Effect of enriched environment on the chronic sleep deprivation mice by NLRP3/Caspase-1 signal pathway

Effect of enriched environment on the chronic sleep deprivation mice by NLRP3/Caspase-1 signal pathway | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Effect of enriched environment on the chronic sleep deprivation mice by NLRP3/Caspase-1 signal pathway Ruiqi Tian, Yingying Zhou, Tang Wei, Dandan Yu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5654841/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Enriched environment (EE) serves as a crucial intervention strategy that enhances central neural plasticity and facilitates motor, sensory, cognitive, and social stimulation. This multifaceted approach ultimately promotes the recovery of cognitive functions in the brain. However, the mechanism is unclear. Neuroinflammation has been proven associated with cognitive impairment. NLRP3 plays a crucial role in neuroinflammation induced sleep deprivation. Our study was to investigate the effect of EE on the chronic sleep deprivation mice by NLRP3/Caspase-1 signal pathway. In this study, the modified multi-platform method (MMPM) was applied to establish the chronic sleep deprivation model. The novel object recognition test (NORT) was used to assess the ability of learning and memory. The evaluation of hippocampal neuronal injury was achieved by performing Nissl staining and immunofluorescence. To evaluate the protein and mRNA expression of NLRP3, Caspase-1, and ASC, western blot and RT-qPCR were utilized. Compared with the SD group, the mice in the SD+ EE group demonstrated longer and more often to explore novel objects and alleviating neuroinflammation in the hippocampal. The mRNA and protein of NLRP3, Caspase-1, and ASC were decreased in the SD+ EE group. Enriched environment can improve the cognitive impairment induced by chronic sleep deprivation through the NLRP3/Caspase-1 signal pathway. enriched environment chronic sleep deprivation cognitive impairment NLRP3/Caspase-1 neuroinflammation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Sleep accounts for approximately one-third of an individual's lifetime and is essential for the normal functioning of the human body. During sleep, various physiological changes occur, including alterations in blood pressure, heart rate, hormone secretion, cellular repair, memory restoration, and cognitive regulation [ 1 ] . With the increasing complexity of social life, heightened work pressures, and the widespread use of electronic devices, individuals' sleep duration has significantly decreased compared to previous generations. Sleep deprivation (SD) has emerged as a recognized health problem in modern society [ 2 ] . SD refers to a series of physical, mental, and behavioral abnormalities, including cognitive impairment [ 3 ] . SD can induce neuroinflammation that contributes to cognitive deficits [ 4 ] . Research has shown that NOD-like receptor thermoprotein domain associated protein 3 (NLRP3) inflammasome is involved in cognitive impairment caused by SD [ 5 ] . As one of the most widely studied members of the NOD-like receptor (NLR) family, NLRP3 inflammasome is mainly composed of three components: nucleotide binding oligomeric domain-like receptor protein 3 (NLRP3), apoptosis associated speck-like protein containing a caspase recurrence domain (ASC), and effector molecule precursor caspase-1 (pro caspase-1) [ 6 ] . Upon stimulation by danger signals recognized by the NLRP3, this complex activates an inflammatory cascade that lead to maturation and secretion of pro-inflammatory cytokines (IL-18, IL-1β), thereby promoting an inflammatory response [ 7 ] . Studies have demonstrated that SD triggers neuronal cell pyroptosis by activating the NLRP3/caspase-1 pathway which results in cognitive impairment in rats [ 8 – 9 ] . Furthermore, previous study has shown that MCC950, as an inhibitor of NLRP3 inflammasome, capable of reversing neuroinflammation [ 10 ] . Enriched environment (EE) was first described by Hebb and was defined as a complex combination of inanimate objects and social stimuli [ 11 ] . Compared to the standard environment, EE enhances physical stimuli, sensory experience, social interactions, exploration opportunities, and the introduction of novel stimuli aimed at improving the sensory, perceptual, and social communication abilities of treatment subjects [ 12 ] . Research has demonstrated that EE can promote the proliferation and differentiation of neurons while supporting the survival of newly formed neurons. Additionally, it enhances synaptic transmission function, regulates synaptic plasticity, and influences the production and expression of neurotrophic factors, neurotransmitters, and hormones through gene regulation associated with nerve regeneration. These ultimately facilitates nerve regeneration processes [ 13 ] . Multiple animal experiments have indicated that EE can ameliorate cognitive impairment in stroke models [ 14 ] . Furthermore, other studies have ravealed that EE can inhibit the production of NLRP3 inflammasomes, this inhibiton could alleviate lipopolysaccharide-induced cognitive deficit [ 15 – 16 ] . The NLRP3/Caspase-1 signaling pathway plays an important role in neuropathy induced by SD. Therefore, we aim to explore whether an EE can mitigate SD-induced cognitive impairment by inhibiting the NLRP3/Caspase-1 signaling pathway. We will conduct research on this topic. 2. Materials and methods 2.1 Experimental animals and grouping All mice were provided by the Liaoning Changsheng biotechnology Co., Ltd. All experimental procedures were followed the guiding principles of mammalian neuroscience research and approved by the Ethics Committee for Animal Experiments at the Dalian University Affiliated Xinhua Hospital (NO. 2022-086-01). A total of 30 female SPF mice (3-month-old) were randomly categorized into four groups: standard environment (Ctrl) group (n = 10), sleep deprivation (SD) group (n = 10), sleep deprivation + enriched environment (SD + EE) group (n = 10). To adapt to the environment, all mice were housed for 1 week in the laboratory where food and water were readily available. The room temperature is (20 ± 2℃)and the humidity is 65–70%. The experimental process is illustrated in Fig. 1 . 2.2 Animal model making 2.2.1 Sleep Deprivation Model The mice were deprived of rapid eye movement (REM) sleep by the modified multi-platform method (MMPM). The device (32×17.5×14.5cm) (five rat per cage), contained 22 platforms (3.5 cm diameter and 4.5 cm height), and the distance between the two platforms is about 4cm. Platforms were located 1cm above the water level. The mice can freely get food and water and move on the platform. During REM sleep, the mice contact the water and awakens due to muscle atonia, causing sleep deprivation. Before the experiment begins, each mouse is familiar with the environment for 3 days to alleviate stress. SD time is from 8:00 to 20:00, with 12 hours of daily sleep deprivation and last 90 days. 2.2.2 Enriched environment Each cage (54×33×20 cm) is equipped with 2 running wheels, balls, mazes, stairs, pipes, and small houses. Each cage has 2–3 levels, increasing the space for mice to exercise. It is replaced and rearranged weekly to add novelty. EE also provided enhanced social stimulation by raising 6 to 8 rats in the cage. The SD + EE group was housed in EE. 2.2.3 Standard environment In order to eliminate the impact of the environmental pressure, standard environment was set up: 6 large platforms (12cm diameter, 4.5cm height, and a diameter spacing of 15cm between platforms) were placed in the device (32×17.5×14.5cm), allowing mice to sleep without falling into the water. The Ctrl and SD groups were housed in standard environment. 2.3 The Novel Object Recognition Test (NORT) The novel object recognition test is used to assess the ability of learning and memory in mice. The mice were placed in a breeding cage and acclimated for 5 days. 3 days before testing, touch the experimental mice for 1 minute every day to avoid stimulation and eliminate any sense of unfamiliarity with the tester. Then, placing the mice in the testing room for 24 hours in order to adapt to the environment. As shown in Fig. 2a, place the rat with its back facing the two objects into the field with the tip of its nose at the same distance as the two objects, and the recording device was immediately turned on. After 10 minutes, put the rat back into their cage and let them rest for 20 minutes. And then replace the A object with the C object (Fig. 2b), place the rat into the field for 10 minutes. After the experiment was completed, the rat was sent back to the cage. The images were analyzed by Smart 3.0 software (Panlab), and the contact between the rat and these two objects, including the number of times the rat's nose or mouth touched the object and the exploration time within a range of 2-3cm from the object. The total exploration time and novel object preference of the familiarization and test phases were calculated separately: RI = exploration time of object C/total exploration time, which was used to judge the cognitive function of the rats. 2.4 Tissue collection 24 hours after the NORT, all mice were deeply anesthetized intraperitoneally with tribromoethanol, and transcardially perfused with normal saline until the limbs of the mice became stiff and the effluent was clear. The hippocampus was carefully removed. The left hippocampus tissues were rapidly fixed in 4% paraformaldehyde at 4°C overnight and cryoprotected for 72 hours in 30% sucrose solution. Subsequently, the tissue was embedded in paraffin and cut into 5 µm standard sections for further morphological analysis. For western blotting, the right hippocampus was stored at -80 ℃. 2.5 Nissl staining Take one brain slice and bake it in a 60 ℃ oven for 1-1.5 hours. After dewaxing with xylene three times, each time for 10 minutes, perform a gradient ethanol hydration treatment with a gradient ethanol concentration of 100% -100% -95% -85% -75% for 5 minutes each. Rinse with distilled water for 1 minute. Place the slices in Toluidine blue stain preheated to 60 ℃ for 35 minutes and rinse with distilled water for 1 minute. Place the slices in 95% ethanol for differentiation treatment, then place them in anhydrous ethanol for dehydration treatment. Use xylene for transparency treatment twice, each time lasting for 5 minutes, and finally, seal the slices with neutral gum. Use Pannoramic DESK, P-ESTA (3D HISTECH, Hungary) scanner to capture images and observe and analyze the staining of the hippocampal CA1 region. 2.6 Immunofluorescence Sections were fixed with 4% paraformaldehyde for half an hour, then blocked it with 10% goat serum at 37°C. Primary antibodies: rabbit anti-NLRP3 (1:500; bs10021R; Bioss), Caspase-1 (1:500; AF5418; Affinity Bioscience), ASC (1: 3000; DF6304; Affinity Bioscience) was left overnight at 4°C. On the next day, the secondary antibody CY5 (1:500; E-AB-1010; Elabscience) containing fluorescein was blocked at room temperature for 1 hour. Staining was performed with 2- (4-acylphenyl) -6-diacylethylamine hydrochloride (DAPI). Use anti-fluorescence quenching tablets containing Hoechst33342 (P0133, Biyuntian) for sealing. Pannoramic DESK and P-ESTA (3D HISTECH, Hungary) scanner were used to capture images. 2.7 Western blot To quantitatively monitor the level of NLRP3, Caspase-1, and ASC, the hippocampus was lysed and the supernatant was collected by centrifugation. After the protein was quantified by the BCA method, 20 µ g of each protein sample was used for 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and then wet transferred to the PVDF membrane (micropore). Seal with 5% skim milk, wash with TBST three times, and then incubate overnight with primary antibody at 4 ° C. Use the following primary antibody: The dilution concentration of primary antibody is as follows: NLRP3 (1:500), Caspase-1 (1:500), ASC (1:3000). Rabbit anti-mouse NLRP3 (bs10021R; Bioss), Caspase-1 (AF5418; Affinity Bioscience), ASC (DF6304; Affinity Bioscience)。 GAPDH is used as an internal reference protein. Wash the membrane three times with TBST and incubate it with the corresponding secondary antibody CY5 (1:500; E-AB-1010; Elabscience) at room temperature for 2 hours, then wash it three times with TBST. According to the product specifications, use the ultra-sensitive ECL chemiluminescence assay kit (4AW011; Four Major Park Biotech) to detect protein bands. ImageJ software was used to quantify the grayscale values of the stripes. 2.8 Quantitative real-time PCR (qRT-PCR) RNA was extracted from hippocampal tissues, and reverse transcribed into cDNA using Line Gene 9600 Fluorescent qPCR. Detection System according to the manufacturer’s instructions. The primer sequences for mRNAs can be found in Table 1 . Table 1 The primer sequences for mRNAs. Primer Forward Reverse NLRP3 5′-AGA AAC TGT GGT TGG TGA GCT G-3′ 5′-GAA TTC ACC AAC CCC AGC TTC TG-3′ Caspase-1 5′-CAA AGG TTT CGG TAC ATC GTC T-3′ 5′-ATC TTG ATT TGA GTG ATT GCA CA-3′ ASC 5′-CAC AGA ACA GGA CAC TTT GTG GA-3′ 5′-CC GAA CTG CCT GGT ACT GTC-3′ 2.9 Statistical analysis Statistical analysis was performed using GraphPad Prism10.0. All data were expressed as mean + standard deviation. Multiple group comparisons were performed by one-way ANOVA, and P < 0.05 was considered a statistically significant difference. 3. Results 3.1 Enriched environment improved cognitive impairment induced by chronic SD The novel object recognition test was employed to assess the impact of EE on the learning and memory of the chronic SD mice. As shown in Fig.3a-b, compared with the Ctrl group, the novel object recognition test index and discrimination ratio were decreased in the SD group (P<0.0001). Compared with the SD group, the new object recognition test index and discrimination ratio were increased in the SD+EE group (P<0.0001). 3.2 Enriched environment alleviated hippocampal injury induced by chronic SD We employed Nissl staining (Fig.4) to examine the structure of the hippocampal. In the hippocampal region in the Ctrl group, the nerve cells were organized neatly, with well-defined cytoplasm, normal morphology, large and round nuclei that were clearly visible, uniform distribution, and abundant Nissl bodies. In contrast, in the SD group, the nerve cells exhibited severe damage characterized by unclear cellular structure, deeply indented nuclei, indistinct nucleoli, significantly reduced cell density, increased vacuole formation, and heightened inflammatory cell infiltration—indicative of neuronal damage to the hippocampus. Conversely, in the SD+EE group, there was disorganization of cell arrangement and a reduction in intercellular density accompanied by enlarged cellular spaces and abnormal morphology, disrupted cell membranes, and irregular nuclei. It suggests that EE alleviated the neuronal damage induced by sleep deprivation in mice. 3.3 Enriched environment decreased the NLRP3 inflammasome in the chronic SD mice To further investigate the impact of EE on NLRP3 inflammasome expression in chronic SD mice, immunofluorescence staining was utilized to determine the expression levels of NLRP3 and Caspase-1 within the hippocampus. As depicted in Fig.5a, there was an elevation of NLRP3 and caspase-1 levels in the SD group when compared with those observed in the Ctrl group; however, a reduction was noted for these markers in the SD+EE group. The Western blot analysis results presented in Fig.5b. Compared with the Ctrl group, the expression of NLRP3, Caspase-1, and ASC were significantly increased in the SD group (P<0.0001). Compared with the SD group, NLRP3, Caspase-1, and ASC expression were decreased in the SD+EE group. 3.4 Enriched environment improved SD induced cognitive impairment by regular NLRP3/caspase-1 signal pathway To determine which inflammasome pathway is implicated in neuronal damage induced by chronic SD, an RT-qPCR array was performed. As shown in Fig.6, the mRNA levels of NLRP3、Caspase-1、ASC were significantly upregulated in the SD group compared with the Ctrl group (P<0.0001, P<0.01), while EE treatment resulted in a notable decrease. 4. Discussion Adequate sleep is crucial for the body to perform normal behavioral activities, and prolonged periods of insufficient sleep can lead to health issues that affect cardiovascular, nervous, gastrointestinal, and other systems [ 17 ] . Both animal and human researches have demonstrated that sleep deprivation (SD) can result in cognitive impairment [ 18 ] . The underlying mechanism involves the induction of an inflammatory response within the central nervous system due to sleep deprivation, which increases the release of pro-inflammatory cytokines (IL-6 and TNF-α) in the brain, leading to neuronal damage and subsequent declines in learning and memory capabilities [ 19 ] . Due to the side effects associated with conventional treatments, oral medications are often unacceptable by many individuals. Recent research has indicated that enriched environment (EE) serve as a rehabilitation model widely utilized in training for post-stroke cognitive impairment [ 20 ] . EE has been shown to ameliorate cognitive deficits by down-regulating neuroinflammation [ 21 ] . Consequently, we investigated the impact of EE on cognitive impairment in mice subjected to chronic SD. Our findings revealed that EE could enhance learning and memory in chronic SD mice by modulating the NLRP3/caspase-1 signaling pathway while alleviating hippocampal neuronal damage. Studies have demonstrated that sleep facilitates spatial learning and memory through the mediation of hippocampal neurogenesis [ 22 ] . Conversely, SD activates glial cells, promotes hippocampal neuroinflammation, and ultimately results in a decline in learning and memory abilities [ 23 ] . To assess the effects of chronic SD on learning and memory in mice, we conducted a novel object recognition experiment. The results indicated that both the duration and frequency of exploration of novel objects were significantly reduced in the SD group, suggesting that chronic SD adversely affects the cognitive abilities of mice. In contrast, both the time spent exploring novel objects and their frequency in the SD + EE group were higher than those observed in the SD group. This finding suggests that EE can ameliorate learning and memory deficits induced by SD. Subsequently, we continued our investigation into the specific mechanisms underlying EE. Previous studies have identified neuroprotective effects associated with EE which can mitigate hippocampal inflammatory response, alleviate pathological conditions affecting damaged hippocampal neurons, and enhance cognitive function in mice [ 24 ] . Our findings indicated that hippocampus neurons in the SD group exhibited damaged, however, these damages were improved in the SD + EE group. These results are consistent with previous research findings [ 25 ] . Furthermore, studies indicate that chronic SD induced reductions in learning and memory are closely linked to neuroinflammation, wherein the NLRP3 inflammasome plays a pivotal role [ 26 ] . The NLRP3 inflammatome comprises NOD-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein containing a caspase-recruitment domain, (ASC), as well as its effector molecule precursor, pro-caspase-1. Upon stimulation, the NLRP3 inflammasome is activated to release pro-inflammatory cytokines (IL-1β, IL-18) which contribute to various non-specific inflammatory processes [ 27 ] . Our study found increased expressions of NLRP3, ASC, and caspase-1 in the hippocampus of mice from the SD group, which was consistent with previous studies [ 28 ] , and confirmed that chronic SD induced neuroinflammation. Enriched environment-an intervention integrating sensory, motor, and social stimuli-has been shown to mitigate hippocampal inflammatory response in stroke models while improving cognitive function [ 29 ] . This suggests that EE can down-regulate expression within the NLRP3/caspase-1 signaling pathway and diminish activation of the NLRP3 inflammasome induced by chronic SD, deprivation thereby ameliorating cognitive impairment. 5. Conclusion Enriched environment enhances learning and memory capabilities in chronic SD mice. Furthermore, EE appears to operate by modulating the NLRP3/caspase-1 signaling pathway. These results suggest that EE may serve as a significant non-pharmacological intervention for treating cognitive impairments associated with SD. Declarations Funding This work was supported by Dalian medical association (Grant Numbers: 2211045). Competing Interests The authors have no relavant financial interests to disclose. Author Contributions Ruiqi Tian: Writing – original draft, Methodology, Data curation, Conceptualization. Yingying Zhou: Writing – original draft, Methodology,Formal analysis,Data curation. Tang Wei: Writing –review & editing. Dandan Y u: Writing –review & editing, Conceptualization. Data availability Data will be made available on request. Ethics approval This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of Dalian University Affiliated Xinhua Hospital (2022.11.20/No.2022-086-01). Clinical trial number: not applicable. Declaration of competing interest All authors declare no conflicts of interest. Acknowledgements Thanks to the BioRender for providing the graphic technical support (figure1-2). References Baranwal, N., Yu, P. K., & Siegel, N. S. (2023). Sleep physiology, pathophysiology, and sleep hygiene. Progress in cardiovascular diseases , 77 , 59–69. https://doi.org/10.1016/j.pcad.2023.02.005. Mason, G. M., Lokhandwala, S., Riggins, T., & Spencer, R. M. C. (2021). Sleep and human cognitive development. Sleep medicine reviews , 57 , 101472. https://doi.org/10.1016/j.smrv.2021.101472. Ramos, A. R., Wheaton, A. 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Translational neurodegeneration , 12 (1), 49. https://doi.org/10.1186/s40035-023-00381-x. Fan, K., Yang, J., Gong, W. Y., Pan, Y. C., Zheng, P., & Yue, X. F. (2021). NLRP3 inflammasome activation mediates sleep deprivation-induced pyroptosis in mice. PeerJ , 9 , e11609. https://doi.org/10.7717/peerj.11609. Gu, J. Y., Xu, Y. W., Feng, L. P., Dong, J., Zhao, L. Q., Liu, C., Wang, H. Y., Zhang, X. Y., Song, C., & Wang, C. H. (2021). Enriched environment mitigates depressive behavior by changing the inflammatory activation phenotype of microglia in the hippocampus of depression model rats. Brain research bulletin , 177 , 252–262. https://doi.org/10.1016/j.brainresbull.2021.10.005. Additional Declarations No competing interests reported. Supplementary Files ASC.png Caspase1.png ASCGAPDH.tiff NLRP3.png NLRP3GAPDH.png Caspase1GAPDH.png Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5654841","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":392130698,"identity":"dbdf4706-72b8-4ae3-8336-b3bbf08ff4e3","order_by":0,"name":"Ruiqi Tian","email":"","orcid":"","institution":"Dalian University Affiliated Xinhua Hospital","correspondingAuthor":false,"prefix":"","firstName":"Ruiqi","middleName":"","lastName":"Tian","suffix":""},{"id":392130700,"identity":"13a86e34-5e8b-4f67-a21f-ed1ac71f9771","order_by":1,"name":"Yingying Zhou","email":"","orcid":"","institution":"Dalian University Affiliated Xinhua Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yingying","middleName":"","lastName":"Zhou","suffix":""},{"id":392130702,"identity":"436bac26-8714-4d6c-a17e-a0c482d1bf03","order_by":2,"name":"Tang Wei","email":"","orcid":"","institution":"Dalian University Affiliated Xinhua Hospital","correspondingAuthor":false,"prefix":"","firstName":"Tang","middleName":"","lastName":"Wei","suffix":""},{"id":392130704,"identity":"5c560546-f326-4d12-a28c-80afc806c81f","order_by":3,"name":"Dandan Yu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAz0lEQVRIiWNgGAWjYBACNvbmAwckKmzk2BgOHyBOCx/PscQHFmfSjPkYjyUQp0VOIsfYoLLtcOI85jMGRDpMIi1N4iZQSxvbmY833jDYyek2ENLC8/iY5Ixz6cZtPGc3W85hSDY2O0BIC3tamrREmbVsm8TZbdI8DAcStxHUwpBjJv2HjZmxTf7NMyK1cAC9L9HmrNjGcIaNSC2gQJYABjIbwzFjyzkGRPhFvh0alfINhx/eeFNhJ0dQCwqQ4CEyapC1kKpjFIyCUTAKRgQAAJSyQ8zHQs4ZAAAAAElFTkSuQmCC","orcid":"","institution":"Dalian University Affiliated Xinhua Hospital","correspondingAuthor":true,"prefix":"","firstName":"Dandan","middleName":"","lastName":"Yu","suffix":""}],"badges":[],"createdAt":"2024-12-16 14:53:05","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5654841/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5654841/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":71995770,"identity":"57121b8b-26d1-4d48-af1b-6cfbfdb84cbb","added_by":"auto","created_at":"2024-12-20 12:32:43","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":79280,"visible":true,"origin":"","legend":"\u003cp\u003eThe time arrangement of the animal experiment.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5654841/v1/efc396cc09dfe14f8f34911b.png"},{"id":71995181,"identity":"eee37734-c323-4d96-9172-3cd1f0043088","added_by":"auto","created_at":"2024-12-20 12:24:44","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":48103,"visible":true,"origin":"","legend":"\u003cp\u003eThe novel object recognition test model.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5654841/v1/615a465b689c63f85d828a19.png"},{"id":71995170,"identity":"38b95451-f3ef-44b4-b785-2b5d4fbaf6ab","added_by":"auto","created_at":"2024-12-20 12:24:43","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":35608,"visible":true,"origin":"","legend":"\u003cp\u003eEE improved cognitive impairment in SD mice. (a) The novel object recognition test index (n=10. ****P\u0026lt;0.0001vs. Ctrl group. ####P \u0026lt; 0.001 vs. SD group). (b) The discrimination ratio (n=10. ****P\u0026lt;0.0001vs. Ctrl group. ####P \u0026lt; 0.001 vs. SD group).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5654841/v1/32eee76ad48c97969846cac6.png"},{"id":71995769,"identity":"ea6c0c24-1581-4aab-aa37-3ff07be06409","added_by":"auto","created_at":"2024-12-20 12:32:43","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":284021,"visible":true,"origin":"","legend":"\u003cp\u003eEE alleviated hippocampal injury in SD mice. Nissl staining, scale bar = 20 μm.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5654841/v1/ca80f7b66e3f71733ea52c72.png"},{"id":71995771,"identity":"282c5cbe-6da9-4dd5-8f11-84b3578771f6","added_by":"auto","created_at":"2024-12-20 12:32:44","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":356322,"visible":true,"origin":"","legend":"\u003cp\u003eEE decreased the NLRP3 inflammasome in SD mice. (a) The Immunofluorescence staining of the hippocampal. Magnification scale bar = 100 μm, other scale bar = 20 μm. (b) NLRP3, Caspase-1, ASC, and GAPDH expressions were determined through Western blot. (c) Analysis of grayscale values of hippocampal NLRP3, Caspase-1, ASC in each group.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5654841/v1/6152b5c6e1710f640fc930b1.png"},{"id":71995781,"identity":"57541685-816d-4ccc-811b-570921426fe2","added_by":"auto","created_at":"2024-12-20 12:32:46","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":63406,"visible":true,"origin":"","legend":"\u003cp\u003eThe mRNA levels of NLRP3、Caspase-1、ASC in each group.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-5654841/v1/18967178ebf2a9351ec6bebf.png"},{"id":73522567,"identity":"a367ee1a-5422-4b86-9a17-c8c626156038","added_by":"auto","created_at":"2025-01-10 19:16:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1530165,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5654841/v1/62368a7b-cbdd-4e31-a93b-d30117fa7db6.pdf"},{"id":71995173,"identity":"c8eb3a48-92ce-4471-95dd-12eb01325144","added_by":"auto","created_at":"2024-12-20 12:24:43","extension":"png","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":16879,"visible":true,"origin":"","legend":"","description":"","filename":"ASC.png","url":"https://assets-eu.researchsquare.com/files/rs-5654841/v1/9dc152648051035bd1225096.png"},{"id":71995174,"identity":"b769ce47-8c86-4a68-9bb1-f95f43997a86","added_by":"auto","created_at":"2024-12-20 12:24:43","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":9998,"visible":true,"origin":"","legend":"","description":"","filename":"Caspase1.png","url":"https://assets-eu.researchsquare.com/files/rs-5654841/v1/b1f8fe7d981e64f0f20fd729.png"},{"id":71995206,"identity":"bb1bde37-4be9-480d-8b45-8c12835530b2","added_by":"auto","created_at":"2024-12-20 12:24:46","extension":"tiff","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":7868,"visible":true,"origin":"","legend":"","description":"","filename":"ASCGAPDH.tiff","url":"https://assets-eu.researchsquare.com/files/rs-5654841/v1/4fb31254d9c302cdd5f41fcc.tiff"},{"id":71995171,"identity":"74b9a7f0-8a83-4d8d-9c6b-98c9f314dc54","added_by":"auto","created_at":"2024-12-20 12:24:43","extension":"png","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":16859,"visible":true,"origin":"","legend":"","description":"","filename":"NLRP3.png","url":"https://assets-eu.researchsquare.com/files/rs-5654841/v1/6e9e49da35a41901f50e80e9.png"},{"id":71995782,"identity":"e338cdf1-1a26-4b35-908f-244014b429c5","added_by":"auto","created_at":"2024-12-20 12:32:46","extension":"png","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":9593,"visible":true,"origin":"","legend":"","description":"","filename":"NLRP3GAPDH.png","url":"https://assets-eu.researchsquare.com/files/rs-5654841/v1/cc07950b529940132c949b26.png"},{"id":71995176,"identity":"1b140a15-a5e0-43d1-a97f-1747f98e361c","added_by":"auto","created_at":"2024-12-20 12:24:43","extension":"png","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":523420,"visible":true,"origin":"","legend":"","description":"","filename":"Caspase1GAPDH.png","url":"https://assets-eu.researchsquare.com/files/rs-5654841/v1/503cba7c3b0e8cdb296c519c.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effect of enriched environment on the chronic sleep deprivation mice by NLRP3/Caspase-1 signal pathway","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eSleep accounts for approximately one-third of an individual's lifetime and is essential for the normal functioning of the human body. During sleep, various physiological changes occur, including alterations in blood pressure, heart rate, hormone secretion, cellular repair, memory restoration, and cognitive regulation\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. With the increasing complexity of social life, heightened work pressures, and the widespread use of electronic devices, individuals' sleep duration has significantly decreased compared to previous generations. Sleep deprivation (SD) has emerged as a recognized health problem in modern society\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. SD refers to a series of physical, mental, and behavioral abnormalities, including cognitive impairment\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. SD can induce neuroinflammation that contributes to cognitive deficits\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. Research has shown that NOD-like receptor thermoprotein domain associated protein 3 (NLRP3) inflammasome is involved in cognitive impairment caused by SD\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAs one of the most widely studied members of the NOD-like receptor (NLR) family, NLRP3 inflammasome is mainly composed of three components: nucleotide binding oligomeric domain-like receptor protein 3 (NLRP3), apoptosis associated speck-like protein containing a caspase recurrence domain (ASC), and effector molecule precursor caspase-1 (pro caspase-1)\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. Upon stimulation by danger signals recognized by the NLRP3, this complex activates an inflammatory cascade that lead to maturation and secretion of pro-inflammatory cytokines (IL-18, IL-1β), thereby promoting an inflammatory response\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. Studies have demonstrated that SD triggers neuronal cell pyroptosis by activating the NLRP3/caspase-1 pathway which results in cognitive impairment in rats\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. Furthermore, previous study has shown that MCC950, as an inhibitor of NLRP3 inflammasome, capable of reversing neuroinflammation\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eEnriched environment (EE) was first described by Hebb and was defined as a complex combination of inanimate objects and social stimuli\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. Compared to the standard environment, EE enhances physical stimuli, sensory experience, social interactions, exploration opportunities, and the introduction of novel stimuli aimed at improving the sensory, perceptual, and social communication abilities of treatment subjects\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. Research has demonstrated that EE can promote the proliferation and differentiation of neurons while supporting the survival of newly formed neurons. Additionally, it enhances synaptic transmission function, regulates synaptic plasticity, and influences the production and expression of neurotrophic factors, neurotransmitters, and hormones through gene regulation associated with nerve regeneration. These ultimately facilitates nerve regeneration processes\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. Multiple animal experiments have indicated that EE can ameliorate cognitive impairment in stroke models\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. Furthermore, other studies have ravealed that EE can inhibit the production of NLRP3 inflammasomes, this inhibiton could alleviate lipopolysaccharide-induced cognitive deficit\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. The NLRP3/Caspase-1 signaling pathway plays an important role in neuropathy induced by SD. Therefore, we aim to explore whether an EE can mitigate SD-induced cognitive impairment by inhibiting the NLRP3/Caspase-1 signaling pathway. We will conduct research on this topic.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003e2.1 Experimental animals and grouping\u003c/h2\u003e\n \u003cp\u003eAll mice were provided by the Liaoning Changsheng biotechnology Co., Ltd. All experimental procedures were followed the guiding principles of mammalian neuroscience research and approved by the Ethics Committee for Animal Experiments at the Dalian University Affiliated Xinhua Hospital (NO. 2022-086-01). A total of 30 female SPF mice (3-month-old) were randomly categorized into four groups: standard environment (Ctrl) group (n\u0026thinsp;=\u0026thinsp;10), sleep deprivation (SD) group (n\u0026thinsp;=\u0026thinsp;10), sleep deprivation\u0026thinsp;+\u0026thinsp;enriched environment (SD\u0026thinsp;+\u0026thinsp;EE) group (n\u0026thinsp;=\u0026thinsp;10). To adapt to the environment, all mice were housed for 1 week in the laboratory where food and water were readily available. The room temperature is (20\u0026thinsp;\u0026plusmn;\u0026thinsp;2℃)and the humidity is 65\u0026ndash;70%. The experimental process is illustrated in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003e2.2 Animal model making\u003c/h2\u003e\n \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e\n \u003ch2\u003e2.2.1 Sleep Deprivation Model\u003c/h2\u003e\n \u003cp\u003eThe mice were deprived of rapid eye movement (REM) sleep by the modified multi-platform method (MMPM). The device (32\u0026times;17.5\u0026times;14.5cm) (five rat per cage), contained 22 platforms (3.5 cm diameter and 4.5 cm height), and the distance between the two platforms is about 4cm. Platforms were located 1cm above the water level.\u003c/p\u003e\n \u003cp\u003eThe mice can freely get food and water and move on the platform. During REM sleep, the mice contact the water and awakens due to muscle atonia, causing sleep deprivation. Before the experiment begins, each mouse is familiar with the environment for 3 days to alleviate stress. SD time is from 8:00 to 20:00, with 12 hours of daily sleep deprivation and last 90 days.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e\n \u003ch2\u003e2.2.2 Enriched environment\u003c/h2\u003e\n \u003cp\u003eEach cage (54\u0026times;33\u0026times;20 cm) is equipped with 2 running wheels, balls, mazes, stairs, pipes, and small houses. Each cage has 2\u0026ndash;3 levels, increasing the space for mice to exercise. It is replaced and rearranged weekly to add novelty. EE also provided enhanced social stimulation by raising 6 to 8 rats in the cage. The SD\u0026thinsp;+\u0026thinsp;EE group was housed in EE.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\n \u003ch2\u003e2.2.3 Standard environment\u003c/h2\u003e\n \u003cp\u003eIn order to eliminate the impact of the environmental pressure, standard environment was set up: 6 large platforms (12cm diameter, 4.5cm height, and a diameter spacing of 15cm between platforms) were placed in the device (32\u0026times;17.5\u0026times;14.5cm), allowing mice to sleep without falling into the water. The Ctrl and SD groups were housed in standard environment.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003e2.3 The Novel Object Recognition Test (NORT)\u003c/h2\u003e\n \u003cp\u003eThe novel object recognition test is used to assess the ability of learning and memory in mice. The mice were placed in a breeding cage and acclimated for 5 days. 3 days before testing, touch the experimental mice for 1 minute every day to avoid stimulation and eliminate any sense of unfamiliarity with the tester. Then, placing the mice in the testing room for 24 hours in order to adapt to the environment. As shown in Fig. 2a, place the rat with its back facing the two objects into the field with the tip of its nose at the same distance as the two objects, and the recording device was immediately turned on. After 10 minutes, put the rat back into their cage and let them rest for 20 minutes. And then replace the A object with the C object (Fig. 2b), place the rat into the field for 10 minutes. After the experiment was completed, the rat was sent back to the cage. The images were analyzed by Smart 3.0 software (Panlab), and the contact between the rat and these two objects, including the number of times the rat\u0026apos;s nose or mouth touched the object and the exploration time within a range of 2-3cm from the object. The total exploration time and novel object preference of the familiarization and test phases were calculated separately: RI\u0026thinsp;=\u0026thinsp;exploration time of object C/total exploration time, which was used to judge the cognitive function of the rats.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003e2.4 Tissue collection\u003c/h2\u003e\n \u003cp\u003e24 hours after the NORT, all mice were deeply anesthetized intraperitoneally with tribromoethanol, and transcardially perfused with normal saline until the limbs of the mice became stiff and the effluent was clear. The hippocampus was carefully removed. The left hippocampus tissues were rapidly fixed in 4% paraformaldehyde at 4\u0026deg;C overnight and cryoprotected for 72 hours in 30% sucrose solution. Subsequently, the tissue was embedded in paraffin and cut into 5 \u0026micro;m standard sections for further morphological analysis. For western blotting, the right hippocampus was stored at -80 ℃.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003e2.5 Nissl staining\u003c/h2\u003e\n \u003cp\u003eTake one brain slice and bake it in a 60 ℃ oven for 1-1.5 hours. After dewaxing with xylene three times, each time for 10 minutes, perform a gradient ethanol hydration treatment with a gradient ethanol concentration of 100% -100% -95% -85% -75% for 5 minutes each. Rinse with distilled water for 1 minute. Place the slices in Toluidine blue stain preheated to 60 ℃ for 35 minutes and rinse with distilled water for 1 minute. Place the slices in 95% ethanol for differentiation treatment, then place them in anhydrous ethanol for dehydration treatment. Use xylene for transparency treatment twice, each time lasting for 5 minutes, and finally, seal the slices with neutral gum. Use Pannoramic DESK, P-ESTA (3D HISTECH, Hungary) scanner to capture images and observe and analyze the staining of the hippocampal CA1 region.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003e2.6 Immunofluorescence\u003c/h2\u003e\n \u003cp\u003eSections were fixed with 4% paraformaldehyde for half an hour, then blocked it with 10% goat serum at 37\u0026deg;C. Primary antibodies: rabbit anti-NLRP3 (1:500; bs10021R; Bioss), Caspase-1 (1:500; AF5418; Affinity Bioscience), ASC (1: 3000; DF6304; Affinity Bioscience) was left overnight at 4\u0026deg;C. On the next day, the secondary antibody CY5 (1:500; E-AB-1010; Elabscience) containing fluorescein was blocked at room temperature for 1 hour. Staining was performed with 2- (4-acylphenyl) -6-diacylethylamine hydrochloride (DAPI). Use anti-fluorescence quenching tablets containing Hoechst33342 (P0133, Biyuntian) for sealing. Pannoramic DESK and P-ESTA (3D HISTECH, Hungary) scanner were used to capture images.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003e2.7 Western blot\u003c/h2\u003e\n \u003cp\u003eTo quantitatively monitor the level of NLRP3, Caspase-1, and ASC, the hippocampus was lysed and the supernatant was collected by centrifugation. After the protein was quantified by the BCA method, 20 \u0026micro; g of each protein sample was used for 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and then wet transferred to the PVDF membrane (micropore). Seal with 5% skim milk, wash with TBST three times, and then incubate overnight with primary antibody at 4 \u0026deg; C. Use the following primary antibody: The dilution concentration of primary antibody is as follows: NLRP3 (1:500), Caspase-1 (1:500), ASC (1:3000). Rabbit anti-mouse NLRP3 (bs10021R; Bioss), Caspase-1 (AF5418; Affinity Bioscience), ASC (DF6304; Affinity Bioscience)。 GAPDH is used as an internal reference protein. Wash the membrane three times with TBST and incubate it with the corresponding secondary antibody CY5 (1:500; E-AB-1010; Elabscience) at room temperature for 2 hours, then wash it three times with TBST. According to the product specifications, use the ultra-sensitive ECL chemiluminescence assay kit (4AW011; Four Major Park Biotech) to detect protein bands. ImageJ software was used to quantify the grayscale values of the stripes.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003e2.8 Quantitative real-time PCR (qRT-PCR)\u003c/h2\u003e\n \u003cp\u003eRNA was extracted from hippocampal tissues, and reverse transcribed into cDNA using Line Gene 9600 Fluorescent qPCR. Detection System according to the manufacturer\u0026rsquo;s instructions. The primer sequences for mRNAs can be found in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eThe primer sequences for mRNAs.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"3\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePrimer\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eForward\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eReverse\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNLRP3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u0026prime;-AGA AAC TGT GGT TGG TGA GCT G-3\u0026prime;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u0026prime;-GAA TTC ACC AAC CCC AGC TTC TG-3\u0026prime;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCaspase-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u0026prime;-CAA AGG TTT CGG TAC ATC GTC T-3\u0026prime;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u0026prime;-ATC TTG ATT TGA GTG ATT GCA CA-3\u0026prime;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eASC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u0026prime;-CAC AGA ACA GGA CAC TTT GTG GA-3\u0026prime;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u0026prime;-CC GAA CTG CCT GGT ACT GTC-3\u0026prime;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003e2.9 Statistical analysis\u003c/h2\u003e\n \u003cp\u003eStatistical analysis was performed using GraphPad Prism10.0. All data were expressed as mean\u0026thinsp;+\u0026thinsp;standard deviation. Multiple group comparisons were performed by one-way ANOVA, and P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered a statistically significant difference.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cstrong\u003e3.1 Enriched environment improved cognitive impairment induced by chronic SD\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe novel object recognition test was employed to assess the impact of EE on the learning and memory of the chronic SD mice. As shown in Fig.3a-b, compared with the Ctrl group, the novel object recognition test index and discrimination ratio were decreased in the SD group (P\u0026lt;0.0001). Compared with the SD group, the new object recognition test index and discrimination ratio were increased in the SD+EE group (P\u0026lt;0.0001).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 Enriched environment alleviated hippocampal injury induced by chronic SD\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe employed Nissl staining (Fig.4) to examine the structure of the hippocampal. In the hippocampal region in the Ctrl group, the nerve cells were organized neatly, with well-defined cytoplasm, normal morphology, large and round nuclei that were clearly visible, uniform distribution, and abundant Nissl bodies. In contrast, in the SD group, the nerve cells exhibited severe damage characterized by unclear cellular structure, deeply indented nuclei, indistinct nucleoli, significantly reduced cell density, increased vacuole formation, and heightened inflammatory cell infiltration\u0026mdash;indicative of neuronal damage to the hippocampus. Conversely, in the SD+EE group, there was disorganization of cell arrangement and a reduction in intercellular density accompanied by enlarged cellular spaces and abnormal morphology, disrupted cell membranes, and irregular nuclei. It suggests that EE alleviated the neuronal damage induced by sleep deprivation in mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3 Enriched environment decreased the NLRP3 inflammasome in the chronic SD mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further investigate the impact of EE on NLRP3 inflammasome expression in chronic SD mice, immunofluorescence staining was utilized to determine the expression levels of NLRP3 and Caspase-1 within the hippocampus. As depicted in Fig.5a, there was an elevation of NLRP3 and caspase-1 levels in the SD group when compared with those observed in the Ctrl group; however, a reduction was noted for these markers in the SD+EE group. The Western blot analysis results presented in Fig.5b. Compared with the Ctrl group, the expression of NLRP3, Caspase-1, and ASC were significantly increased in the SD group (P\u0026lt;0.0001). Compared with the SD group, NLRP3, Caspase-1, and ASC expression were decreased in the SD+EE group.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4 Enriched environment improved SD induced cognitive impairment by regular NLRP3/caspase-1 signal pathway\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine which inflammasome pathway is implicated in neuronal damage induced by chronic SD, an RT-qPCR array was performed. As shown in Fig.6, the mRNA levels of NLRP3、Caspase-1、ASC were significantly upregulated in the SD group compared with the Ctrl group (P\u0026lt;0.0001, P\u0026lt;0.01), while EE treatment resulted in a notable decrease.\u0026nbsp;\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eAdequate sleep is crucial for the body to perform normal behavioral activities, and prolonged periods of insufficient sleep can lead to health issues that affect cardiovascular, nervous, gastrointestinal, and other systems\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. Both animal and human researches have demonstrated that sleep deprivation (SD) can result in cognitive impairment\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. The underlying mechanism involves the induction of an inflammatory response within the central nervous system due to sleep deprivation, which increases the release of pro-inflammatory cytokines (IL-6 and TNF-α) in the brain, leading to neuronal damage and subsequent declines in learning and memory capabilities\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. Due to the side effects associated with conventional treatments, oral medications are often unacceptable by many individuals. Recent research has indicated that enriched environment (EE) serve as a rehabilitation model widely utilized in training for post-stroke cognitive impairment\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. EE has been shown to ameliorate cognitive deficits by down-regulating neuroinflammation\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. Consequently, we investigated the impact of EE on cognitive impairment in mice subjected to chronic SD. Our findings revealed that EE could enhance learning and memory in chronic SD mice by modulating the NLRP3/caspase-1 signaling pathway while alleviating hippocampal neuronal damage.\u003c/p\u003e \u003cp\u003eStudies have demonstrated that sleep facilitates spatial learning and memory through the mediation of hippocampal neurogenesis\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. Conversely, SD activates glial cells, promotes hippocampal neuroinflammation, and ultimately results in a decline in learning and memory abilities\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. To assess the effects of chronic SD on learning and memory in mice, we conducted a novel object recognition experiment. The results indicated that both the duration and frequency of exploration of novel objects were significantly reduced in the SD group, suggesting that chronic SD adversely affects the cognitive abilities of mice. In contrast, both the time spent exploring novel objects and their frequency in the SD\u0026thinsp;+\u0026thinsp;EE group were higher than those observed in the SD group. This finding suggests that EE can ameliorate learning and memory deficits induced by SD. Subsequently, we continued our investigation into the specific mechanisms underlying EE. Previous studies have identified neuroprotective effects associated with EE which can mitigate hippocampal inflammatory response, alleviate pathological conditions affecting damaged hippocampal neurons, and enhance cognitive function in mice\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. Our findings indicated that hippocampus neurons in the SD group exhibited damaged, however, these damages were improved in the SD\u0026thinsp;+\u0026thinsp;EE group. These results are consistent with previous research findings\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFurthermore, studies indicate that chronic SD induced reductions in learning and memory are closely linked to neuroinflammation, wherein the NLRP3 inflammasome plays a pivotal role\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. The NLRP3 inflammatome comprises NOD-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein containing a caspase-recruitment domain, (ASC), as well as its effector molecule precursor, pro-caspase-1. Upon stimulation, the NLRP3 inflammasome is activated to release pro-inflammatory cytokines (IL-1β, IL-18) which contribute to various non-specific inflammatory processes\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. Our study found increased expressions of NLRP3, ASC, and caspase-1 in the hippocampus of mice from the SD group, which was consistent with previous studies\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e, and confirmed that chronic SD induced neuroinflammation. Enriched environment-an intervention integrating sensory, motor, and social stimuli-has been shown to mitigate hippocampal inflammatory response in stroke models while improving cognitive function\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. This suggests that EE can down-regulate expression within the NLRP3/caspase-1 signaling pathway and diminish activation of the NLRP3 inflammasome induced by chronic SD, deprivation thereby ameliorating cognitive impairment.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eEnriched environment enhances learning and memory capabilities in chronic SD mice. Furthermore, EE appears to operate by modulating the NLRP3/caspase-1 signaling pathway. These results suggest that EE may serve as a significant non-pharmacological intervention for treating cognitive impairments associated with SD.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Dalian medical association (Grant Numbers: 2211045).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relavant financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRuiqi Tian:\u0026nbsp;\u003c/strong\u003eWriting \u0026ndash; original draft, Methodology, Data curation, Conceptualization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eYingying Zhou:\u0026nbsp;\u003c/strong\u003eWriting \u0026ndash; original draft, Methodology,Formal analysis,Data curation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTang Wei:\u0026nbsp;\u003c/strong\u003eWriting \u0026ndash;review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDandan Y\u003c/strong\u003e\u003cstrong\u003eu:\u003c/strong\u003e Writing \u0026ndash;review \u0026amp; editing, Conceptualization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be made available on request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of Dalian University Affiliated Xinhua Hospital (2022.11.20/No.2022-086-01).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number: not applicable.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors declare no conflicts of interest.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThanks to the BioRender for providing the graphic technical support (figure1-2).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eBaranwal, N., Yu, P. K., \u0026amp; Siegel, N. S. (2023). 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Enriched environment-induced neuroplasticity in ischemic stroke and its underlying mechanisms. \u003cem\u003eFrontiers in cellular neuroscience\u003c/em\u003e, \u003cem\u003e17\u003c/em\u003e, 1210361. https://doi.org/10.3389/fncel.2023.1210361.\u003c/li\u003e\n \u003cli\u003eZhang, X., Yuan, M., Yang, S., Chen, X., Wu, J., Wen, M., Yan, K., \u0026amp; Bi, X. (2021). Enriched environment improves post-stroke cognitive impairment and inhibits neuroinflammation and oxidative stress by activating Nrf2-ARE pathway. \u003cem\u003eThe International journal of neuroscience\u003c/em\u003e, \u003cem\u003e131\u003c/em\u003e(7), 641\u0026ndash;649. https://doi.org/10.1080/00207454.2020.1797722.\u003c/li\u003e\n \u003cli\u003eHavekes, R., \u0026amp; Abel, T. (2017). 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(2023). NLRP3 inflammasome in cognitive impairment and pharmacological properties of its inhibitors. \u003cem\u003eTranslational neurodegeneration\u003c/em\u003e, \u003cem\u003e12\u003c/em\u003e(1), 49. https://doi.org/10.1186/s40035-023-00381-x.\u003c/li\u003e\n \u003cli\u003eFan, K., Yang, J., Gong, W. Y., Pan, Y. C., Zheng, P., \u0026amp; Yue, X. F. (2021). NLRP3 inflammasome activation mediates sleep deprivation-induced pyroptosis in mice. \u003cem\u003ePeerJ\u003c/em\u003e, \u003cem\u003e9\u003c/em\u003e, e11609. https://doi.org/10.7717/peerj.11609.\u003c/li\u003e\n \u003cli\u003eGu, J. Y., Xu, Y. W., Feng, L. P., Dong, J., Zhao, L. Q., Liu, C., Wang, H. Y., Zhang, X. Y., Song, C., \u0026amp; Wang, C. H. (2021). Enriched environment mitigates depressive behavior by changing the inflammatory activation phenotype of microglia in the hippocampus of depression model rats. \u003cem\u003eBrain research bulletin\u003c/em\u003e, \u003cem\u003e177\u003c/em\u003e, 252\u0026ndash;262. https://doi.org/10.1016/j.brainresbull.2021.10.005.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"enriched environment, chronic sleep deprivation, cognitive impairment NLRP3/Caspase-1, neuroinflammation","lastPublishedDoi":"10.21203/rs.3.rs-5654841/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5654841/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eEnriched environment (EE) serves as a crucial intervention strategy that enhances central neural plasticity and facilitates motor, sensory, cognitive, and social stimulation. This multifaceted approach ultimately promotes the recovery of cognitive functions in the brain. However, the mechanism is unclear. Neuroinflammation has been proven associated with cognitive impairment. NLRP3 plays a crucial role in neuroinflammation induced sleep deprivation. Our study was to investigate the effect of EE on the chronic sleep deprivation mice by NLRP3/Caspase-1 signal pathway. In this study, the modified multi-platform method (MMPM) was applied to establish the chronic sleep deprivation model. The novel object recognition test (NORT) was used to assess the ability of learning and memory. The evaluation of hippocampal neuronal injury was achieved by performing Nissl staining and immunofluorescence. To evaluate the protein and mRNA expression of NLRP3, Caspase-1, and ASC, western blot and RT-qPCR were utilized. Compared with the SD group, the mice in the SD+ EE group demonstrated longer and more often to explore novel objects and alleviating neuroinflammation in the hippocampal. The mRNA and protein of NLRP3, Caspase-1, and ASC were decreased in the SD+ EE group. Enriched environment can improve the cognitive impairment induced by chronic sleep deprivation through the NLRP3/Caspase-1 signal pathway.\u003c/p\u003e","manuscriptTitle":"Effect of enriched environment on the chronic sleep deprivation mice by NLRP3/Caspase-1 signal pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-20 12:24:37","doi":"10.21203/rs.3.rs-5654841/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"712ee901-2840-4342-b1e5-84ce93fdc79e","owner":[],"postedDate":"December 20th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-01-16T14:38:36+00:00","versionOfRecord":[],"versionCreatedAt":"2024-12-20 12:24:37","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5654841","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5654841","identity":"rs-5654841","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}