Dexmedetomidine prevents spatial learning and memory impairment induced by chronic REM sleep deprivation in rats

Abstract

The study was attempted to investigate the effect on and mechanisms of action of dexmedetomidine with regard to learning and memory impairment in rats with chronic rapid eye movement (REM) sleep deprivation. A total of 50 male Sprague Dawley rats were randomly divided into five groups. Modified multiple platform method was conducted to cause the sleep deprivation of rats. Dexmedetomidine and midazolam were administered by intraperitoneal injection. Learning and memory ability was assessed through Morris water maze. Morphological changes of rat hippocampal neurons and synaptic were detected by transmission electron microscope and Golgi staining. The gene expression in hippocampus of each group was detected by RNA-seq and verified by RT-PCR and western blot. REM Sleep-deprived rats exhibited spatial learning and memory deficits. Furthermore, there was decreased density of synaptic spinous in the hippocampal CA1 region of the sleep deprivation group compared with the control. Additionally, transmission electron microscopy showed that the synaptic gaps of hippocampal neurons in REM sleep deprivation group were loose and fuzzy. Interestingly, dexmedetomidine treatment normalized these events to control levels following REM sleep deprivation. Molecular biological methods showed that Alox15 expression increased significantly after REM sleep deprivation as compared to control, while dexmedetomidine administration reversed the expression of Alox15. Dexmedetomidine alleviated the spatial learning and memory dysfunction induced with chronic REM sleep deprivation in rats. This protective effect may be related to the down-regulation of Alox15 expression and thereby the enhancement of synaptic structural plasticity in the hippocampal CA1 area of rats.

Supplementary Information

The online version contains supplementary material available at 10.1007/s41105-023-00450-8.

Introduction

Sleep is a biologic process that is essential for life, which plays a critical role in consolidating learning and memory [1, 2]. Sleep is divided into rapid eye movement (REM) sleep and non-rapid eye movement (NREM) sleep. REM sleep can support cortical plasticity [3] and facilitate integration memories [4] and episodic and emotional memory consolidation [5, 6]. Insomnia is the most common sleep problem, affecting between 10 and 20% in the general population, with approximately 50% having a chronic course [7]. Insomnia is linked to significant distress or impairment in key areas of functioning, especially cognitive performance [8]. Benzodiazepines are still the most frequently prescribed hypnotics in improving both sleep onset and sleep maintenance because of their wide availability [9]. However, long-term treatment with benzodiazepines has also been described as impairing cognitive performance [10].

Dexmedetomidine (Dex), a highly selective, short-acting alpha 2 adrenoreceptor agonist, is frequently used during perioperative anesthesia. It is reported that Dex not only improves postoperative sleep quality, but also ameliorates the incidence of postoperative delirium and cognitive dysfunction [11–13]. Our previous work proposed that Dex might be a potential treatment for patients with chronic insomnia [14, 15]. However, the efficacy of Dex in the treatment of cognitive dysfunction caused by insomnia needs further clarification.

Sleep deprivation animal research has been vital to the elucidation of mechanisms that underlie sleep as well as the pathology related to sleep loss. We used the modified multiple platform method (MMPM) to induce rapid eye movement (REM) sleep deprivation of 20 h for 21 consecutive days in rats [16, 17]. The rats were regularly exposed to stresses that artificially reduced their sleep time, to maximally simulate the state of long-term lack of sleep associated with chronic insomnia.

A considerable body of evidence suggests that REM sleep deprivation can impair forms of synaptic plasticity [18], subsequently reducing spatial learning and memory. In this study, we investigated whether Dex can counteract the learning and memory impairment caused by chronic REM sleep deprivation as well as potential mechanisms for that protection. A water-soluble benzodiazepine, midazolam, was used as a comparator to Dex as it is commonly used in humans in the treatment of chronic insomnia.

Materials and methods

Animals

Fifty 5-month-old male Sprague Dawley rats weighing 300–350 g were provided by Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China). Rats were housed under controlled ambient environmental conditions (food and water ad libitum, temperature 22 ± 2 °C, relative humidity 50 ± 5%, 12-h light/12-h dark cycle, lights on at 8 a.m.). After adaption for one week, the rats were randomly assigned to five groups: normal control group (Con), wide platform control group (WPF), sleep deprivation group (SD), sleep deprivation with Dex supplement group (DEX), and sleep deprivation with midazolam supplement group (MID) (n = 10, respectively).

REM sleep deprivation

REM sleep deprivation was conducted using the modified multiple platform method (MMPM) [19]. Fifteen small rounded platforms (6.5 cm in diameter, 8 cm in height) were evenly fixed in a plastic water box (110 cm in length, 60 cm in width, 40 cm in height). The water level was controlled to just below the platform for a depth of 1 cm and was maintained at a temperature of 22 ± 2 °C. The distance between the two rounded platforms (edge to edge) was 15 cm in order to allow the rats free movement from one platform to another, but would fall into the water if they fell asleep. Food and water were placed in the top of the box and available ad libitum. The container was cleaned every day. Before REM sleep deprivation, rats were placed in the modified multiple platform water box for 2 h every day, for 3 consecutive days to adapt to the environment. Rats were sleep deprived 20 h per day (from 20:00 PM to 16:00 PM of the next day), after which they were allowed to sleep 4 h in their home cages during the last of the light phase (from 16:00 PM to 20:00 PM). The REM sleep deprivation process lasted for a total of 21 days [16]. For the WPF group, wide platforms with 18 cm diameter, which permitted animals to sleep without falling into the water, were used with the aim of assessing possible stresses of the aquarium environment.

Drug administration

Dex (Yangtze River Pharmaceutical Group Co., Ltd., Jiangsu, China) was diluted in 0.9% normal saline to a final concentration of 4 μg/mL and was administered intraperitoneally according to the weight of rats (20 μg/kg) [20, 21]. Midazolam (Jiangsu Nhwa Pharmaceutical Co., Ltd., Jiangsu, China) was administered at a concentration of 2 mg/kg body weight [22, 23]. Rats in the DEX group were treated with Dex once a day at 12:00 am, consecutively for 21 days, while WPF and SD groups received intraperitoneal administration of normal saline.

Morris water maze

The Morris water maze (MWM) task was used to measure spatial learning and memory [24]. The experimental apparatus consisted of a cylindrical tank (150 cm diameter, wall depth 60 cm) filled with opaque water (21 ± 1 °C) and divided into four quadrants of equal area. The tank was painted black and surrounded by permanent visual cues. An invisible platform (15 cm diameter and 35 cm high) was submerged 1 cm beneath the water surface and placed in the center of one quadrant. In the training trial 1, rats were trained four trials per day with 20 min intertrial intervals and allowed to find the hidden platform. Rats that failed to reach the escape platform within 60 s were manually guided to the platform and allowed to remain on it for 15 s. After that, the rat’s hair was dried, and the rat returned to the cage. Escape latency represented spatial learning ability. We also conducted a probe test on the last day of each water maze test set, which was 24 h after training. In the probe test 1, the platform was removed, and each rat was allowed to swim for 60 s. The time spent in the target quadrant and the number of crossings in the target quadrant were analyzed as a criterion for spatial memory retention. After 16-day sleep deprivation, the reversal MWM test, including training 2 and probe test 2, was conducted. The platform was placed in the opposite quadrant from the previous location that housed the hidden platform. Data collection was automated by computerized tracking/image analyzer system. The flow diagram of this experiment is shown in Fig. 1.

Fig. 1.

Schematic diagram of the experimental procedure

Golgi staining

To obtain samples for Golgi staining, after being anesthetized with pentobarbital sodium, the rats were perfused with 0.9% saline, followed by 4% paraformaldehyde. After that, the brains were removed. A Golgi Staining Kit (FD Neuro Technologies) was used for subsequent tissue preparation and staining procedures, and the entire procedure was performed in strict accordance with the manufacturer’s user manual and material safety data sheet. The extracted brain was immersed in the Rapid Golgi-cox solution (solution A/B) prepared one day in advance (the solution was changed after 24 h) for 14 days and then transferred to the Solution C (the solution was changed after 24 h) for three days. The whole process was carried out at room temperature and protected from light. A series of 150-micron coronal sections were cut with a vibratome, and the sections were then applied to gelatin-coated slides. The slides were further processed in accordance with the manufacturer’s instructions and finally covered with Permount Mounting Medium. The pyramidal neurons in the CA1 regions were selected for dendritic spines analysis. The Image software was used to analyze the images. The number of spines was counted by double-blind hand counter (n = 3 rats per group, 5 images per animal).

Transmission electron microscopy

The rats were killed after being anesthetized with pentobarbital sodium at the end of the studies. The hippocampal CA1 region was rapidly dissected free and immersed in 3% glutaraldehyde for 24 h and then rinsed with 0.1 M phosphate buffer 3 times. The tissues were then fixed with 1% osmium tetroxide (Sigma, St. Louis, MO) for 2 h, dehydrated, embedded in araldite for 24 h, and cut into 1 μm plastic sections. The sections were observed under a transmission electron microscope (TEM; H-9000NARIbaraki, Hitachi Ltd., Tokyo, Japan) after staining in uranyl acetate. The experimenters were blind to the experimental groups during the reading of TEM images.

Next-generation sequencing (RNA-Seq)

The rats were killed immediately at the end of the studies. Brain tissues were rapidly dissected on ice to separate the hippocampus and then stored in tubes at -80 °C until assayed. Total RNA was extracted from hippocampus of rats in the Con, SD, and DEX groups (five rats in each group), and the quality of the RNA was assessed by Bioanalyzer. Samples with A260/A280 > 1.8 and RNA integrity number (RIN) > 8 were used for library construction. The mRNA was purified using oligo dT magnetic beads and fragmented into small pieces and then followed by reverse transcription to double-stranded cDNA. End repair was then performed on these cDNA fragments as well as adaptor ligation, and 5–6 cycles of PCR enrichment. Sequencing was performed by Illumina Hi-Seq 2500 according to standard manufacturer instructions (Illumina, San Diego, California, USA). The raw reads were first filtered into clean reads, followed by mapping to the rat reference genome (build GRCm38) using hisat2, and quantification of gene level expression was performed using featureCounts. Sequencing and quantification of transcription were mainly completed by the Beijing BerryGene Co. (Beijing, China). The NOISeq method was adopted to screen the differentially expressed genes (DEGs) between the Con and SD groups, as well as the SD and DEX groups according to the criteria of a fold change ≥ 1.5 and p < 0.05. Functional processes significantly overrepresented in the data set were determined using the Database for Annotation, Visualization and Integrated Discovery (DAVID) overrepresentation tool on the Gene Ontology (GO) database.

RNA extraction and real-time RT-PCR

Total RNA was isolated from the hippocampus using the RNeasy Mini kit (Qiagen, Alameda, CA), and then reverse transcribed by use of a one-step RT-PCR kit (TaKaRa Bio, Shiga, Japan). Sequence-specific primers were synthesized as follows:

GAPDH forward, 5′-CTGGAGAAACCTGCCAAGTATG-3′;

GAPDH reverse, 5′-GGTGGAAGAATGGGAGTTGCT-3′;

ALOX15 forward, 5′-GTTCAGGAAACATAGGGAAGAGG-3′;

and ALOX15 reverse, 5′-CGTTGGTCTACAGGGAGGTCAG-3′.

GAPDH was used as a control for RNA loading and reverse transcription efficiency.

Western blot

The hippocampal tissues were homogenized using mechanical grinding in RIPA lysis buffer containing a protease inhibitor cocktail (Thermo Pierce, Rockford, IL, USA). The homogenates were centrifuged at 10,000 rpm for 20 min at 4 °C, and the supernatant was collected and quantified using the BCA Protein Assay Kit (Thermo Pierce, Rockford, IL, USA) with bovine serum albumin as the standard. Protein samples (50 μg) were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto 0.45 μm polyvinylidene fluoride Membrane (Millipore, MA, USA). This membrane was blocked with 5% skim milk and then incubated at 4 °C overnight with the following primary antibodies: rabbit anti-GAPDH antibody, anti-β-actin (1:1000; Abmart, Shanghai, China). Next, the membranes were washed in 0.1% TBST buffer and treated with secondary antibody (1:2000; Vector Laboratories) for 2 h at 37 °C. The bands were visualized by use of enhanced chemiluminescence detection kit (Santa Cruz Biotechnology), and data were analyzed by using Image J software. GAPDH was used as control to obtain the target-protein/GAPDH ratio.

Primary neuron culture

The 1-day neonatal Sprague Dawley rats were euthanatized, immersed in 75% alcohol, and then decapitated. The hippocampus were dissected in HBSS under a stereo microscope in a sterile environment. The harvested hippocampi were digested by 0.25% trypsin at 37 °C for 30 min and dissociated into single cells by gentle trituration. Cells were resuspended and seeded onto poly-lysine-coated plates at a density of 4 × 105 cells/mL in DMEM (Gibco, NY, USA) containing 10% F12 and 10% FBS, 1% glutamine at 37 °C in a humidified 5% CO₂ atmosphere. Eight hours after plating, the plating medium was changed to neurobasal medium (Gibco, NY, USA) supplemented with 2% B27, 1% glutamine and cytosine arabinoside 10 μmol/L, and half of the medium was replaced every 2 days. Experiments were performed at 14 days in vitro. The cells were pretreated with different concentrations of Dex (1 μM, 10 μM, 20 μM) for 1 h to observe the length of dendrites and the growth state of neurons, in order to select the appropriate concentration to continue the next experiment. The hippocampal neuron incubated with Dex was collected to assess the levels of Alox15 proteins. Samples for detection in each group were repeated at least three times, and each group had at least three independent batch samples.

Statistics

The data were shown as mean ± SEM. SPSS 22.0 software (SPSS Inc., Chicago, Illinois, USA) and GraphPad Prism 7.0 were used for the analysis of all data. Data on the escape latency in the MWM test were subjected to a repeated-measures two-way ANOVA followed by the Bonferroni post hoc test. The Kruskal–Wallis with Dunn’s multiple comparison test was used to determine the difference on platform crossing times. In the western blotting and PCR experiments, we used one-way ANOVA followed by LSD multiple comparison tests to compare expressions between groups. A P value < 0.05 was considered statistically significant.

Results

Dex ameliorates spatial learning and memory deficits caused by REM sleep deprivation

To assess if Dex has a positive effect on cognitive function in sleep-deprived rats, we used MWM to assess the spatial learning and memory. During training stage 1, there were no differences among all groups in escape latency (Fig. 2A), which confirmed the equal learning ability prior to REM sleep deprivation. After 16-day REM sleep deprivation, the reversal MWM test was conducted. In training stage 2, Con rats showed similar performance to the WPF group (Fig. 2B). However, when compared with the WPF group, the SD group demonstrated significantly increased escape latencies. The performance of the DEX group was significantly improved when compared with the SD group. These results suggest that 20 μg/ml Dex administration ameliorated the spatial learning impairment of SD rats; however, there are no significant differences between SD group and the MID group.

Fig. 2.

REM sleep deprivation-induced learning and memory impairment was prevented by dexmedetomidine. A The performance of rats in finding the hidden platform prior to REM sleep deprivation. B Escape latency to find the hidden platform in the Morris water maze in each group after sleep deprivation. C The numbers of rats crossing through platform. D The time spend in platform area. E The mean speed of rats swimming in the probe test. F Typical swimming patterns showed the swimming path of the rats in the second probe test. ANOVAs were followed by Tukey–Kramer corrections. *P < 0.05, **P < 0.01. Data are expressed as the mean ± SEM (n = 10 in each group)

For the analysis of the probe trials, which evaluated the retention of spatial memory, we recorded the number of crossings in platform areas and the percent time spent in the target quadrant. The results of probe test 1 demonstrated that there was no difference in the number of platform crossings, nor the average time spent in the target quadrant for all groups prior to sleep deprivation (Fig. 2C). Probe test 2 was performed on the following day of training 2. The crossing frequency of the SD group was reduced as was the time spent in the target quadrant, when compared to the WPF group. DEX groups showed increased crossing numbers compared to the SD group and had more crossing numbers in the platform areas. The MID group, however, showed no significant differences compared with the SD group (Fig. 2C, D). No significant differences were observed in swimming path length among the groups (Fig. 2E). Typical swimming path of each group in the probe test 2 is shown in Fig. 2F. These results demonstrate that Dex administration significantly improves spatial learning and memory deficits in REM sleep-deprived rats, while Mid does not.

DEX prevents the reduction of dendritic spines and regulates the ultrastructure of synapses in hippocampal CA1 neurons following REM sleep deprivation

The hippocampus CA1 region is one of the brain regions in rodents in charge of higher nervous activities, including spatial learning and memory. It is well established that synaptic function is closely associated with the integrity of synaptic structure. We further explored whether DEX could change the morphology of dendritic spines enumerated by Golgi staining. When the number of dendritic spines per 20 μm was counted (Fig. 3A, B), we found that when compared with the Con group, there were fewer spines on CA1 neuronal dendrites in the SD group (**P < 0.01), whereas the DEX group demonstrated significant attenuation in the decrease of dendritic spines on CA1 neuronal dendrites (*P < 0.05), suggesting an increase in synaptic transmission efficiency.

Fig. 3.

The changes of synaptic spine density and synaptic ultrastructure in hippocampal CA1 neurons. A Alteration of synaptic spine density on hippocampal CA1 neurons visualized with Golgi staining, (a–c) Golgi staining of hippocampal neurons in indicated groups of rats, (d–f) Magnified images of a–c. Golgi staining of dendritic spines in hippocampal CA1 neurons in indicated groups of rats. A magnified view of the black boxed areas is shown on the bottom side of the group. B Number of dendritic spines per 20 μm on hippocampal CA1 neurons in indicated groups of rats. Data are expressed as the mean ± SME (n = 3), *P < 0.05, **P < 0.01. C The changes in the synaptic ultrastructure of the hippocampus CA1 neurons were observed under TEM, (g–i) Ultrastructure of synapses in each group, (j–l) Magnified images of g–i

The changes in the synaptic ultrastructure of the CA1 neurons in each group were observed under TEM (Fig. 3C). In the Con group, the synaptic cleft was clearly visible, and the presynaptic membrane was clear and uniform with a complete outline. Numerous synaptic vesicles were observed in the anterior membrane region, and abundant postsynaptic densities were detected in the posterior membrane. On the other hand, the synaptic spaces in the SD group were widened and blurred. Administration of DEX restored the indicators of morphological changes to the synapse.

Transcriptome profiles in the hippocampus

To gain a more comprehensive insight into the gene expression changes in the hippocampus, RNA-Seq and differentially expressed genes (DEGs) screening analyses were performed between the Con group, the SD group, and the DEX group (five rats in each group). According to the cutoff criteria, 443 genes were differentially expressed following sleep deprivation, and 567 DEGs were identified in the DEX group relative to SD group. The volcano plot showed the distribution of all DEGs (Fig. 4A). Notably, when parallel clustering the DEGs in the three groups, we found the expression levels of 184 DEGs changed in the SD group relative to the Con group, while these genes tended toward normal in the DEX group (Fig. 4B). Of the 184 genes identified, the majority (153) were increased in the SD group (fold change ≥ 2; P value ≤ 0.05), while 30 genes were decreased (fold change ≤ 2; P value ≤ 0.05). These data suggest that the genes altered during REM sleep deprivation were protected by the administration of DEX.

Fig. 4.

The difference in gene expression among the groups. A The volcano plot of differentially expressed genes. The red dots represent upregulated genes, and the green dots represent down-regulated genes. The abscissa represents the expression difference multiple of DEGs, and the ordinate represents the level of significance difference. B Venn diagram of the number of mRNAs overlapped between the SD group vs. the Con group and the DEX group vs. SD group. C KEGG pathway analysis of DEGs. The top 20 items of potential biological function analysis were shown with the parameter gene number and P value. D The transcript levels of Alox15 were determined by qRT-PCR. Results are shown by mean ± SEM (n = 3). Data were analyzed by one-way ANOVA with LSD post hoc test. *P < 0.05. DEGs, differentially expressed genes. DEX Dexmedetomidine

Next, GO functional enrichment analyzing and KEGG pathway analysis were employed to further understand gene biological functions of DEGs. The results revealed that metabolic process, response to stimulation, membrane part, binding, and catalytic activity were significantly enriched GO terms. KEGG pathway analysis revealed that several neurogenic disease-associated pathways were identified to be in the top enriched pathway terms, including neuroactive ligand–receptor interaction, serotonergic synapses, and cholinergic synapses. We also found that metabolic pathways enriched the most genes (Fig. 4C).

Validation of the RNA sequencing data by qRT-PCR

Alox15, one of the DEGs, is involved in the regulation of serotonergic synapses and metabolic pathways and plays an important role in cognitive function in the progression of Alzheimer’s disease. RNA-Seq showed that the mRNA expression of alox15 genes increased significantly after REM sleep deprivation as compared to Con (fold change ≥ 2; P value ≤ 0.05), and the transcript level of alox15 was significantly down-regulated after administration of DEX (fold change ≥ 2; P value ≤ 0.05). This prompted us to select Alox15 as a gene of interest for further exploration. Results of qRT-PCR exhibited similar changes to the RNA-Seq analyses (Fig. 4D), with the Alox15 gene significantly upregulated in the SD group as compared with the Con group (*P < 0.05), and this trend was reversed in the DEX group (*P < 0.05). This suggested that the reliability of the transcriptomic data was validated by qRT-PCR for the selected genes.

DEX reverses SD-induced upregulation of Alox15 expression

Given the findings that the sleep deprivation-induced spatial memory deficit was accompanied by the alterations in Alox15 mRNA expression, we selected the Alox15 from RNA-Seq analyses and qRT-PCR for western blot. As can be seen in Fig. 5A, B, compared to the Con group, Alox15 expression was significantly increased in the hippocampus following REM sleep deprivation. DEX reversed REM sleep deprivation-induced Alox15 upregulation. These results suggest that improvement in spatial memory resulting from DEX administration may, in part, be related to the regulation of the expression of the Alox15 protein in the rat.

Fig. 5.

Verification of differently expressed of Alox15. A Western blot assay of Alox15 protein levels in hippocampal. B A representative of 3 independent experiments is shown. GAPDH was used as a control. Data represent means ± SEM (n = 3). Data were analyzed by one-way ANOVA with LSD post hoc test. **P < 0.01. C Effects of dexmedetomidine on changes in the morphology of primary hippocampal neurons viewed under a light microscope. D Western blot assay of Alox15 protein levels in rat hippocampus incubated with the indicated doses of Dexmedetomidine for 1 h. E A representation of 3 independent experiments is shown. GAPDH was used as a control. Data represent means ± SEM (n = 3). One-way ANOVA followed by t test. *P < 0.05

Changes in the morphology of primary hippocampal neurons were recorded after treatment with the previously described doses of DEX for 1 h (Fig. 5C). In the concentration of 0, 1, and10 μM, the hippocampal neuronal cell bodies, dendrites, and axons were integrity under the light microscope. The dendritic connections between hippocampal neurons were closely linked, with interwoven protrusions that constituted a rich and dense neural network. The aggregation of neuronal cell bodies was also more obvious. In the 20 μM, neurons gradually degenerated, the cell body shrunk, deformed and aggregated, the refractive index weakened, and the neural network became sparse. So, we excluded the 20 μM concentration in the next experiment. As shown in Fig. 5 D and E, the expression of Alox15 in the 1.10 μM DEX group was significantly decreased compared with that of 0 μM group.

Discussion

Chronic sleep loss/fragmentation that is prevalent in our current society is associated with serious consequences on the cognitive functioning of individuals. Our results showed that DEX administration effectively prevented the 21-day chronic REM sleep deprivation-induced deficit in learning and memory in rats.

In the current study, we designed a WPF group that exposed rats to water environment without REM sleep deprivation to eliminate the potential confounding effects of the stress caused by water on behavioral performance. It is notable that the WPF group rats showed a similar learning and memory performance to those seen in the Con group. That strongly suggests that the water environment used in this study does not independently effect learning and memory function, which is consistent with previous work. We also found that spatial memory function was similar in appearance between the Con and DEX groups, suggesting that DEX administration reverses the spatial memory deficit that occurs with REM sleep deprivation. It was previously reported that DEX may prevent postoperative neurocognitive dysfunction by reducing the abnormal expression of Alzheimer’s disease biomarkers. In this study, the dose of DEX administration was 20 μg/kg. This dose was used in several published studies and has been shown to be effective in protecting against cognitive function impairment in conditions other than sleep deprivation. Our behavioral test data provided evidence that DEX administration mitigates the deleterious effects of REM sleep deprivation on the performance of rats in the MWM test.

In the behavioral test, MID could not ameliorate the learning and memory deficits induced by REM sleep deprivation. The classic hypnotics act on gamma-aminobutyric acid type A (GABA) receptors, mediating inhibition of the limbic, thalamic, and hypothalamic regions of the central nervous system and improving sleep-related problems [25]. Administration of MID caused neuronal damage with ultrastructural abnormalities [26] and triggered apoptotic neurodegeneration [27, 28]. Studies have shown that MID administration impairs the retention of different types of memory by producing specific deleterious effects on learning or by including state-dependent memory deficits. Indeed, in vitro work demonstrated that the administration of the MID could selectivity inhibit long-time potentiation and subsequently influence memory. Given these inadequacies, an alternative treatment option that promotes sleep via a different mechanism of action is needed. DEX exerts its hypnotic action through selective activation of alpha-2 adrenergic receptors in the locus coeruleus. It induces a state mimicking natural sleep accompanied by an increase in slow-wave activity [29]. It has also been reported to exert neuroprotective effects against various brain injuries by inhibiting neuronal cell damage, inflammatory response, and neuronal apoptosis.

The hippocampus is one of the brain regions in rodents in charge of higher nervous activities, including emotional integration, cognition, and memory. It is especially vulnerable to the negative consequences of sleep loss. Reports have suggested that sleep deprivation compromises hippocampal function probably through the modification of synaptic plasticity at both the electrophysiological and molecular levels as well as at a structural level. Observations of the morphological changes of synapses on hippocampal pyramidal neurons of the CA1 region showed histopathological and ultrastructural abnormalities, including dendritic spine reduction and synaptic space blurring observed in the SD group. Our results suggest that DEX administration reverses this synaptic structural damage that occurs with sleep deprivation. It is also reported that DEX alleviates hypoxia-induced synaptic loss and cognitive impairment and increases the expression of postsynaptic density protein 95 and synaptophysin protein [30]. Thus, the mechanism by which dexmedetomidine protects synaptic plasticity needs to be further explored.

To better elucidate the molecular mechanisms of a suggested DEX-induced protective effect on spatial memory, we studied transcriptomic changes among the three groups. In the over 184 DEGs identified by this analysis, we discovered that Alox15 was involved in the regulation of several related pathways and might play an important role in cognitive function. Alox15, one type of the 15-lipoxygenase (LOX) enzyme which can express in monocytes [31], macrophages [31, 32], endothelial cells [33] and so on. 15-LOX is key neuromolecular factor essential in lipid-mediated signaling, neurotrophic support, defense against reactive oxygen and nitrogen species and neuroprotection in the CNS [34]. Alox15 can catalyze the generation of DHA and whose reaction products have been shown to play a role in protecting from amyloid and dendritic pathology in AD model mice [35], enabling working memory and consolidation information [36, 37].

To verify the results of RNA-seq analyses, the interested DEGs were selected for qRT-PCR. Gene expression measured by qRT-PCR revealed similar changes to the gene expression analyses. We showed that the overexpression of Alox15 mRNA is strongly associated with spatial memory deficit. This observation confirms previous findings, suggesting that the spatial memory deficits are predominantly associated with an increased Alox15 mRNA expression level in the hippocampus. Upregulated Alox15 mRNA level is observed in the brain tissues of AD patients [38]. In agreement with this conclusion, Alox15 knockout mice have an enhanced working spatial and recognition memory compared to wild-type mice [39].

In this study, we found Dex administration not only improved REM sleep deprivation-induced spatial learning and memory deficits but also reversed the expression of Alox15 protein levels. Our data suggest that Alox15 could be a key target for reducing the incidence of cognitive dysfunction. This raises the hypothesis that DEX’s protective effect on spatial learning and memory deficits may be achieved by suppressing the expression level of Alox15, thereby enhancing synaptic structural plasticity in the hippocampal CA1 areas associated with REM sleep deprivation. Nonetheless, no inhibitors or gene knockout was used, so we do not know whether DEX can improve the surgical phenotype through regulating Alox15 protein.

Conclusion

REM sleep deprivation can cause spatial learning and memory deficits. DEX but not MID alleviates the learning and memory dysfunction induced by chronic REM sleep deprivation in rats. Our findings support the hypothesis that the DEX administration may prevent spatial learning and memory deficits induced by REM sleep deprivation, possibly through suppressing the expression of Alox15 and enhancing synaptic structural plasticity in the hippocampal CA1 areas of rats.

Supplementary Information

Below is the link to the electronic supplementary material.

Funding

This research was funded by the National Natural Science Foundation of China (No. 82072086) and Research funding of difficult disease of Beijing Institute of Translational Medicine, Chinese Academy of Sciences (No. TB2019-011).

Declarations

Conflict of interest

The authors declare no conflicts of interest.

Ethical Committee Permission

Animal protocols were approved by The Animal Care and Use Committee of Aviation General Hospital of China Medical University Laboratory (Approval No. HK2019-08-20)