λ⊥ indicates that the myelin of axons is damaged (Sen & Baser, 2005). In this experiment, there was no difference in λ‖ between the experimental groups, but the increase in λ⊥ suggests that pathologic changes may have occurred in the plasma membrane and myelin of neurons. Nissl staining revealed that CRD model WKY and SHR rats had fewer hippocampal CA1 and CA3 neurons. In SHR rats, CRD had a greater impact on the number of CA1 and CA3 neurons. Although CRD and hypertension did not affect the number of DG neurons, a downward trend was still observed. The CA1 and CA3 areas and DG of the hippocampus have distinct structure and function (Kazu, Thomas, McHugh, Matthew, & Susumu, 2004; Vera, Erika, Nozomu, Motoharu, & Magdalena, 2018; Chi-Wing, Gabriel, Judith, Timothy, & Norbert, 2018; Herb, Wisden, Catania, Maréchal, Dresse, & Seeburg, 1997). Neurons in the DG exhibit greater resistance to insults such as ischemia compared to those in the CA1 and CA3 areas (Liam, Stefano, & René, 2013) through activation of multiple signaling pathways (Diego et al., 2014). The CA3 area and DG act cooperatively in functions related to memory (Yassa & Stark, 2011). Electrophysiological recordings have shown that electrical activity in the hippocampus begins in CA1 before moving to CA3 (Kazu, Thomas, Matthew, & Susumu, 2004). Thus, a decrease in the number of neurons in any of the three regions caused by CRD or hypertension can lead to decreased memory function. To determine whether the decrease in our study was due to neuronal death, we used FJB, an anionic fluorescent dye with high affinity for degenerated neurons (Schmued, Albertso, & Sliker, 1997; Schmued & Hopkins, 2000; Yu et al., 2012). We found that FJB staining intensity was enhanced in the hippocampus of CRD rats, confirming that neuronal degeneration was increased (Andreone, Larhammar, & Lewcock, 2019; Wu, Dejanovic, & Gandham, 2019). We also used an antibody

against Iba-1, a marker of microglia (Salter & Stevens 2017), to assess the damage to brain tissue caused by CRD. Under pathologic conditions, the number of microglia is increased (Fekete et al., 2019). NSE is an enzyme in neurons; elevated serum levels of NSE imply its release into the circulation from damaged neurons (Gójska-Grymajło, Zieliński, Wardowska, Gąsecki, Pikuła, & Karaszewski, 2018). Neuronal death is a highly detrimental pathological change in the nervous system (Prusiner, 2001). As neurons in adult animals are mostly non-renewable (Werner & Nave , 2019; Schmued, Stowers, Scallet, & Xu, 2005), an increase in neuronal death can lead to a reduction in the number of neurons. Given that neurons are the basic structural and functional unit of the nervous system (Goldman-Rakic, 1999; Toran-Allerand, Miranda, Bentham, Sohrabji, Brown, Hochberg, & MacLusky, 1992) and that the hippocampus is important for memory function, loss of neurons in this area will inevitably lead to cognitive decline. In this study, we observed by Nissl staining that CRD significantly reduced the number of neurons in the hippocampus, which was associated with cognitive impairment. In contrast, fewer degenerated neurons were observed by FJB staining. This discrepancy can be explained as follows. In this experiment, we only observed changes in the number of degenerated neurons in the rat hippocampus in the third month and saw no changes at other times (eg, in the first or second month). Given that rats were exposed to an abnormal photoperiod for 3 months, we speculate that neuronal death occurred continuously and phasically during this period. Thus, although a quantitative analysis of FJB staining revealed that neuronal degeneration in the hippocampus was increased in the third month, this was not representative of the entire experimental period. Additionally, the FJB staining protocol has certain limitations such as a weaker staining intensity than FJC staining (Schmued, Stowers, Scallet, & Xu, 2005); therefore, some degenerated neurons may have been overlooked, leading to an underestimation of neuronal death. Nonetheless, the results demonstrate that after 3 months of CRD, the rate of neuronal death in the hippocampus was increased relative to rats with a normal CR, suggesting that CRD causes the death of

hippocampal neurons. CRD may induce neuronal degeneration in the hippocampus by reducing blood flow to the brain. ASL allows the measurement of blood flow in specific brain regions. We found by ASL that blood flow was significantly reduced in the hippocampus of CRD rats. CRD also triggers inflammation, as evidenced by the upregulation of NF-κB in the hippocampus and of IL-6 in serum. NF-κB plays an important role in the immune and inflammatory responses and in apoptosis (Ghanizade, 2011). Increased inflammation can lead to a variety of neurologic diseases (Yamaguchi et al, 2019; Gomes, Cunha, Nascimento, Ribeiro, Vaz, & Brites, 2019). Our observations suggest that the inflammatory response induced by CRD contributed to hippocampal injury. Increased caspase levels triggered by CRD can also cause neuronal death (Narkilahti, Pirttilä, Lukasiuk, Tuunanen, & Pitkänen, 2003). Caspase-3 is a downstream regulator of the mitochondrial and Fas signalingmediated apoptosis pathways (Didonna, Sussman, Benetti, & Legname, 2012; Beckham, Tuttle, & Tyler, 2010). Taken together, these are some of the possible mechanisms that may be responsible for the decreased number of neurons in CRD. Astrocytes, which express GFAP, play an important role in hippocampal plasticity (Pixley, Kobayashi, & de Vellis, 1984) by promoting synapse formation (Sofroniew & Vinters, 2010; Nedergaard, Ransom, & Goldman, 2003) and neuron excitability (Shigetomi, Bowser, Sofroniew, & Khakh, 2008). A reduction in the number of astrocytes in the hippocampal CA1 and CA3 areas has been linked to spatial memory decline in rats (Perea, Navarrete, & Araque, 2009; Zappa, López, Falomir, Trípodi, Morel, & Reggiani, 2018) accompanied by structural and functional degradation (Lana, Ugolini, & Nosi, 2017). The number of astrocytes is higher in the DG than in other areas of the hippocampus (Larsson, Wilhelmsson, Pekna, & Pekny, 2004). Our results demonstrate that CRD affects hippocampal plasticity by affecting astrocytes. To evaluate hippocampal plasticity, we examined the expression of BDNF and Syn-1. BDNF plays

important roles in the survival, differentiation, growth, and development of neurons as well as in plasticity (Cowansage, LeDoux, & Monfils, 2010; Fleitas et al., 2018), synapse formation (Lipsky & Marini, 2007), and long-term potentiation (Leal, Comprido, & de Luca, 2017). Syn-1 is a marker for synaptic plasticity (Farisello, Boido, & Nieus, 2013) whose downregulation has been linked to cognitive dysfunction (Froula et al., 2018). We found that CRD reduced BDNF and Syn-1 levels and that expression of the latter was lower in SHR rats under CRD than in rats on a normal LD cycle. These results indicate that CRD and hypertension decrease hippocampal plasticity by inhibiting BDNF and Syn-1 expression. We used 90 rats in our experiments, of which 36 were subjected to the Morris water maze test (9 pm group) and 54 were subjected to all experiments (main group). Unlike traditional behavioral experiments, our strategy was to use CRD rats, which exhibit more marked phase changes than NCR rats. This situation was complicated by the lack of relevant information on when CRD rats should be tested. According to some reports, under NCR conditions, the Morris water maze test conducted under different ZTs on the same day does not reveal differences in behavioral performance (Howard, Ana, Martin, & Theresa, 2012). In order to ensure that all rats were subjected to the Morris water maze test under the same phase and ZT, those in the main experimental group were tested under a normal LD cycle and at the same ZT (ZT1, 09:00). However, we also considered that after 90 days of abnormal LD entrainment, CRD rats may have developed a set biorhythm and phase every 3 days. We therefore added the 9 pm group, which was exposed to an abnormal LD cycle on days 94–96. This experiment was also carried out at the same ZT (ZT1, 21:00). Our results showed no difference between the main and 9 pm groups, indicating that circadian phase had no effect on the behavior of CRD rats. ClockLab behavioral analysis confirmed that the CR amplitude of CRD rats was decreased—that is, they did not exhibit the characteristic oscillations observed in NCR in a typical 24-h cycle. Thus, the results of the Morris water maze test were a true reflection of cognitive performance.

This study had some limitations. After statistical analysis, DTI and brain biopsies showed that the structure and plasticity of the hippocampus were similar in the CRD16/8 and CRD12/12 groups, which was unexpected but suggests that CRD16/8 is less disruptive than CRD12/12, as confirmed by the behavioral experiments. It is possible that our observation period of 3 months was too short to detect alterations in cognitive function. In summary, our results demonstrate that CRD has adverse effects on the hippocampus in WKY and SHR rats, which are manifested as memory loss, changes in hippocampal plasticity, and reduced blood flow in the brain. These effects of CRD were more pronounced in hypertensive rats. Additionally, rats in the CRD16/8 group showed superior performance in the behavioral tests to those in the CRD12/12 group. These findings suggest that prolonging rest time can mitigate the extent of cognitive decline caused by CRD, which has important clinical implications for sleep and neurodegenerative disorders in humans.

Contributions YunLei Wang and Tong Zhang designed the experiments. YunLei Wang conducted the experiments. YunLei Wang, YuGe Zhang, WenZhu Wang, and Xu Liu analyzed the data. YunLei Wang and Tong Zhang wrote the manuscript. Tong Zhang, YaFei Chi, and BaoGui Zhang supervised the project.

Declaration of Competing Interest The author(s) declare no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Acknowledgments

This work was supported by the Special Fund for Basic Scientific Research of Central Public Research Institutes (grant no. 2017CZ-1).

Figure legends: Figure 1. A–C. CR modeling and testing times of the NCR, CRD12/12, and CRD16/8 groups for both the main and 9PM experimental groups.

Figure 2. A–C. Statistical analysis of CR period, amplitude, and rest time. D. 24-h Rhythm activity profiles in the six groups of rats. *P < 0.05, **P < 0.01, ***P < 0.001 (least significant difference t test).

Figure 3. Figures A-D show the Morris water maze results for the rats in the main experimental group. A shows the changes in navigation training of the six groups of rats during the 5 days of training. Statistical significance is shown in Figure A. * represents the difference in blood pressure factors, *p < 0.05, **p < 0.01, ***p < 0.001; # represents the difference in the values for different circadian rhythms, #p < 0.05, ##p < 0.01, ###p < 0.001. The statistical significance of quadrant memory tests in the six groups of rats is shown in Figure B, *p < 0.05, **p < 0.01, ***p < 0.001 (least significant difference t test). Two-way heatmap results for quadrant memory training of the six groups of rats are shown in Figure C. The trajectories of quadrant memory training in the six groups of rats are shown in Figure D. The recognition coefficients and statistical results of the novel object recognition experiments in the six groups of rats are shown in Figure E. The preference index and statistical results of the novel object recognition experiments in the six groups of rats are shown in Figure F. The bidirectional thermogram statistical analysis for the recognition coefficients of the six groups of rats are shown in Figure G. *p < 0.05, **p < 0.01, ***p < 0.001 (least significant difference t test).

Figure 4. This figure shows the Morris water maze results for the rats in the “9 PM” experimental group. We copied data from NCR rats of the main test group to compare with the data for CRD rats in the “9 PM” group.

Figure A shows the changes in navigation training of the six groups of rats during the 5-day training period and their statistical significance. * represents the difference in blood pressure factors, *p < 0.05, **p < 0.01, ***p < 0.001; # represents the difference in values for different circadian rhythms, #p < 0.05, ##p < 0.01, ###p < 0.001. The statistical significance of quadrant memory tests in the six groups of rats is shown in Figure B, *p < 0.05, **p < 0.01, ***p < 0.001 (least significant difference t test). Two-way heatmap results for quadrant memory training in the six groups of rats are shown in Figure C. The trajectories of the quadrant memory training in the six groups of rats are shown in Figure D. Figure E shows the comparison of quadrant memory times for the “9 PM” and main groups. Independent sample t-test was used to compare the difference in quadrant memory time between “9 PM” and main groups. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5. A–D. FA value, λ⊥, λ‖, and blood flow in the hippocampus. E. Blood flow in the hippocampus (arrow) in the six groups of rats. Red and blue represent high and low blood flow, respectively. *P < 0.05, **P < 0.01, ***P < 0.001 (least significant difference t test).

Figure 6. The statistical results for hippocampal CA1, CA3, and dentate gyrus neurons (Nissl+ cells) are shown in Figures A–C. *p<0.05, **p<0.01, ***p<0.001 (least significant difference t test). Images from all six groups of Nissl-stained CA1 regions are shown in Figure D. Scale bar = 50 μm. The statistical results for the hippocampal CA1, CA3, and dentate gyrus astrocytes (GFAP+ cells) are shown in Figures E–G, respectively. *p<0.05, **p<0.01, ***p<0.001 (least significant difference t test). Images of astrocytes in the CA1 region of the six groups of rats are shown in Figure H. Scale bar = 50 μm. The statistical results for the hippocampal BDNF expression are shown in Figure I. *p<0.05, **p<0.01, ***p<0.001 (least significant difference t test). Representative images of BDNF-stained hippocampus from each of the six groups are shown in Figure J. Scale

bar = 500 μm. The statistical results for the hippocampal Syn expression are shown in Figure K. *p<0.05, **p<0.01, ***p<0.001 (least significant difference t test). Representative images of Syn expression from the six groups of rats are shown in Figure L. Scale bar = 500 μm.

Figure 7. A–C. Quantification of cells positive for FJB staining in the hippocampus CA1 and CA3 areas and DG. *P < 0.05, **P < 0.01, ***P < 0.001 (least significant difference t test). D. Representative images of FJBpositive hippocampus (Scale bar = 500 μm), CA1 (Scale bar = 50 μm), CA3 (Scale bar = 50 μm) and –DG (Scale bar = 50 μm) area in the six groups.

Figure 8. A–C. Quantification of cells positive for Iba-1 immunolabeling in the hippocampus CA1 and CA3 areas and DG. *P < 0.05, **P < 0.01, ***P < 0.001 (least significant difference t test). D. Representative images of microglia (Iba-1+ cells) in the CA1 area in the six groups of rats. Scale bar = 50 μm. The statistical results for the hippocampal Caspase expression are shown in Figure E. *p<0.05, **p<0.01, ***p<0.001 (least significant difference t test). Representative images of Caspase in the hippocampal expression from each of the six groups are shown in Figure G. Scale bar = 500 μm. The statistical results for the hippocampal NF-κB expression are shown in Figure F. *p<0.05, **p<0.01, ***p<0.001 (least significant difference t test). Representative images of NF-κB in the hippocampal expression from each of the six groups are shown in Figure H. Scale bar = 500 μm. I and J, serum NSE and IL-6 data of the six groups of rats. *p<0.05, **p<0.01, ***p<0.001 (least significant difference t test).

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Table 1: Statistical analysis of mean arterial pressure before the experiment in six groups of rats (X±S). Group

Normal circadian rhythm

Circadian Rhythm Disorder (16/8)

Circadian Rhythm Disorder (12/12)

SHR

160.34±12.1 mmHg

162±12.84 mmHg

161.45±12.04 mmHg

WKY

78.75±17.48 mmHg

80.78±12.25 mmHg

81.11±13.48 mmHg

Highlights 1.

CRD can induce memory loss.

2.

The numbers of neurons and astrocytes in the hippocampus are decreased in CRD.

3.

Hypertension aggravates the adverse effects of CRD.

4.

Extending rest time mitigates the adverse effects of CRD.